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Sulfation pathways from red to green

  • Süleyman Günal
    Affiliations
    Botanical Institute, Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne 50674, Germany
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  • Rebecca Hardman
    Affiliations
    Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom
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  • Stanislav Kopriva
    Correspondence
    To whom correspondence may be addressed. Tel.:49-211-4708530
    Affiliations
    Botanical Institute, Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Cologne 50674, Germany
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  • Jonathan Wolf Mueller
    Correspondence
    To whom correspondence may be addressed. Tel.:44-121-4158819
    Affiliations
    Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

    Centre for Endocrinology, Diabetes and Metabolism (CEDAM), Birmingham Health Partners, Birmingham B15 2TH, United Kingdom
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Open AccessPublished:July 02, 2019DOI:https://doi.org/10.1074/jbc.REV119.007422
      Sulfur is present in the amino acids cysteine and methionine and in a large range of essential coenzymes and cofactors and is therefore essential for all organisms. It is also a constituent of sulfate esters in proteins, carbohydrates, and numerous cellular metabolites. The sulfation and desulfation reactions modifying a variety of different substrates are commonly known as sulfation pathways. Although relatively little is known about the function of most sulfated metabolites, the synthesis of activated sulfate used in sulfation pathways is essential in both animal and plant kingdoms. In humans, mutations in the genes encoding the sulfation pathway enzymes underlie a number of developmental aberrations, and in flies and worms, their loss-of-function is fatal. In plants, a lower capacity for synthesizing activated sulfate for sulfation reactions results in dwarfism, and a complete loss of activated sulfate synthesis is also lethal. Here, we review the similarities and differences in sulfation pathways and associated processes in animals and plants, and we point out how they diverge from bacteria and yeast. We highlight the open questions concerning localization, regulation, and importance of sulfation pathways in both kingdoms and the ways in which findings from these “red” and “green” experimental systems may help reciprocally address questions specific to each of the systems.

      Introduction

      Sulfur (S) is an essential nutrient for all life forms. It is present in a plethora of metabolites of primary and secondary metabolism, most prominently in the amino acids cysteine and methionine, and cofactors such as iron–sulfur clusters, lipoic acid, and CoA. In the majority of these metabolites, sulfur is present in its reduced form of organic thiols; however, some compounds contain S in its oxidized form of sulfate (
      • Beinert H.
      A tribute to sulfur.
      ,
      • Takahashi H.
      • Kopriva S.
      • Giordano M.
      • Saito K.
      • Hell R.
      Sulfur assimilation in photosynthetic organisms: molecular functions and regulations of transporters and assimilatory enzymes.
      ). Sulfate is transferred to suitable substrates onto hydroxyl or amino groups by sulfotransferases (
      • Coughtrie M.W.H.
      Function and organization of the human cytosolic sulfotransferase (SULT) family.
      ,
      • Hirschmann F.
      • Krause F.
      • Papenbrock J.
      The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions.
      ). These biological sulfation reactions as well as desulfation catalyzed by sulfatases are often denoted as sulfation pathways (Fig. 1) (
      • Mueller J.W.
      • Gilligan L.C.
      • Idkowiak J.
      • Arlt W.
      • Foster P.A.
      The regulation of steroid action by sulfation and desulfation.
      ,
      • Mueller J.W.
      • Shafqat N.
      Adenosine-5′-phosphosulfate–a multifaceted modulator of bifunctional 3′-phospho-adenosine-5′-phosphosulfate synthases and related enzymes.
      ).
      Figure thumbnail gr1
      Figure 1Red and green sulfation pathways. A, sulfate is taken up by various sulfate transporters; in plants, some of them transport sulfate into the chloroplast (1). Sulfate activation occurs via animal bifunctional PAPS synthases (2) that shuttle between cytoplasm and nucleus or plant ATP sulfurylase (3) and APS kinase (4) isoforms that are localized in cytoplasm and the chloroplast. PAPS serves as a substrate for cytoplasmic sulfation pathways (5), where PAP is produced. Sulfated compounds can then be de-sulfated by sulfatases (6), enzymes that are absent in plants, or they are secreted via OATPs (7). Two animal PAPS transporters (8) channel PAPS into the Golgi apparatus where many carbohydrate and protein sulfotransferases modify macromolecules for secretion. Although plant protein sulfotransferases are known that reside in the Golgi, an analogous transporter (8) has not yet been identified. Human PAP phosphatases (9) are in the Golgi and the cytoplasm; plant PAP phosphatases are, however, localized in the mitochondrion and the chloroplast. Dedicated PAP(S) transporter in the chloroplast (10) and the mitochondrion (11) deliver PAPS to the cytoplasm and play an important role in the degradation of PAP. In plants, APS represents a branching point where reductive biosynthetic pathways diverge (12). B, examples of structures of sulfated metabolites.
      The activated sulfate for the sulfation pathways, 3′-phosphoadenosine 5-phosphosulfate (PAPS),
      The abbreviations used are: PAPS
      3′-phosphoadenosine 5′-phosphosulfate
      APS
      adenosine 5′-phosphosulfate
      ATPS
      ATP sulfurylase
      PAPSS
      PAPS synthase
      TPST
      tyrosylprotein sulfotransferase
      SOT
      sulfotransferase
      SULT
      SULT, cytosolic sulfotransferase
      DHEA
      dehydroepiandrosterone
      DHEAS
      dehydroepiandrosterone sulfate
      STS
      steroid sulfatase
      PDB
      Protein Data Bank
      PAP
      3′-phosphoadenosine-5′-phosphate
      gPAPP
      Golgi PAP phosphatase
      MSD
      multiple sulfatase deficiency
      OATP
      organic anion transporter
      SNP
      single-nucleotide polymorphism.
      is formed from sulfate by two ATP-dependent steps: adenylation, i.e. the transfer of the AMP moiety of ATP to sulfate to form adenosine 5′-phosphosulfate (APS) by ATP sulfurylase (ATPS), and the phosphorylation of APS at its 3′-OH group by APS kinase. The two enzymes are either fused into a single enzyme PAPS synthase (PAPSS) in the animal kingdom or occur as independent proteins in the green lineage (
      • Patron N.J.
      • Durnford D.G.
      • Kopriva S.
      Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.
      ). The by-product of PAPS-dependent sulfation reactions, 3′-phosphoadenosine 5-phosphate (PAP), is finally dephosphorylated to AMP by 3′-nucleotidases. This reaction to remove PAP is important beyond the sulfation pathways, as PAP accumulation has many additional physiological effects (
      • Chan K.X.
      • Wirtz M.
      • Phua S.Y.
      • Estavillo G.M.
      • Pogson B.J.
      Balancing metabolites in drought: the sulfur assimilation conundrum.
      ,
      • Lee B.R.
      • Huseby S.
      • Koprivova A.
      • Chételat A.
      • Wirtz M.
      • Mugford S.T.
      • Navid E.
      • Brearley C.
      • Saha S.
      • Mithen R.
      • Hell R.
      • Farmer E.E.
      • Kopriva S.
      Effects of fou8/fry1 mutation on sulfur metabolism: is decreased internal sulfate the trigger of sulfate starvation response?.
      ).
      Sulfate activation to APS or PAPS is a prerequisite not only for sulfation pathways but also for primary sulfate assimilation in plants, algae, bacteria, and fungi (
      • Takahashi H.
      • Kopriva S.
      • Giordano M.
      • Saito K.
      • Hell R.
      Sulfur assimilation in photosynthetic organisms: molecular functions and regulations of transporters and assimilatory enzymes.
      ). Particularly fungi and some bacteria require PAPS for sulfate reduction and synthesis of cysteine. In these organisms, the activated sulfate in PAPS is reduced to sulfite by PAPS reductase, and after further reduction to sulfide, it is incorporated into cysteine (
      • Carroll K.S.
      • Gao H.
      • Chen H.
      • Stout C.D.
      • Leary J.A.
      • Bertozzi C.R.
      A conserved mechanism for sulfonucleotide reduction.
      ). The green lineage as well as a large number of bacterial taxa, however, use APS for sulfate reduction by APS reductase, whereas Metazoa do not possess the ability to reduce sulfate and are dependent on sulfur-containing amino acids in their diet (
      • Patron N.J.
      • Durnford D.G.
      • Kopriva S.
      Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.
      ). In sulfate-reducing organisms, sulfation pathways compete with the primary sulfate reduction for activated sulfate, and the two branches of sulfur metabolism must be well-coordinated (
      • Mugford S.G.
      • Lee B.R.
      • Koprivova A.
      • Matthewman C.
      • Kopriva S.
      Control of sulfur partitioning between primary and secondary metabolism.
      ). The ability to reduce sulfate is thus the major difference in sulfur metabolism between animals and plants and impacts on other metabolic branches, including sulfation pathways.
      In plants, traditionally, studies of sulfur metabolism concentrated on reductive, primary sulfur metabolism. For the red kingdom, in his scholarly “Tribute to Sulfur,” Helmut Beinert (
      • Beinert H.
      A tribute to sulfur.
      ) wrote that sulfate “is of limited use to higher organisms except for sulfation and detoxification reactions,” without any further discussion of the topic. Since then, things have dramatically changed with growing evidence of the importance of sulfation pathways in both kingdoms. In addition, convergent findings in the green (plant) and red (animal) biochemistry of sulfur, e.g. recognition of hydrogen sulfide as a gaseous signal (
      • Calderwood A.
      • Kopriva S.
      Hydrogen sulfide in plants: from dissipation of excess sulfur to signaling molecule.
      ,
      • Kimura H.
      The physiological role of hydrogen sulfide and beyond.
      ), revealed the value of comparative analysis of the same pathways in very different models. Here, we compare the mechanisms the two lineages, red and green, evolved to perform and control sulfation pathways. Given their importance for the metabolism of specific compounds and for the general sulfur metabolism, we extend the scope of our comparison to the enzymes providing the active sulfate and removing the by-product PAP. We aim to identify open questions common to both humans and plants as well as questions where knowledge from one lineage might be useful to inform research in the other.

      Activation of sulfate to PAPS

      The organification or activation of sulfate to PAPS by ATPS and APS kinase initiates sulfation pathways (
      • Lipmann F.
      Biological sulfate activation and transfer.
      ). The catalytic and substrate-binding sites of the ATP sulfurylases from plants and animals are highly conserved (
      • Jez J.M.
      • Ravilious G.E.
      • Herrmann J.
      Structural biology and regulation of the plant sulfation pathway.
      ); however, subsequent reactions and the enzymatic blueprints vary greatly between different lineages (
      • Patron N.J.
      • Durnford D.G.
      • Kopriva S.
      Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.
      ). Also, the localization and regulation of ATP sulfurylase and APS kinase show lineage-specific differences.

      Plant ATP sulfurylase and APS kinase

      In plants, ATPS occurs as a homodimer, consisting of two 48-kDa monomers (
      • Herrmann J.
      • Ravilious G.E.
      • McKinney S.E.
      • Westfall C.S.
      • Lee S.G.
      • Baraniecka P.
      • Giovannetti M.
      • Kopriva S.
      • Krishnan H.B.
      • Jez J.M.
      Structure and mechanism of soybean ATP sulfurylase and the committed step in plant sulfur assimilation.
      ). Plants and algae have multiple ATPS isoforms localized in chloroplast and cytosol; the model plant Arabidopsis thaliana possesses four (
      • Rotte C.
      • Leustek T.
      Differential subcellular localization and expression of ATP sulfurylase and 5′-adenylylsulfate reductase during ontogenesis of Arabidopsis leaves indicates that cytosolic and plastid forms of ATP sulfurylase may have specialized functions.
      ). In some plants, such as potato, distinct cytosolic and plastidic isoforms can be identified (
      • Klonus D.
      • Höfgen R.
      • Willmitzer L.
      • Riesmeier J.W.
      Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant.
      ), whereas all four Arabidopsis isoforms possess N-terminal chloroplast-targeting peptides. Cytosolic activity is caused by alternative translation of the ATPS2 transcript, producing two different proteins: one with the target peptide transported into the chloroplast and one without the peptide located in the cytosol (
      • Bohrer A.S.
      • Yoshimoto N.
      • Sekiguchi A.
      • Rykulski N.
      • Saito K.
      • Takahashi H.
      Alternative translational initiation of ATP sulfurylase underlying dual localization of sulfate assimilation pathways in plastids and cytosol in Arabidopsis thaliana.
      ). The reason for the dual localization of the ATPS seems to be the need for both APS for sulfate reduction in plastids and PAPS for sulfotransferases in the cytosol (
      • Bohrer A.S.
      • Kopriva S.
      • Takahashi H.
      Plastid-cytosol partitioning and integration of metabolic pathways for APS/PAPS biosynthesis in Arabidopsis thaliana.
      ). However, given the major role of plastids for synthesis of PAPS and the presence of PAPS transporters in plastid envelopes, the role of cytosolic ATPS is not obvious. Interestingly, both human PAPS synthases are also regulated on the level of cellular localization of the enzyme between the nucleus and cytosol, even though a function of PAPS in the nucleus is completely unknown (
      • Schröder E.
      • Gebel L.
      • Eremeev A.A.
      • Morgner J.
      • Grum D.
      • Knauer S.K.
      • Bayer P.
      • Mueller J.W.
      Human PAPS synthase isoforms are dynamically regulated enzymes with access to nucleus and cytoplasm.
      ).
      Because of its position at the beginning of the pathway, ATPS is a good candidate for controlling sulfate assimilation. Indeed, early findings in Brassica napus showed that ATPS activity and transcript levels were down-regulated by downstream products of sulfate assimilation, cysteine and GSH, and were up-regulated by sulfate starvation (
      • Lappartient A.G.
      • Vidmar J.J.
      • Leustek T.
      • Glass A.D.
      • Touraine B.
      Inter-organ signaling in plants: regulation of ATP sulfurylase and sulfate transporter genes expression in roots mediated by phloem-translocated compound.
      ). These findings played a key role in formulating the concept of demand-driven regulation of sulfur metabolism in plants (
      • Koprivova A.
      • Kopriva S.
      Molecular mechanisms of regulation of sulfate assimilation: first steps on a long road.
      ,
      • Lappartient A.G.
      • Touraine B.
      Demand-driven control of root ATP sulfurylase activity and SO42− uptake in intact canola (the role of phloem-translocated glutathione).
      ). However, the subsequent enzyme in the primary sulfate assimilation pathway in plants, the APS reductase, is regulated more strongly and was shown by metabolic flux control analysis to be the major control point of the pathway (
      • Vauclare P.
      • Kopriva S.
      • Fell D.
      • Suter M.
      • Sticher L.
      • von Ballmoos P.
      • Krähenbuhl U.
      • den Camp R.O.
      • Brunold C.
      Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine 5′-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols.
      ,
      • Scheerer U.
      • Haensch R.
      • Mendel R.R.
      • Kopriva S.
      • Rennenberg H.
      • Herschbach C.
      Sulphur flux through the sulphate assimilation pathway is differently controlled by adenosine 5′-phosphosulphate reductase under stress and in transgenic poplar plants overexpressing gamma-ECS, SO, or APR.
      • Kopriva S.
      • Koprivova A.
      Plant adenosine 5′-phosphosulphate reductase: the past, the present, and the future.
      ). Indeed, a recent modeling approach showed that the pattern of flux control is dynamic and not static (
      • Feldman-Salit A.
      • Veith N.
      • Wirtz M.
      • Hell R.
      • Kummer U.
      Distribution of control in the sulfur assimilation in Arabidopsis thaliana depends on environmental conditions.
      ); changes occur with differing environmental conditions, and control resides mostly at APS reductase or downstream sulfite reductase and not ATPS (
      • Feldman-Salit A.
      • Veith N.
      • Wirtz M.
      • Hell R.
      • Kummer U.
      Distribution of control in the sulfur assimilation in Arabidopsis thaliana depends on environmental conditions.
      ). ATPS, however, still contributes to the control of sulfate accumulation in Arabidopsis (
      • Koprivova A.
      • Giovannetti M.
      • Baraniecka P.
      • Lee B.R.
      • Grondin C.
      • Loudet O.
      • Kopriva S.
      Natural variation in the ATPS1 isoform of ATP sulfurylase contributes to the control of sulfate levels in Arabidopsis.
      ) and to the response to sulfate starvation as a target of microRNA miR395 (
      • Kawashima C.G.
      • Matthewman C.A.
      • Huang S.
      • Lee B.R.
      • Yoshimoto N.
      • Koprivova A.
      • Rubio-Somoza I.
      • Todesco M.
      • Rathjen T.
      • Saito K.
      • Takahashi H.
      • Dalmay T.
      • Kopriva S.
      Interplay of SLIM1 and miR395 in the regulation of sulfate assimilation in Arabidopsis.
      ). Arabidopsis ATPS1 and ATPS3 isoforms are part of the regulatory network of glucosinolate synthesis, and atps1 mutants show a lower concentration of these sulfated secondary compounds (
      • Koprivova A.
      • Giovannetti M.
      • Baraniecka P.
      • Lee B.R.
      • Grondin C.
      • Loudet O.
      • Kopriva S.
      Natural variation in the ATPS1 isoform of ATP sulfurylase contributes to the control of sulfate levels in Arabidopsis.
      ,
      • Yatusevich R.
      • Mugford S.G.
      • Matthewman C.
      • Gigolashvili T.
      • Frerigmann H.
      • Delaney S.
      • Koprivova A.
      • Flügge U.I.
      • Kopriva S.
      Genes of primary sulfate assimilation are part of the glucosinolate biosynthetic network in Arabidopsis thaliana.
      ). Glucosinolates are part of the plant immune response to pathogens and herbivores as well as plant natural products responsible for smell, taste, and health effects of cruciferous vegetables, but also anti-nutrients for animal feed (
      • Halkier B.A.
      • Gershenzon J.
      Biology and biochemistry of glucosinolates.
      ).
      Although essential and sufficient for sulfate reduction, ATPS has to be coupled with the APS kinase for sulfation pathways. This enzyme, ubiquitous in nature and highly conserved in structure and sequence, shows the same localization in plants as ATPS. Arabidopsis possesses four APS kinase genes, which encode three plastidic and one (APK3) cytosolic isoform (
      • Mugford S.G.
      • Yoshimoto N.
      • Reichelt M.
      • Wirtz M.
      • Hill L.
      • Mugford S.T.
      • Nakazato Y.
      • Noji M.
      • Takahashi H.
      • Kramell R.
      • Gigolashvili T.
      • Flügge U.I.
      • Wasternack C.
      • Gershenzon J.
      • Hell R.
      • et al.
      Disruption of adenosine-5′-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites.
      ). APS kinase phosphorylates APS produced by ATPS and thus competes with APS reductase for this substrate. The two enzymes represent entries into the two branches of sulfate assimilation: a primary reductive assimilation pathway and a secondary oxidized sulfur metabolism involving sulfation pathways (
      • Kopriva S.
      • Mugford S.G.
      • Baraniecka P.
      • Lee B.R.
      • Matthewman C.A.
      • Koprivova A.
      Control of sulfur partitioning between primary and secondary metabolism in Arabidopsis.
      ). The secondary pathway has been rarely investigated, because PAPS production is not necessary for the primary sulfate reduction and synthesis of cysteine and GSH (
      • Kopriva S.
      • Mugford S.G.
      • Baraniecka P.
      • Lee B.R.
      • Matthewman C.A.
      • Koprivova A.
      Control of sulfur partitioning between primary and secondary metabolism in Arabidopsis.
      ). However, even though APS kinase is part of the secondary sulfate assimilation pathway, it is vital for plant survival (
      • Mugford S.G.
      • Yoshimoto N.
      • Reichelt M.
      • Wirtz M.
      • Hill L.
      • Mugford S.T.
      • Nakazato Y.
      • Noji M.
      • Takahashi H.
      • Kramell R.
      • Gigolashvili T.
      • Flügge U.I.
      • Wasternack C.
      • Gershenzon J.
      • Hell R.
      • et al.
      Disruption of adenosine-5′-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites.
      ,
      • Mugford S.G.
      • Matthewman C.A.
      • Hill L.
      • Kopriva S.
      Adenosine-5′-phosphosulfate kinase is essential for Arabidopsis viability.
      ). Interestingly, it is the loss of two plastidic APS kinase isoforms APK1 and APK2 that results in strongly reduced accumulation of sulfated metabolites, such as glucosinolates, and not the disruption of the cytosolic enzyme APK3 (
      • Mugford S.G.
      • Yoshimoto N.
      • Reichelt M.
      • Wirtz M.
      • Hill L.
      • Mugford S.T.
      • Nakazato Y.
      • Noji M.
      • Takahashi H.
      • Kramell R.
      • Gigolashvili T.
      • Flügge U.I.
      • Wasternack C.
      • Gershenzon J.
      • Hell R.
      • et al.
      Disruption of adenosine-5′-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites.
      ). This, on the one hand, again challenges the significance of cytosolic APS and PAPS synthesis; on the other hand, it shows the necessity of intracellular PAPS transport. Indeed, a PAPS transporter has been identified in chloroplast envelope membranes, part of the glucosinolate co-expression network, whose mutation shows a phenotype similar to apk1 apk2 mutants (see below and Ref.
      • Gigolashvili T.
      • Geier M.
      • Ashykhmina N.
      • Frerigmann H.
      • Wulfert S.
      • Krueger S.
      • Mugford S.G.
      • Kopriva S.
      • Haferkamp I.
      • Flügge U.I.
      The Arabidopsis thylakoid ADP/ATP carrier TAAC has an additional role in supplying plastidic phosphoadenosine 5′-phosphosulfate to the cytosol.
      ).
      The apk1 apk2 double knockout turned out to be an excellent tool to dissect the importance of secondary sulfate assimilation (
      • Mugford S.G.
      • Lee B.R.
      • Koprivova A.
      • Matthewman C.
      • Kopriva S.
      Control of sulfur partitioning between primary and secondary metabolism.
      ,
      • Mugford S.G.
      • Yoshimoto N.
      • Reichelt M.
      • Wirtz M.
      • Hill L.
      • Mugford S.T.
      • Nakazato Y.
      • Noji M.
      • Takahashi H.
      • Kramell R.
      • Gigolashvili T.
      • Flügge U.I.
      • Wasternack C.
      • Gershenzon J.
      • Hell R.
      • et al.
      Disruption of adenosine-5′-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites.
      ). The reduced synthesis of PAPS in apk1 apk2 results in a shift of sulfur flux from the secondary to the primary sulfur assimilation pathway, increased accumulation of reduced sulfur compounds, and highly-reduced glucosinolate levels (
      • Mugford S.G.
      • Lee B.R.
      • Koprivova A.
      • Matthewman C.
      • Kopriva S.
      Control of sulfur partitioning between primary and secondary metabolism.
      ,
      • Mugford S.G.
      • Yoshimoto N.
      • Reichelt M.
      • Wirtz M.
      • Hill L.
      • Mugford S.T.
      • Nakazato Y.
      • Noji M.
      • Takahashi H.
      • Kramell R.
      • Gigolashvili T.
      • Flügge U.I.
      • Wasternack C.
      • Gershenzon J.
      • Hell R.
      • et al.
      Disruption of adenosine-5′-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites.
      ). Furthermore, all components of the glucosinolate synthesis pathway were coordinately up-regulated leading to substantial accumulation of the desulfo-precursors of glucosinolates (
      • Mugford S.G.
      • Yoshimoto N.
      • Reichelt M.
      • Wirtz M.
      • Hill L.
      • Mugford S.T.
      • Nakazato Y.
      • Noji M.
      • Takahashi H.
      • Kramell R.
      • Gigolashvili T.
      • Flügge U.I.
      • Wasternack C.
      • Gershenzon J.
      • Hell R.
      • et al.
      Disruption of adenosine-5′-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites.
      ). Although glucosinolates and other sulfated secondary metabolites seem not to be essential for Arabidopsis growth, the apk1 apk2 mutants are significantly smaller than the WT plants (
      • Mugford S.G.
      • Yoshimoto N.
      • Reichelt M.
      • Wirtz M.
      • Hill L.
      • Mugford S.T.
      • Nakazato Y.
      • Noji M.
      • Takahashi H.
      • Kramell R.
      • Gigolashvili T.
      • Flügge U.I.
      • Wasternack C.
      • Gershenzon J.
      • Hell R.
      • et al.
      Disruption of adenosine-5′-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites.
      ). When additional APS kinase genes, APK3 or APK4, are mutated, the semi-dwarf phenotype is even stronger (
      • Mugford S.G.
      • Matthewman C.A.
      • Hill L.
      • Kopriva S.
      Adenosine-5′-phosphosulfate kinase is essential for Arabidopsis viability.
      ). The generation of multiple mutations in APS kinase genes revealed that the enzyme is essential for Arabidopsis growth (
      • Mugford S.G.
      • Matthewman C.A.
      • Hill L.
      • Kopriva S.
      Adenosine-5′-phosphosulfate kinase is essential for Arabidopsis viability.
      ). Which acceptors of PAPS are essential remains to be determined, as neither the glucosinolates nor the sulfated peptide hormones (such as the phytosulfokines (
      • Matsubayashi Y.
      • Sakagami Y.
      Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L.
      ), root growth factors (
      • Matsuzaki Y.
      • Ogawa-Ohnishi M.
      • Mori A.
      • Matsubayashi Y.
      Secreted peptide signals required for maintenance of root stem cell niche in Arabidopsis.
      ), or Casparian strip integrity factors (
      • Nakayama T.
      • Shinohara H.
      • Tanaka M.
      • Baba K.
      • Ogawa-Ohnishi M.
      • Matsubayashi Y.
      A peptide hormone required for Casparian strip diffusion barrier formation in Arabidopsis roots.
      )) discovered so far, seem to be crucial.
      APS kinase is regulated on both transcriptional and post-transcriptional levels. The genes are part of the glucosinolate transcriptional network, under control by a family of six MYB transcription factors in Arabidopsis and thus co-expressed with genes providing the main substrate for PAPS (
      • Yatusevich R.
      • Mugford S.G.
      • Matthewman C.
      • Gigolashvili T.
      • Frerigmann H.
      • Delaney S.
      • Koprivova A.
      • Flügge U.I.
      • Kopriva S.
      Genes of primary sulfate assimilation are part of the glucosinolate biosynthetic network in Arabidopsis thaliana.
      ). In addition, according to the demand-driven concept, sulfate starvation represses APS kinase to channel the scarce sulfur to the primary sulfate assimilation. Excitingly, redox regulation of APS kinase enzyme activity through dimerization of the protein and formation of disulfide bridges has been revealed in a structural analysis (
      • Ravilious G.E.
      • Nguyen A.
      • Francois J.A.
      • Jez J.M.
      Structural basis and evolution of redox regulation in plant adenosine-5′-phosphosulfate kinase.
      ). Reducing conditions leading to monomerization of the protein increase the catalytic efficiency, including alleviation of enzyme inhibition by its substrate APS (
      • Ravilious G.E.
      • Nguyen A.
      • Francois J.A.
      • Jez J.M.
      Structural basis and evolution of redox regulation in plant adenosine-5′-phosphosulfate kinase.
      ). This is particularly interesting as it complements the redox regulation important for control of the reductive branch of sulfate assimilation (
      • Takahashi H.
      • Kopriva S.
      • Giordano M.
      • Saito K.
      • Hell R.
      Sulfur assimilation in photosynthetic organisms: molecular functions and regulations of transporters and assimilatory enzymes.
      ). APS reductase is activated by oxidation, e.g. during abiotic stress, which leads to higher activity and synthesis of cysteine and GSH (
      • Bick J.A.
      • Setterdahl A.T.
      • Knaff D.B.
      • Chen Y.
      • Pitcher L.H.
      • Zilinskas B.A.
      • Leustek T.
      Regulation of the plant-type 5′-adenylyl sulfate reductase by oxidative stress.
      ). Accordingly, recombinant APS reductase is inactivated by incubation with reductants (
      • Kopriva S.
      • Koprivova A.
      Plant adenosine 5′-phosphosulphate reductase: the past, the present, and the future.
      ). APS reductase and APS kinase occupy the opposite branches of sulfate assimilation from APS (
      • Mugford S.G.
      • Lee B.R.
      • Koprivova A.
      • Matthewman C.
      • Kopriva S.
      Control of sulfur partitioning between primary and secondary metabolism.
      ). Considering that APS reductase is activated by oxidation (
      • Bick J.A.
      • Setterdahl A.T.
      • Knaff D.B.
      • Chen Y.
      • Pitcher L.H.
      • Zilinskas B.A.
      • Leustek T.
      Regulation of the plant-type 5′-adenylyl sulfate reductase by oxidative stress.
      ), the reciprocal activation of APS kinase by reduction indicates that this redox mechanism may control the distribution of sulfur fluxes between primary and secondary sulfur metabolism (
      • Jez J.M.
      • Ravilious G.E.
      • Herrmann J.
      Structural biology and regulation of the plant sulfation pathway.
      ).

      Bifunctional PAPS synthases in animals

      In contrast to plants with separate proteins possessing ATPS and APS kinase activities, vertebrate and invertebrate genomes feature these activities in single polypeptides with a C-terminal ATPS domain and an N-terminal APS kinase domain (
      • Patron N.J.
      • Durnford D.G.
      • Kopriva S.
      Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.
      ,
      • van den Boom J.
      • Heider D.
      • Martin S.R.
      • Pastore A.
      • Mueller J.W.
      3′-Phosphoadenosine 5′-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding.
      ). These so-called PAPS synthases are strictly conserved within animal genomes with a single gene in invertebrates and a PAPSS1 and PAPSS2 gene pair in vertebrates (
      • van den Boom J.
      • Heider D.
      • Martin S.R.
      • Pastore A.
      • Mueller J.W.
      3′-Phosphoadenosine 5′-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding.
      ). An additional PAPSS2 gene copy in teleost fish and mammalian-specific splice forms of PAPSS2 are minor extensions to this rule (
      • van den Boom J.
      • Heider D.
      • Martin S.R.
      • Pastore A.
      • Mueller J.W.
      3′-Phosphoadenosine 5′-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding.
      ). It is interesting to ask why this second sulfate-activating complex has evolved and has been strictly maintained in animals. Possibly, this has been a requirement for the expansion of sulfation pathways in animals. Two PAPS synthase genes would allow us to selectively support different sulfation pathways, either via transcriptional co-regulation (
      • Foster P.A.
      • Mueller J.W.
      Sulfation pathways: insights into steroid sulfation and desulfation pathways.
      ), transient protein interaction (
      • Mueller J.W.
      • Idkowiak J.
      • Gesteira T.F.
      • Vallet C.
      • Hardman R.
      • van den Boom J.
      • Dhir V.
      • Knauer S.K.
      • Rosta E.
      • Arlt W.
      Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.
      ), or yet-to-be described regulatory mechanisms.
      Such a subfunctionalization is also indicated by the fact that PAPS synthases 1 and 2 cannot complement each other. Genetic defects for PAPSS1 have never been reported so far. However, mutations in the gene coding for PAPSS2 are associated with bone and cartilage malformations as well as a steroid sulfation defect (
      • Oostdijk W.
      • Idkowiak J.
      • Mueller J.W.
      • House P.J.
      • Taylor A.E.
      • O’Reilly M.W.
      • Hughes B.A.
      • de Vries M.C.
      • Kant S.G.
      • Santen G.W.
      • Verkerk A.J.
      • Uitterlinden A.G.
      • Wit J.M.
      • Losekoot M.
      • Arlt W.
      PAPSS2 deficiency causes androgen excess via impaired DHEA sulfation–in vitro and in vivo studies in a family harboring two novel PAPSS2 mutations.
      ); this is discussed in detail below.
      The mechanistic question is what makes the two PAPS synthase enzymes so different. Certainly, the two genes are differentially regulated to a certain extent, and transcriptional co-regulation with certain sulfotransferases has been reported (
      • Sonoda J.
      • Xie W.
      • Rosenfeld J.M.
      • Barwick J.L.
      • Guzelian P.S.
      • Evans R.M.
      Regulation of a xenobiotic sulfonation cascade by nuclear pregnane X receptor (PXR).
      ,
      • Kim M.S.
      • Shigenaga J.
      • Moser A.
      • Grunfeld C.
      • Feingold K.R.
      Suppression of DHEA sulfotransferase (Sult2A1) during the acute-phase response.
      ). However, correlations of transcript levels between PAPS synthases and sulfotransferases are only weak (Fig. 2), and both PAPS synthases are expressed at the same time in certain tissues (
      • Fagerberg L.
      • Hallström B.M.
      • Oksvold P.
      • Kampf C.
      • Djureinovic D.
      • Odeberg J.
      • Habuka M.
      • Tahmasebpoor S.
      • Danielsson A.
      • Edlund K.
      • Asplund A.
      • Sjöstedt E.
      • Lundberg E.
      • Szigyarto C.A.
      • Skogs M.
      • et al.
      Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics.
      ). Different subcellular localization was believed to be of functional significance (
      • Besset S.
      • Vincourt J.B.
      • Amalric F.
      • Girard J.P.
      Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.
      ), but the conserved nuclear localization and export signals were identified both in PAPSS1 and PAPSS2 (
      • Schröder E.
      • Gebel L.
      • Eremeev A.A.
      • Morgner J.
      • Grum D.
      • Knauer S.K.
      • Bayer P.
      • Mueller J.W.
      Human PAPS synthase isoforms are dynamically regulated enzymes with access to nucleus and cytoplasm.
      ). Diverse catalytic activities were purported to explain the observed functional difference, based on only a 5-fold difference in kcat/Km values when treating bifunctional PAPS synthases as pseudo one-step Michaelis-Menten enzymes (
      • Fuda H.
      • Shimizu C.
      • Lee Y.C.
      • Akita H.
      • Strott C.A.
      Characterization and expression of human bifunctional 3′-phosphoadenosine 5′-phosphosulphate synthase isoforms.
      ). This characteristic could not be reproduced when assaying APS kinase only, which catalyzes the rate-limiting step of overall PAPS biosynthesis (
      • Mueller J.W.
      • Idkowiak J.
      • Gesteira T.F.
      • Vallet C.
      • Hardman R.
      • van den Boom J.
      • Dhir V.
      • Knauer S.K.
      • Rosta E.
      • Arlt W.
      Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.
      ,
      • Grum D.
      • van den Boom J.
      • Neumann D.
      • Matena A.
      • Link N.M.
      • Mueller J.W.
      A heterodimer of human 3′-phosphoadenosine-5′-phosphosulphate (PAPS) synthases is a new sulphate activating complex.
      ). A difference in protein stability of the two recombinant human PAPS synthases as described (
      • van den Boom J.
      • Heider D.
      • Martin S.R.
      • Pastore A.
      • Mueller J.W.
      3′-Phosphoadenosine 5′-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding.
      ). PAPSS2 was less stable than PAPSS1 toward chemical or thermal unfolding (
      • van den Boom J.
      • Heider D.
      • Martin S.R.
      • Pastore A.
      • Mueller J.W.
      3′-Phosphoadenosine 5′-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding.
      ). ATP sulfurylase and APS kinase activity assays, run after incubation at an elevated temperature, indicated that the sulfurylase domain is less stable than the APS kinase domain (
      • van den Boom J.
      • Heider D.
      • Martin S.R.
      • Pastore A.
      • Mueller J.W.
      3′-Phosphoadenosine 5′-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding.
      ). This is probably due to ligand binding as the APS kinase may pull an ADP molecule from the bacterial host via several purification steps (
      • Mueller J.W.
      • Shafqat N.
      Adenosine-5′-phosphosulfate–a multifaceted modulator of bifunctional 3′-phospho-adenosine-5′-phosphosulfate synthases and related enzymes.
      ,
      • Harjes S.
      • Bayer P.
      • Scheidig A.J.
      The crystal structure of human PAPS synthetase 1 reveals asymmetry in substrate binding.
      ). It will be interesting to see how these results translate into protein stability within the living cell (
      • Brylski O.
      • Ebbinghaus S.
      • Mueller J.W.
      Melting down protein stability: PAPS synthase 2 in patients and in a cellular environment.
      ). In light of these findings, the PAPSS gene fusion could also be thought of as a solubility anchor of a more stable domain for another less stable domain, among other factors.
      Figure thumbnail gr2
      Figure 2PAPS synthases and human sulfotransferases are weakly transcriptionally correlated. Expression profiles for PAPS synthases and sulfotransferases from 27 different human tissues were derived from Fagerberg et al. (
      • Fagerberg L.
      • Hallström B.M.
      • Oksvold P.
      • Kampf C.
      • Djureinovic D.
      • Odeberg J.
      • Habuka M.
      • Tahmasebpoor S.
      • Danielsson A.
      • Edlund K.
      • Asplund A.
      • Sjöstedt E.
      • Lundberg E.
      • Szigyarto C.A.
      • Skogs M.
      • et al.
      Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics.
      ). PAPSS1 and PAPSS2 expression profiles were compared with each other and against different sulfotransferases. Top panel: PAPSS1 and PAPSS2 expression seem to be weakly anti-correlated. Comparing PAPSS1 or PAPSS2 with SULT1A1 shows a weak positive correlation between PAPSS2 and SULT1A1, but a negative correlation for PAPSS1 with SULT1A1. All units in these panels are in RPKM (reads per kb of transcript per million mapped reads). Bottom panel: to illustrate the positive or negative correlation of the tissue-specific expression, the correlation coefficient R is plotted for all 52 sulfotransferases versus PAPSS1 (black) or PAPSS2 (red). There is a tendency for PAPSS2 to be co-expressed with cytosolic sulfotransferases.

      Benefit of being fused together

      For bifunctional PAPS synthases, answering questions on whether and how the individual domains functionally interact with each other continue to drive our understanding in the field. Channeling of the APS intermediate between the two domains of human PAPS synthases was initially hypothesized but was subsequently ruled out based on kinetic (
      • Lansdon E.B.
      • Fisher A.J.
      • Segel I.H.
      Human 3′-phosphoadenosine 5′-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5′-phosphosulfate kinase domains.
      ,
      • Sun M.
      • Leyh T.S.
      Channeling in sulfate activating complexes.
      ) and structural data (
      • Harjes S.
      • Bayer P.
      • Scheidig A.J.
      The crystal structure of human PAPS synthetase 1 reveals asymmetry in substrate binding.
      ). The crystal structure of full-length human PAPSS1 shows dimers of APS kinase and ATP sulfurylase each with large interacting surfaces but only a weak interaction between those sulfurylase and kinase domain dimers (
      • Harjes S.
      • Bayer P.
      • Scheidig A.J.
      The crystal structure of human PAPS synthetase 1 reveals asymmetry in substrate binding.
      ). The large dimer interface is conserved between PAPS synthase isoforms; hence, they can form high-affinity homo- and heterodimers (
      • Grum D.
      • van den Boom J.
      • Neumann D.
      • Matena A.
      • Link N.M.
      • Mueller J.W.
      A heterodimer of human 3′-phosphoadenosine-5′-phosphosulphate (PAPS) synthases is a new sulphate activating complex.
      ). APS channeling was excluded as there was no channel visible in the structure (
      • Harjes S.
      • Bayer P.
      • Scheidig A.J.
      The crystal structure of human PAPS synthetase 1 reveals asymmetry in substrate binding.
      ); APS produced by the sulfurylase domain exchanges freely with bulk APS (
      • Lansdon E.B.
      • Fisher A.J.
      • Segel I.H.
      Human 3′-phosphoadenosine 5′-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5′-phosphosulfate kinase domains.
      ) and APS kinase and reverse sulfurylase assays can be run without problems starting from APS (
      • Mueller J.W.
      • Idkowiak J.
      • Gesteira T.F.
      • Vallet C.
      • Hardman R.
      • van den Boom J.
      • Dhir V.
      • Knauer S.K.
      • Rosta E.
      • Arlt W.
      Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.
      ). Because no metabolic need for APS is known in animals, the PAPS synthases may represent a way for stoichiometric gene expression and protein localization of its two enzymatic components.
      The formation of a bifunctional enzyme even without the added effect of substrate channeling can be an answer to the low-catalytic efficiency of the forward reaction of ATPS with a Keq of ∼10−8 (
      • Leyh T.S.
      The physical biochemistry and molecular genetics of sulfate activation.
      ). Therefore, the equilibrium can be shifted by the removal of the products, e.g. linking with inorganic pyrophosphatase to remove pyrophosphate or with enzymes utilizing APS. The animal PAPS synthase is clearly a mechanism for the latter, but not the only one in nature (
      • Patron N.J.
      • Durnford D.G.
      • Kopriva S.
      Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.
      ).
      Another mechanism to increase the efficiency is employed by most bacteria, which possess a variant of ATPS that couples APS production with GTP hydrolysis (
      • Leyh T.S.
      • Suo Y.
      GTPase-mediated activation of ATP sulfurylase.
      ), and the need for increased catalytic efficiency may have led to other gene fusions. In filamentous fungi, ATPS is also fused with APS kinase; however, the kinase domain of the fusion protein is at the C-terminal end and functions only as an activation domain to modulate activity of ATPS without having a kinase activity (
      • Foster B.A.
      • Thomas S.M.
      • Mahr J.A.
      • Renosto F.
      • Patel H.C.
      • Segel I.H.
      Cloning and sequencing of ATP sulfurylase from Penicillium chrysogenum. Identification of a likely allosteric domain.
      ,
      • Gay S.C.
      • Segel I.H.
      • Fisher A.J.
      Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity.
      ). A number of gene fusions have been found in the eukaryotic microalgae, which as secondary or tertiary endosymbionts were likely more prone to genome rearrangements (
      • Patron N.J.
      • Durnford D.G.
      • Kopriva S.
      Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.
      ). ATPS in the diatom Thalassiosira pseudonana and other Protozoans is fused to both APS kinase and inorganic pyrophosphatase, whereas in the dinoflagellate Heterocapsa triquetra ATPS is fused with the other APS-utilizing enzyme, APS reductase (
      • Patron N.J.
      • Durnford D.G.
      • Kopriva S.
      Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.
      ). In addition to this diversity, at least three different ATP sulfurylase enzymes have evolved independently, the plant and animal enzyme, the bacterial GTPase-linked enzyme, and one mainly found in cyanobacteria and green algae (
      • Patron N.J.
      • Durnford D.G.
      • Kopriva S.
      Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.
      ,
      • Mougous J.D.
      • Lee D.H.
      • Hubbard S.C.
      • Schelle M.W.
      • Vocadlo D.J.
      • Berger J.M.
      • Bertozzi C.R.
      Molecular basis for G protein control of the prokaryotic ATP sulfurylase.
      ). The ATPS domains from the fusion proteins are phylogenetically related to the ATPS from plants/animals or green algae (
      • Patron N.J.
      • Durnford D.G.
      • Kopriva S.
      Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.
      ).
      In plants, the need for APS for primary assimilation is greater that for the sulfation pathways, and therefore, a mechanism shifting the equilibrium from APS to PAPS is not advantageous. Another interesting evolutionary aspect of plant ATPS is that although it is largely a plastidic enzyme, it is in no way related to the ATPS in cyanobacteria, the precursors of plant plastids (
      • Patron N.J.
      • Durnford D.G.
      • Kopriva S.
      Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.
      ). Because chlorophyte ATPS is of cyanobacterial origin, it seems that the common precursor must have contained both forms, plastidic and Eukaryotic, and the sister lineages, plants and green algae, each retained a different one.

      Core sulfation pathways

      Sulfotransferases are the first enzymes of the core sulfation pathways. They transfer sulfate from PAPS to the hydroxyl or amino group of a wide variety of acceptors: carbohydrates, lipids, peptides, hormone precursors, xenobiotics, and other molecules (
      • Coughtrie M.W.H.
      Function and organization of the human cytosolic sulfotransferase (SULT) family.
      ,
      • Dias I.H.K.
      • Ferreira R.
      • Gruber F.
      • Vitorino R.
      • Rivas-Urbina A.
      • Sanchez-Quesada J.L.
      • Vieira Silva J.
      • Fardilha M.
      • de Freitas V.
      • Reis A.
      Sulfate-based lipids: analysis of healthy human fluids and cell extracts.
      ). There are also PAPS-independent aryl sulfotransferases from some bacteria, which use phenolic sulfates as donors (
      • Malojčić G.
      • Owen R.L.
      • Glockshuber R.
      Structural and mechanistic insights into the PAPS-independent sulfotransfer catalyzed by bacterial aryl sulfotransferase and the role of the DsbL/Dsbl system in its folding.
      ). These aryl sulfotransferases display a different fold, but retain a similar spatial arrangement of the active-site residues, indicative of convergent evolution. They are covered in more detail elsewhere (
      • Malojčić G.
      • Owen R.L.
      • Glockshuber R.
      Structural and mechanistic insights into the PAPS-independent sulfotransfer catalyzed by bacterial aryl sulfotransferase and the role of the DsbL/Dsbl system in its folding.
      ). The other components, sulfatases, are hydrolytic enzymes, part of the alkaline phosphatase superfamily, that cleave biological sulfate esters (
      • Mohamed M.F.
      • Hollfelder F.
      Efficient, crosswise catalytic promiscuity among enzymes that catalyze phosphoryl transfer.
      ). A post-translational modification dramatically enhances the catalytic activity of sulfatase enzymes—a cysteine or serine residue within the catalytic center is converted to a formyl-glycine (
      • Dierks T.
      • Schmidt B.
      • von Figura K.
      Conversion of cysteine to formylglycine: a protein modification in the endoplasmic reticulum.
      ).
      Lipmann (
      • Lipmann F.
      Biological sulfate activation and transfer.
      ) referred to sulfotransferases as sulfokinases, and a common evolutionary origin of sulfotransferases and kinases has been purported, with subsequent phylogenetic divergence of enzyme activity (
      • Leipe D.D.
      • Koonin E.V.
      • Aravind L.
      Evolution and classification of P-loop kinases and related proteins.
      ). There are four conserved regions of sulfotransferases used for the characterization of the enzymes (
      • Varin L.
      • DeLuca V.
      • Ibrahim R.K.
      • Brisson N.
      Molecular characterization of two plant flavonol sulfotransferases.
      ), including a P-loop for catalysis (
      • Allali-Hassani A.
      • Pan P.W.
      • Dombrovski L.
      • Najmanovich R.
      • Tempel W.
      • Dong A.
      • Loppnau P.
      • Martin F.
      • Thonton J.
      • Edwards A.M.
      • Bochkarev A.
      • Plotnikov A.N.
      • Vedadi M.
      • Arrowsmith C.H.
      Structural and chemical profiling of the human cytosolic sulfotransferases.
      ,
      • Hirschmann F.
      • Krause F.
      • Baruch P.
      • Chizhov I.
      • Mueller J.W.
      • Manstein D.J.
      • Papenbrock J.
      • Fedorov R.
      Structural and biochemical studies of sulphotransferase 18 from Arabidopsis thaliana explain its substrate specificity and reaction mechanism.
      ). This protein structure is highly conserved, except for plant tyrosylprotein sulfotransferase (
      • Komori R.
      • Amano Y.
      • Ogawa-Ohnishi M.
      • Matsubayashi Y.
      Identification of tyrosylprotein sulfotransferase in Arabidopsis.
      ). Based on the conserved regions, sulfotransferases are found in all kingdoms of life (
      • Hirschmann F.
      • Krause F.
      • Baruch P.
      • Chizhov I.
      • Mueller J.W.
      • Manstein D.J.
      • Papenbrock J.
      • Fedorov R.
      Structural and biochemical studies of sulphotransferase 18 from Arabidopsis thaliana explain its substrate specificity and reaction mechanism.
      ). Different research communities abbreviate mammalian cytosolic sulfotransferases to SULT, plant enzymes to SOT (or SULT), and Golgi enzymes according to their main activity/substrate (e.g. HS6ST for heparan sulfate-6-O-sulfotransferase, and TPST for tyrosylprotein sulfotransferase). For consistency, we will keep the different abbreviations.
      The main differences between animal and plant sulfation pathways are the number of genes with more than 50 genes for human SULTs and 21 SOT genes in Arabidopsis, whereas the 17 human sulfatases do not have counterparts in plant genomes.

      Arabidopsis and the human sulfotransferase repertoire

      Sulfotransferases are grouped into categories, such as soluble or membrane-bound, cytosolic or Golgi-located, substrate preference for low-molecular-weight substrates, or the larger carbohydrates, proteins, or proteoglycans (
      • Coughtrie M.W.H.
      Function and organization of the human cytosolic sulfotransferase (SULT) family.
      ).
      Langford et al. (
      • Langford R.
      • Hurrion E.
      • Dawson P.A.
      Genetics and pathophysiology of mammalian sulfate biology.
      ) listed 13 cytoplasmic and 37 Golgi-residing SULTs in the human genome. Ensembl lists several additional entries for the human genome (
      • Zerbino D.R.
      • Achuthan P.
      • Akanni W.
      • Amode M.R.
      • Barrell D.
      • Bhai J.
      • Billis K.
      • Cummins C.
      • Gall A.
      • Girón C.G.
      • Gil L.
      • Gordon L.
      • Haggerty L.
      • Haskell E.
      • Hourlier T.
      • et al.
      Ensembl 2018.
      ), but most of them seem to be pseudogenes (
      • Mueller J.W.
      • Gilligan L.C.
      • Idkowiak J.
      • Arlt W.
      • Foster P.A.
      The regulation of steroid action by sulfation and desulfation.
      ). The only proteins annotated having sulfotransferase activity at Ensembl that should append Langford’s list are DSEL, a dermatan sulfate epimerase, and the WSCD1 protein (
      • Zerbino D.R.
      • Achuthan P.
      • Akanni W.
      • Amode M.R.
      • Barrell D.
      • Bhai J.
      • Billis K.
      • Cummins C.
      • Gall A.
      • Girón C.G.
      • Gil L.
      • Gordon L.
      • Haggerty L.
      • Haskell E.
      • Hourlier T.
      • et al.
      Ensembl 2018.
      ). Hirschmann et al. (
      • Hirschmann F.
      • Krause F.
      • Papenbrock J.
      The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions.
      ) list 22 genes for sulfotransferases in Arabidopsis (but one of them is annotated as a pseudogene). Phylogenetic analysis of protein sequences from the final 21 and 52 genes representing the Arabidopsis and human sulfotransferase repertoires, respectively, reveals that Arabidopsis SOTs 1–18 share high-sequence similarity with human cytoplasmic SULTs. In fact, these two groups share a higher degree of similarity with each other than human Golgi and cytoplasmic SULTs (Fig. 3). This is illustrated by a structural overlay of AtSOT18 and human SULT1A1 (Fig. 4). Hence, any new insight on one class of these sulfotransferases may have direct applicability to the other (
      • Foster P.A.
      • Mueller J.W.
      Sulfation pathways: insights into steroid sulfation and desulfation pathways.
      ).
      Figure thumbnail gr3
      Figure 3Alignment of sulfotransferases. Protein sequences from 52 human and 21 Arabidopsis sulfotransferases were derived from RefSeq entries from the nucleotide database at www.ncbi.nlm.nih.gov. From multiple splice forms, the one selected was assigned the major isoform. These sequences were subjected to multiple sequence alignments using Clustal Omega and MAFFT (
      • Li W.
      • Cowley A.
      • Uludag M.
      • Gur T.
      • McWilliam H.
      • Squizzato S.
      • Park Y.M.
      • Buso N.
      • Lopez R.
      The EMBL-EBI bioinformatics web and programmatic tools framework.
      ). From the MAFFT tree, a neighbor-joining tree without distance corrections, a collapsed tree was manually curated. A striking finding was that AtTPST (RefSeq NP_563804) was not grouped with the human TPSTs but with heparan-6-O-sulfotransferases 1–3 (NCBI RefSeq NM_004807, NM_001077188, and NM_153456). The abbreviations used are as follows: SULT, cytosolic sulfotransferase; SOT, plant sulfotransferase; TPST, tyrosylprotein sulfotransferase; CHST, chondroitin sulfotransferase; HS(2/3/6)ST, heparan-(2/3/6)-O-sulfotransferase; NDST, N-deacetylase and N-sulfotransferase; Gal3ST, galactose-3-O-sulfotransferase.
      Figure thumbnail gr4
      Figure 4Structural representation of different sulfotransferases. A, all structures are shown in the same orientation with the bound PAP nucleotide shown in blue and a substrate in orange. Please note the central β-sheet in all structures and the PAP co-factor are bound exactly at the same position. Structures shown are human sulfotransferase SULT1A1 bound to PAP and 3-cyano-7-hydroxycoumarin (Protein Data Bank code 3U3M), A. thaliana SOT18 complexed to PAP and sinigrin (Protein Data Bank code 5MEX), Danio rerio heparan sulfate 6-O sulfotransferase HS6ST3 with PAP and part of its heptasaccharide displayed (Protein Data Bank code 5T0A), as well as human TPST2 with bound PAP and C4 peptide (Protein Data Bank code 3AP1). Structural visualizations were done using YASARA (
      • Krieger E.
      • Vriend G.
      YASARA View–molecular graphics for all devices–from smartphones to workstations.
      ). B, these complexes were structurally aligned using MUSTANG (
      • Konagurthu A.S.
      • Whisstock J.C.
      • Stuckey P.J.
      • Lesk A.M.
      MUSTANG: a multiple structural alignment algorithm.
      ). Root mean square deviation (RMSD) values for structural alignment, the number of aligned residues, and the percentage of amino acid identity are listed.
      These insights mainly include discoveries of novel mechanisms of regulation (
      • Foster P.A.
      • Mueller J.W.
      Sulfation pathways: insights into steroid sulfation and desulfation pathways.
      ). For example, the flexibility of the main substrate-binding loops—elucidated in part with new analytical and computational tools—is the molecular basis for the broad specificity of the sulfotransferase SULT1A1 (
      • Berger I.
      • Guttman C.
      • Amar D.
      • Zarivach R.
      • Aharoni A.
      The molecular basis for the broad substrate specificity of human sulfotransferase 1A1.
      ), with possible implication also for AtSOT12 described below. This flexibility makes it difficult to search for pharmacologically useful and isoform-specific inhibitors of sulfotransferases using computational docking (
      • Rohn-Glowacki K.J.
      • Falany C.N.
      The potent inhibition of human cytosolic sulfotransferase 1A1 by 17α-ethinylestradiol is due to interactions with isoleucine 89 on loop 1.
      ), except the flexibility is built into the structural models (
      • Cook I.
      • Wang T.
      • Falany C.N.
      • Leyh T.S.
      High accuracy in silico sulfotransferase models.
      ). Such inhibitors are useful because human sulfotransferases metabolize many drugs and may thus interfere with various pharmacological interventions. Another regulatory mechanism, allosteric regulation, has only recently been described in sulfation pathways (
      • Foster P.A.
      • Mueller J.W.
      Sulfation pathways: insights into steroid sulfation and desulfation pathways.
      ). Recently, an allosteric site in human SULT1A3 was discovered that may be targeted for isoform-specific SULT1A3 allosteric inhibitors (
      • Darrah K.
      • Wang T.
      • Cook I.
      • Cacace M.
      • Deiters A.
      • Leyh T.S.
      Allosteres to regulate neurotransmitter sulfonation.
      )
      Dimers of human cytoplasmic SULTs are believed to form via an unusually small dimer interface containing the amino acids KTVE (
      • Petrotchenko E.V.
      • Pedersen L.C.
      • Borchers C.H.
      • Tomer K.B.
      • Negishi M.
      The dimerization motif of cytosolic sulfotransferases.
      ) that are conserved in cytoplasmic SULTs (
      • Weitzner B.
      • Meehan T.
      • Xu Q.
      • Dunbrack Jr., R.L.
      An unusually small dimer interface is observed in all available crystal structures of cytosolic sulfotransferases.
      ). Biochemical data and molecular dynamics simulation suggest that dimer formation is of functional significance as it modulates flexibility of the catalytic loop 3 (
      • Tibbs Z.E.
      • Rohn-Glowacki K.J.
      • Crittenden F.
      • Guidry A.L.
      • Falany C.N.
      Structural plasticity in the human cytosolic sulfotransferase dimer and its role in substrate selectivity and catalysis.
      ). Recently, a crystal structure of the AtSOT18–sinigrin–PAP complex elucidated the functional domain and residues for the substrate-binding site of the enzyme (
      • Hirschmann F.
      • Krause F.
      • Baruch P.
      • Chizhov I.
      • Mueller J.W.
      • Manstein D.J.
      • Papenbrock J.
      • Fedorov R.
      Structural and biochemical studies of sulphotransferase 18 from Arabidopsis thaliana explain its substrate specificity and reaction mechanism.
      ). The structure demonstrated evolutionary conservation of the sulfotransferases between humans and plants (Fig. 4) and suggested a loop-gating mechanism as responsible for substrate specificity for the sulfotransferase in plants (
      • Hirschmann F.
      • Krause F.
      • Baruch P.
      • Chizhov I.
      • Mueller J.W.
      • Manstein D.J.
      • Papenbrock J.
      • Fedorov R.
      Structural and biochemical studies of sulphotransferase 18 from Arabidopsis thaliana explain its substrate specificity and reaction mechanism.
      ). Noteworthy, AtSOT18 and the other AtSOTs do not contain the KTVE motif, and dimer formation has not been reported for plant sulfotransferases.
      Plant SOTs are still not completely characterized even in the model plant Arabidopsis (
      • Hirschmann F.
      • Krause F.
      • Papenbrock J.
      The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions.
      ). Some Arabidopsis SOTs have a broad substrate specificity, and some are very specific, such as AtSOT15 catalyzing only sulfation of 11- and 12-hydroxyl-jasmonate (
      • Gidda S.K.
      • Miersch O.
      • Levitin A.
      • Schmidt J.
      • Wasternack C.
      • Varin L.
      Biochemical and molecular characterization of a hydroxyjasmonate sulfotransferase from Arabidopsis thaliana.
      ); however, the substrates for almost half of all SOTs are unknown (
      • Hirschmann F.
      • Krause F.
      • Papenbrock J.
      The multi-protein family of sulfotransferases in plants: composition, occurrence, substrate specificity, and functions.
      ). Most attention has been paid to three Arabidopsis SOTs, AtSOT16, AtSOT17, and AtSOT18, which transfer sulfate to desulfo-glucosinolate precursors as the final step in glucosinolate synthesis (
      • Hirai M.Y.
      • Klein M.
      • Fujikawa Y.
      • Yano M.
      • Goodenowe D.B.
      • Yamazaki Y.
      • Kanaya S.
      • Nakamura Y.
      • Kitayama M.
      • Suzuki H.
      • Sakurai N.
      • Shibata D.
      • Tokuhisa J.
      • Reichelt M.
      • Gershenzon J.
      • et al.
      Elucidation of gene-to-gene and metabolite-to-gene networks in Arabidopsis by integration of metabolomics and transcriptomics.
      ). This is due to the important role of glucosinolates in defense against biotic and abiotic stress and their anti-carcinogenic and neuroprotective properties in the human diet (
      • Halkier B.A.
      • Gershenzon J.
      Biology and biochemistry of glucosinolates.
      ,
      • Traka M.
      • Mithen R.
      Glucosinolates, isothiocyanates and human health.
      ). The three desulfo-glucosinolate SOTs have different affinities to various classes of the precursors, i.e. aliphatic and indolic, at least in vitro (
      • Klein M.
      • Reichelt M.
      • Gershenzon J.
      • Papenbrock J.
      The three desulfoglucosinolate sulfotransferase proteins in Arabidopsis have different substrate specificities and are differentially expressed.
      ). Interestingly, it seems that SOTs from different natural Arabidopsis accessions possess different specificity to these precursors (
      • Klein M.
      • Papenbrock J.
      Kinetics and substrate specificities of desulfo-glucosinolate sulfotransferases in Arabidopsis thaliana.
      ); however, whether these in vitro data are relevant in vivo needs to be confirmed.
      Similar to the desulfo-glucosinolate SOTs, only in vitro substrate specificities are known for other SOTs. While AtSOT5, AtSOT8, and AtSOT13 transfer sulfate to flavonoids, AtSOT10 modifies the plant hormones, brassinosteroids (
      • Hashiguchi T.
      • Sakakibara Y.
      • Hara Y.
      • Shimohira T.
      • Kurogi K.
      • Akashi R.
      • Liu M.C.
      • Suiko M.
      Identification and characterization of a novel kaempferol sulfotransferase from Arabidopsis thaliana.
      ,
      • Marsolais F.
      • Boyd J.
      • Paredes Y.
      • Schinas A.M.
      • Garcia M.
      • Elzein S.
      • Varin L.
      Molecular and biochemical characterization of two brassinosteroid sulfotransferases from Arabidopsis, AtST4a (At2g14920) and AtST1 (At2g03760).
      ). In contrast to these SOTs with relatively narrow specificity, the AtSOT12 accepts a variety of substrates for the synthesis of sulfated flavonoids, salicylic acid and brassinosteroids (
      • Baek D.
      • Pathange P.
      • Chung J.S.
      • Jiang J.
      • Gao L.
      • Oikawa A.
      • Hirai M.Y.
      • Saito K.
      • Pare P.W.
      • Shi H.
      A stress-inducible sulphotransferase sulphonates salicylic acid and confers pathogen resistance in Arabidopsis.
      ). The activity with salicylic acid, a phytohormone involved in defense against pathogens, seems to be responsible for the increased pathogen susceptibility of sot12 mutants (
      • Baek D.
      • Pathange P.
      • Chung J.S.
      • Jiang J.
      • Gao L.
      • Oikawa A.
      • Hirai M.Y.
      • Saito K.
      • Pare P.W.
      • Shi H.
      A stress-inducible sulphotransferase sulphonates salicylic acid and confers pathogen resistance in Arabidopsis.
      ). The functions of other SOTs remain to be elucidated, particularly given the large variety of so far unknown sulfur-containing metabolites in Arabidopsis (
      • Gläser K.
      • Kanawati B.
      • Kubo T.
      • Schmitt-Kopplin P.
      • Grill E.
      Exploring the Arabidopsis sulfur metabolome.
      ).

      Protein sulfation by TPSTs

      Tyrosine sulfation is a major post-translational regulation of secreted proteins and peptides in both animals and plants. However, this modification seems to be confined to multicellular Eukaryotes, as TPSTs have not been found in either bacteria or yeast (
      • Kehoe J.W.
      • Bertozzi C.R.
      Tyrosine sulfation: a modulator of extracellular protein-protein interactions.
      ). TPST catalyzes the transfer of sulfate from PAPS to the phenolic group of the amino acid tyrosine in the Golgi (
      • Komori R.
      • Amano Y.
      • Ogawa-Ohnishi M.
      • Matsubayashi Y.
      Identification of tyrosylprotein sulfotransferase in Arabidopsis.
      ,
      • Hartmann-Fatu C.
      • Bayer P.
      Determinants of tyrosylprotein sulfation coding and substrate specificity of tyrosylprotein sulfotransferases in metazoans.
      ,
      • Goettsch S.
      • Badea R.A.
      • Mueller J.W.
      • Wotzlaw C.
      • Schoelermann B.
      • Schulz L.
      • Rabiller M.
      • Bayer P.
      • Hartmann-Fatu C.
      Human TPST1 transmembrane domain triggers enzyme dimerisation and localisation to the Golgi compartment.
      ). It is estimated that one-third of all secreted human proteins are tyrosine-sulfated (
      • Monigatti F.
      • Gasteiger E.
      • Bairoch A.
      • Jung E.
      The sulfinator: predicting tyrosine sulfation sites in protein sequences.
      ). Human TPST1 and TPST2 are type II transmembrane proteins with a C-terminal globular domain within the Golgi lumen (
      • Goettsch S.
      • Badea R.A.
      • Mueller J.W.
      • Wotzlaw C.
      • Schoelermann B.
      • Schulz L.
      • Rabiller M.
      • Bayer P.
      • Hartmann-Fatu C.
      Human TPST1 transmembrane domain triggers enzyme dimerisation and localisation to the Golgi compartment.
      ). They share 67% amino acid identity with each other. As Caenorhabditis elegans and Drosophila only contain one TPST gene, a gene duplication may have occurred at the invertebrate–vertebrate transition.
      Elucidating the biological roles of individual TPST isoforms through biochemical and structural studies of recombinant TPST proteins was a challenge for a long time. In 2013, Teramoto et al. (
      • Teramoto T.
      • Fujikawa Y.
      • Kawaguchi Y.
      • Kurogi K.
      • Soejima M.
      • Adachi R.
      • Nakanishi Y.
      • Mishiro-Sato E.
      • Liu M.C.
      • Sakakibara Y.
      • Suiko M.
      • Kimura M.
      • Kakuta Y.
      Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.
      ) reported the crystal structure of human TPST2, followed by the structure of human TPST1 in 2017 (
      • Tanaka S.
      • Nishiyori T.
      • Kojo H.
      • Otsubo R.
      • Tsuruta M.
      • Kurogi K.
      • Liu M.C.
      • Suiko M.
      • Sakakibara Y.
      • Kakuta Y.
      Structural basis for the broad substrate specificity of the human tyrosylprotein sulfotransferase-1.
      ). These structures are remarkable for two reasons. First, the core protein fold and the 5′-phosphosulfate–binding (5′-PSB) and the 3′-phosphate–binding (3′-PB) motifs involved in PAPS co-factor binding are structurally conserved even in these sulfotransferases only distantly related to their cytoplasmic counterparts (
      • Teramoto T.
      • Fujikawa Y.
      • Kawaguchi Y.
      • Kurogi K.
      • Soejima M.
      • Adachi R.
      • Nakanishi Y.
      • Mishiro-Sato E.
      • Liu M.C.
      • Sakakibara Y.
      • Suiko M.
      • Kimura M.
      • Kakuta Y.
      Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.
      ,
      • Tanaka S.
      • Nishiyori T.
      • Kojo H.
      • Otsubo R.
      • Tsuruta M.
      • Kurogi K.
      • Liu M.C.
      • Suiko M.
      • Sakakibara Y.
      • Kakuta Y.
      Structural basis for the broad substrate specificity of the human tyrosylprotein sulfotransferase-1.
      ). Second, they explain the mechanism of substrate recognition. Protein substrates need to locally unfold to bind to TPSTs in a deep active-site cleft, a process similar to the one known for tyrosine kinases (
      • Teramoto T.
      • Fujikawa Y.
      • Kawaguchi Y.
      • Kurogi K.
      • Soejima M.
      • Adachi R.
      • Nakanishi Y.
      • Mishiro-Sato E.
      • Liu M.C.
      • Sakakibara Y.
      • Suiko M.
      • Kimura M.
      • Kakuta Y.
      Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.
      ). Both human TPST enzymes target peptidic motifs with negatively charged residues around the acceptor tyrosine (
      • Hartmann-Fatu C.
      • Bayer P.
      Determinants of tyrosylprotein sulfation coding and substrate specificity of tyrosylprotein sulfotransferases in metazoans.
      ) with very similar or identical recognition mechanisms (
      • Tanaka S.
      • Nishiyori T.
      • Kojo H.
      • Otsubo R.
      • Tsuruta M.
      • Kurogi K.
      • Liu M.C.
      • Suiko M.
      • Sakakibara Y.
      • Kakuta Y.
      Structural basis for the broad substrate specificity of the human tyrosylprotein sulfotransferase-1.
      ). This leaves open how the observed functional differences of the TPST isoforms (see below and Ref.
      • Danan L.M.
      • Yu Z.
      • Ludden P.J.
      • Jia W.
      • Moore K.L.
      • Leary J.A.
      Catalytic mechanism of Golgi-resident human tyrosylprotein sulfotransferase-2: a mass spectrometry approach.
      ) are caused on the protein level.
      Indeed, despite structural and mechanistic similarities, the two TPST isoforms display notable functional differences. TPST1-knockout mice show reduced body weight and fewer litters due to increased fetal death in uterus, whereas male fertility is not affected (
      • Ouyang Y.B.
      • Crawley J.T.
      • Aston C.E.
      • Moore K.L.
      Reduced body weight and increased postimplantation fetal death in tyrosylprotein sulfotransferase-1-deficient mice.
      ). This suggests that TPST1 reaction products which would not be sulfated in the TPST1 knockout, have a role in females during development of embryos. TPST2-knockout mice, however, primarily show male infertility (
      • Borghei A.
      • Ouyang Y.B.
      • Westmuckett A.D.
      • Marcello M.R.
      • Landel C.P.
      • Evans J.P.
      • Moore K.L.
      Targeted disruption of tyrosylprotein sulfotransferase-2, an enzyme that catalyzes post-translational protein tyrosine O-sulfation, causes male infertility.
      ), due to compromised egg–sperm interaction (
      • Marcello M.R.
      • Jia W.
      • Leary J.A.
      • Moore K.L.
      • Evans J.P.
      Lack of tyrosylprotein sulfotransferase-2 activity results in altered sperm-egg interactions and loss of ADAM3 and ADAM6 in epididymal sperm.
      ). Taken together with the sulfated proteins from the blood-clotting cascade and the sulfated co-receptors on the host cells for HIV infections (
      • Hartmann-Fatu C.
      • Bayer P.
      Determinants of tyrosylprotein sulfation coding and substrate specificity of tyrosylprotein sulfotransferases in metazoans.
      ), the picture emerges that tyrosine sulfation acts as macromolecular glue to strengthen interactions of proteins with other proteins or other biopolymers. A recent biophysical study clearly illustrates this point. In the complex of an N-terminal sulfated part of the chemokine receptor CCR5 and its CCL5 ligand NMR revealed that high-affinity binding is attributed to sulfate-mediated twisting of the two N termini (
      • Abayev M.
      • Rodrigues J.P.G.L.M.
      • Srivastava G.
      • Arshava B.
      • Jaremko Ł.
      • Jaremko M.
      • Naider F.
      • Levitt M.
      • Anglister J.
      The solution structure of monomeric CCL5 in complex with a doubly sulfated N-terminal segment of CCR5.
      ). Identifying more and more sulfated proteins is expected in the near future due to advances in MS that allow better recovery of sulfated peptides and unambiguous distinction from their isobaric phosphorylated counterparts (
      • Chen G.
      • Zhang Y.
      • Trinidad J.C.
      • Dann 3rd., C.
      Distinguishing sulfotyrosine containing peptides from their phosphotyrosine counterparts using mass Spectrometry.
      ).
      The importance of tyrosine sulfation in plants has been known for a long time because of the number of sulfated growth-regulating peptides (
      • Matsubayashi Y.
      • Sakagami Y.
      Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L.
      ,
      • Amano Y.
      • Tsubouchi H.
      • Shinohara H.
      • Ogawa M.
      • Matsubayashi Y.
      Tyrosine-sulfated glycopeptide involved in cellular proliferation and expansion in Arabidopsis.
      ). Despite the importance of the tyrosine sulfation, however, the corresponding sulfotransferase remained elusive in plants, as no homologous proteins to the animal enzyme could be found. AtTPST was identified in Arabidopsis after isolation of the enzyme from the microsomal fraction and proteomics analyses (
      • Komori R.
      • Amano Y.
      • Ogawa-Ohnishi M.
      • Matsubayashi Y.
      Identification of tyrosylprotein sulfotransferase in Arabidopsis.
      ). AtTPST is a 62-kDa transmembrane protein located in the Golgi that lacks the characteristic cytosolic sulfotransferase domain (
      • Komori R.
      • Amano Y.
      • Ogawa-Ohnishi M.
      • Matsubayashi Y.
      Identification of tyrosylprotein sulfotransferase in Arabidopsis.
      ). The importance of plant tyrosine sulfation is confirmed by the semi-dwarf phenotype of the Arabidopsis tpst1 mutant with early senescence, light green leaves, and diminutive roots (
      • Komori R.
      • Amano Y.
      • Ogawa-Ohnishi M.
      • Matsubayashi Y.
      Identification of tyrosylprotein sulfotransferase in Arabidopsis.
      ).

      Human sulfatase enzymes

      The human genome contains 17 genes for sulfatases (
      • Langford R.
      • Hurrion E.
      • Dawson P.A.
      Genetics and pathophysiology of mammalian sulfate biology.
      ,
      • Zerbino D.R.
      • Achuthan P.
      • Akanni W.
      • Amode M.R.
      • Barrell D.
      • Bhai J.
      • Billis K.
      • Cummins C.
      • Gall A.
      • Girón C.G.
      • Gil L.
      • Gordon L.
      • Haggerty L.
      • Haskell E.
      • Hourlier T.
      • et al.
      Ensembl 2018.
      ), which hydrolyze a range of biological sulfate esters. They are all grouped into the alkaline phosphatase superfamily (
      • Mohamed M.F.
      • Hollfelder F.
      Efficient, crosswise catalytic promiscuity among enzymes that catalyze phosphoryl transfer.
      ).
      The activity of human (and bacterial or fungal) sulfatases depends on enzymatic oxidation of a cysteine to formylglycine (
      • Mueller J.W.
      • Gilligan L.C.
      • Idkowiak J.
      • Arlt W.
      • Foster P.A.
      The regulation of steroid action by sulfation and desulfation.
      ). The corresponding enzyme is encoded by the sulfatase-modifying factor 1 (SUMF1) gene. This formylglycine is further hydrated to form the active site of sulfatase (
      • Thomas M.P.
      • Potter B.V.
      The structural biology of oestrogen metabolism.
      ). The resulting geminal diol can be interpreted as activated water for the hydrolysis reaction (
      • Thomas M.P.
      • Potter B.V.
      The structural biology of oestrogen metabolism.
      ). A recent crystal structure of the formylglycine-generating enzyme clearly shows its catalytic copper co-factor coordinated by two active-site cysteine residues, explaining its mechanism (
      • Appel M.J.
      • Meier K.K.
      • Lafrance-Vanasse J.
      • Lim H.
      • Tsai C.L.
      • Hedman B.
      • Hodgson K.O.
      • Tainer J.A.
      • Solomon E.I.
      • Bertozzi C.R.
      Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation.
      ). As this oxidase creates an aldehyde, it has emerged as an enabling biotechnology tool for bio-conjugation reactions (
      • Krüger T.
      • Dierks T.
      • Sewald N.
      Formylglycine-generating enzymes for site-specific bioconjugation.
      ). There is also a SUMF2 gene in the human genome that encodes a protein devoid of the oxidase activity, but interacting and modulating the function of SUMF1 by forming inhibitory heterodimers (
      • Zito E.
      • Fraldi A.
      • Pepe S.
      • Annunziata I.
      • Kobinger G.
      • Di Natale P.
      • Ballabio A.
      • Cosma M.P.
      Sulfatase activities are regulated by the interaction of the sulfatase-modifying factor 1 with SUMF2.
      ).
      Sulfatase genes encode proteins with a broad range of substrate specificity. The extracellular endoglucosamine 6-sulfatases, SULF-1 and SULF-2, target highly sulfated extracellular heparan sulfate domains, which are involved in growth factor signaling, tumor progression, and protein aggregation diseases (
      • Nishitsuji K.
      Heparan sulfate S-domains and extracellular sulfatases (Sulfs): their possible roles in protein aggregation diseases.
      ). Some sulfatases (such as arylsulfatase A and B) are cytoplasmic, whereas others are membrane-bound (
      • Mueller J.W.
      • Gilligan L.C.
      • Idkowiak J.
      • Arlt W.
      • Foster P.A.
      The regulation of steroid action by sulfation and desulfation.
      ). Noteworthy is the steroid sulfatase (STS), with a very rare and unusual membrane topology; its membrane stem is made up of two α-helices (
      • Hernandez-Guzman F.G.
      • Higashiyama T.
      • Pangborn W.
      • Osawa Y.
      • Ghosh D.
      Structure of human estrone sulfatase suggests functional roles of membrane association.
      ). STS has been described as an enzyme in the lumen of the endoplasmic reticulum (
      • Thomas M.P.
      • Potter B.V.
      The structural biology of oestrogen metabolism.
      ), but it was also detected in the Golgi apparatus (
      • Willemsen R.
      • Kroos M.
      • Hoogeveen A.T.
      • van Dongen J.M.
      • Parenti G.
      • van der Loos C.M.
      • Reuser A.J.
      Ultrastructural localization of steroid sulphatase in cultured human fibroblasts by immunocytochemistry: a comparative study with lysosomal enzymes and the mannose 6-phosphate receptor.
      ).
      From an endocrine point of view, STS is highly interesting as it renders steroid sulfation a reversible and dynamic process (
      • Mueller J.W.
      • Gilligan L.C.
      • Idkowiak J.
      • Arlt W.
      • Foster P.A.
      The regulation of steroid action by sulfation and desulfation.
      ). STS is highly expressed in the placenta and forms, together with the fetal adrenal and fetal liver, the fetoplacental unit (
      • Greaves R.F.
      • Jevalikar G.
      • Hewitt J.K.
      • Zacharin M.R.
      A guide to understanding the steroid pathway: new insights and diagnostic implications.
      ). This unit produces the placental estrogens, estradiol and estriol, from fetal adrenal androgens via fetal adrenal sulfation, fetal hepatic hydroxylation, and placental desulfation, further downstream conversion, and release into the maternal circulation (
      • Rainey W.E.
      • Rehman K.S.
      • Carr B.R.
      The human fetal adrenal: making adrenal androgens for placental estrogens.
      ). In adults, STS is also expressed in many other tissues allowing for the uptake of sulfated steroid precursors and their desulfation.
      Plants do not seem to possess sulfatase activity. This poses an obvious question for the catabolism of sulfated secondary metabolites. Glucosinolates are an important pool of sulfur, which can be recycled during sulfur starvation. Glucosinolate degradation is part of their anti-herbivore activity, which is initiated by tissue damage, bringing the glucosinolates into contact with thioglucosidases (myrosinases). Removal of the sugar moiety leads to chemical rearrangement of the aglycones to form volatile isothiocyanates or nitriles and release of sulfate (
      • Ratzka A.
      • Vogel H.
      • Kliebenstein D.J.
      • Mitchell-Olds T.
      • Kroymann J.
      Disarming the mustard oil bomb.
      ). Glucosinolates can, however, be degraded also without tissue damage by atypical PEN2 myrosinase as a part of innate immunity (
      • Bednarek P.
      • Pislewska-Bednarek M.
      • Svatos A.
      • Schneider B.
      • Doubsky J.
      • Mansurova M.
      • Humphry M.
      • Consonni C.
      • Panstruga R.
      • Sanchez-Vallet A.
      • Molina A.
      • Schulze-Lefert P.
      A glucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal defense.
      ). Nothing is known about the catabolism of other sulfated compounds in plants.
      Plants can also profit from microbial sulfatase activity in the rhizosphere. Soil contains a great portion of organic sulfur, up to 90%, which is not available to plants (
      • Kertesz M.A.
      • Mirleau P.
      The role of soil microbes in plant sulphur nutrition.
      ). Soil bacteria, however, can metabolize these compounds by sulfatases, releasing the sulfate, and thus improving plant sulfur nutrition. Hence, releasing sulfate via sulfatase activity is the mechanism of some plant growth-promoting bacteria (
      • Kertesz M.A.
      • Mirleau P.
      The role of soil microbes in plant sulphur nutrition.
      ). Attempts to engineer intracellular or excreted sulfatase in plants, to make the organic sulfate available to plants, failed so far, most likely because of the need for the post-translational activation by production of formylglycine.

      PAP metabolism

      The nucleotide PAP is produced during PAPS-dependent sulfation pathways. It is also formed during CoA-dependent fatty acid synthetase activation (
      • Moolman W.J.
      • de Villiers M.
      • Strauss E.
      Recent advances in targeting coenzyme A biosynthesis and utilization for antimicrobial drug development.
      ), although how or whether these two pathways interconnect is currently unclear. As a reaction product, PAP strongly inhibits sulfotransferase activity (
      • Rens-Domiano S.S.
      • Roth J.A.
      Inhibition of M and P phenol sulfotransferase by analogues of 3′-phosphoadenosine-5′-phosphosulfate.
      ). With its two phosphate moieties, PAP may be regarded as the shortest possible RNA strand, and consequently, PAP interferes with RNA metabolism, inhibiting the XRN RNA-degrading exoribonucleases (
      • Gy I.
      • Gasciolli V.
      • Lauressergues D.
      • Morel J.B.
      • Gombert J.
      • Proux F.
      • Proux C.
      • Vaucheret H.
      • Mallory A.C.
      Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors.
      ). To prevent the toxic effects of PAP, dedicated PAP phosphatases are found in all kingdoms of life. Most of the enzymes from higher Eukaryotes show multiple specificity toward PAP or PAPS, and they also impact inositol signaling by removing phosphate from inositol bis- and triphosphates (
      • Quintero F.J.
      • Garciadeblás B.
      • Rodríguez-Navarro A.
      The SAL1 gene of Arabidopsis, encoding an enzyme with 3′(2′),5′-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities increases salt tolerance in yeast.
      ,
      • López-Coronado J.M.
      • Bellés J.M.
      • Lesage F.
      • Serrano R.
      • Rodríguez P.L.
      A novel mammalian lithium-sensitive enzyme with a dual enzymatic activity, 3′-phosphoadenosine 5′-phosphate phosphatase and inositol-polyphosphate 1-phosphatase.
      ), all representing small and negatively charged substrates.
      Lithium is known to influence many different proteins, and PAP phosphatases belong to the most sensitive targets for lithium inhibition. Mechanistically, lithium inhibition is well-understood for the PAP phosphatase CysQ from Mycobacterium tuberculosis. Lithium replaces one of a cluster of magnesium ions bound in the active site of the enzyme (
      • Erickson A.I.
      • Sarsam R.D.
      • Fisher A.J.
      Crystal structures of Mycobacterium tuberculosis CysQ, with substrate and products bound.
      ), due to the diagonal relationship between magnesium and lithium; these elements, diagonally adjacent in 2nd and 3rd periods of the periodic table, display a number of similar properties. As the negative amino acids in the catalytic center are highly conserved, it is highly likely that the same mechanism is in place in other PAP phosphatases.
      In many microorganisms PAP phosphatases are strongly associated with sulfate assimilation, because accumulation of PAP also inhibits PAPS reductase, an essential enzyme in sulfate reduction. In yeast, loss of PAP phosphatase Met-22 leads to methionine auxotrophy (
      • Thomas D.
      • Barbey R.
      • Surdin-Kerjan Y.
      Gene-enzyme relationship in the sulfate assimilation pathway of Saccharomyces cerevisiae. Study of the 3′-phosphoadenylylsulfate reductase structural gene.
      ). Defects in PAP catabolism result in severe growth inhibition; in animals they are mainly due to inhibition of sulfation-dependent processes (
      • Nizon M.
      • Alanay Y.
      • Tuysuz B.
      • Kiper P.O.
      • Genevieve D.
      • Sillence D.
      • Huber C.
      • Munnich A.
      • Cormier-Daire V.
      IMPAD1 mutations in two Catel-Manzke like patients.
      ,
      • Frederick J.P.
      • Tafari A.T.
      • Wu S.M.
      • Megosh L.C.
      • Chiou S.T.
      • Irving R.P.
      • York J.D.
      A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation.
      ), and in plants the defects are much more complex, because of the involvement of PAP in additional signaling pathways (
      • Estavillo G.M.
      • Crisp P.A.
      • Pornsiriwong W.
      • Wirtz M.
      • Collinge D.
      • Carrie C.
      • Giraud E.
      • Whelan J.
      • David P.
      • Javot H.
      • Brearley C.
      • Hell R.
      • Marin E.
      • Pogson B.J.
      Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis.
      ,
      • Robles P.
      • Fleury D.
      • Candela H.
      • Cnops G.
      • Alonso-Peral M.M.
      • Anami S.
      • Falcone A.
      • Caldana C.
      • Willmitzer L.
      • Ponce M.R.
      • Van Lijsebettens M.
      • Micol J.L.
      The RON1/FRY1/SAL1 gene is required for leaf morphogenesis and venation patterning in Arabidopsis.
      ).

      Plant PAP phosphatases and PAP-dependent stress signaling

      Plant PAP phosphatase SAL1 belongs to the most pleiotropic plant genes. It was first identified in rice as a protein complementing an inability to grow on sulfate in cysQ mutants of E. coli and met22 yeast mutants (
      • Peng Z.
      • Verma D.P.
      A rice HAL2-like gene encodes a Ca2+-sensitive 3′(2′),5′-diphosphonucleoside 3′(2′)-phosphohydrolase and complements yeast met22 and Escherichia coli cysQ mutations.
      ). It was subsequently shown to catalyze conversion of PAPS to APS and PAP to AMP, and this function was speculated to regulate sulfur fluxes (
      • Neuwald A.F.
      • Krishnan B.R.
      • Brikun I.
      • Kulakauskas S.
      • Suziedelis K.
      • Tomcsanyi T.
      • Leyh T.S.
      • Berg D.E.
      cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth.
      ). A homologue from Arabidopsis was identified in a screen for genes improving salt sensitivity and was named SAL1 (
      • Quintero F.J.
      • Garciadeblás B.
      • Rodríguez-Navarro A.
      The SAL1 gene of Arabidopsis, encoding an enzyme with 3′(2′),5′-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities increases salt tolerance in yeast.
      ). Since then, SAL1 has been found in numerous genetic screens for a number of unrelated phenotypes and is therefore described under many different names. A common denomination, FIERY1 or FRY1, comes from a screen for mutants in abscisic acid and stress signaling, where its loss-of-function resulted in hyperinduction of the luciferase reporter gene driven by stress-responsible promoter (
      • Xiong L.
      • Lee Bh.
      • Ishitani M.
      • Lee H.
      • Zhang C.
      • Zhu J.K.
      FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis.
      ). The phenotypes observed in the various alleles of sal1 mutants include cold and drought tolerance and signaling (
      • Estavillo G.M.
      • Crisp P.A.
      • Pornsiriwong W.
      • Wirtz M.
      • Collinge D.
      • Carrie C.
      • Giraud E.
      • Whelan J.
      • David P.
      • Javot H.
      • Brearley C.
      • Hell R.
      • Marin E.
      • Pogson B.J.
      Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis.
      ,
      • Xiong L.
      • Lee H.
      • Huang R.
      • Zhu J.K.
      A single amino acid substitution in the Arabidopsis FIERY1/HOS2 protein confers cold signaling specificity and lithium tolerance.
      ), leaf shape and venation pattern (
      • Robles P.
      • Fleury D.
      • Candela H.
      • Cnops G.
      • Alonso-Peral M.M.
      • Anami S.
      • Falcone A.
      • Caldana C.
      • Willmitzer L.
      • Ponce M.R.
      • Van Lijsebettens M.
      • Micol J.L.
      The RON1/FRY1/SAL1 gene is required for leaf morphogenesis and venation patterning in Arabidopsis.
      ), RNA silencing (
      • Gy I.
      • Gasciolli V.
      • Lauressergues D.
      • Morel J.B.
      • Gombert J.
      • Proux F.
      • Proux C.
      • Vaucheret H.
      • Mallory A.C.
      Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors.
      ), increased jasmonate levels (
      • Rodríguez V.M.
      • Chételat A.
      • Majcherczyk P.
      • Farmer E.E.
      Chloroplastic phosphoadenosine phosphosulfate metabolism regulates basal levels of the prohormone jasmonic acid in Arabidopsis leaves.
      ), glucosinolate and sulfur accumulation (
      • Lee B.R.
      • Huseby S.
      • Koprivova A.
      • Chételat A.
      • Wirtz M.
      • Mugford S.T.
      • Navid E.
      • Brearley C.
      • Saha S.
      • Mithen R.
      • Hell R.
      • Farmer E.E.
      • Kopriva S.
      Effects of fou8/fry1 mutation on sulfur metabolism: is decreased internal sulfate the trigger of sulfate starvation response?.
      ), lateral root formation (
      • Chen H.
      • Xiong L.
      The bifunctional abiotic stress signalling regulator and endogenous RNA silencing suppressor FIERY1 is required for lateral root formation.
      ), increasing circadian period (
      • Litthauer S.
      • Chan K.X.
      • Jones M.A.
      3′-Phosphoadenosine 5′-phosphate accumulation delays the circadian system.
      ), and many others. Initially it was believed that these phenotypes are caused by defects in inositol phosphate signaling (
      • Xiong L.
      • Lee Bh.
      • Ishitani M.
      • Lee H.
      • Zhang C.
      • Zhu J.K.
      FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis.
      ), but current evidence points to PAP being the main factor (
      • Chan K.X.
      • Wirtz M.
      • Phua S.Y.
      • Estavillo G.M.
      • Pogson B.J.
      Balancing metabolites in drought: the sulfur assimilation conundrum.
      ,
      • Lee B.R.
      • Huseby S.
      • Koprivova A.
      • Chételat A.
      • Wirtz M.
      • Mugford S.T.
      • Navid E.
      • Brearley C.
      • Saha S.
      • Mithen R.
      • Hell R.
      • Farmer E.E.
      • Kopriva S.
      Effects of fou8/fry1 mutation on sulfur metabolism: is decreased internal sulfate the trigger of sulfate starvation response?.
      ,
      • Kim B.H.
      • von Arnim A.G.
      FIERY1 regulates light-mediated repression of cell elongation and flowering time via its 3′(2′),5′-bisphosphate nucleotidase activity.
      ,
      • Chan K.X.
      • Phua S.Y.
      • Breusegem F.V.
      Secondary sulfur metabolism in cellular signalling and oxidative stress responses.
      ), thus linking sulfation pathways with a number of cellular processes.
      In contrast to animal PAP phosphatases in the Golgi and the cytoplasm, the plant SAL1 enzyme in chloroplasts and mitochondria has a different localization than the sulfotransferases forming PAP (
      • Estavillo G.M.
      • Crisp P.A.
      • Pornsiriwong W.
      • Wirtz M.
      • Collinge D.
      • Carrie C.
      • Giraud E.
      • Whelan J.
      • David P.
      • Javot H.
      • Brearley C.
      • Hell R.
      • Marin E.
      • Pogson B.J.
      Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis.
      ). The number of phenotypes described in sal1 mutants resemble those of loss-of-function mutants in XRN exoribonucleases (
      • Hirsch J.
      • Misson J.
      • Crisp P.A.
      • David P.
      • Bayle V.
      • Estavillo G.M.
      • Javot H.
      • Chiarenza S.
      • Mallory A.C.
      • Maizel A.
      • Declerck M.
      • Pogson B.J.
      • Vaucheret H.
      • Crespi M.
      • Desnos T.
      • et al.
      A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5′ → 3′ exoribonuclease (XRN) activities in Arabidopsis thaliana roots.
      ) and can be complemented by expression of SAL1 in the nucleus, implying that one mode of action of PAP is inhibition of XRNs (
      • Estavillo G.M.
      • Crisp P.A.
      • Pornsiriwong W.
      • Wirtz M.
      • Collinge D.
      • Carrie C.
      • Giraud E.
      • Whelan J.
      • David P.
      • Javot H.
      • Brearley C.
      • Hell R.
      • Marin E.
      • Pogson B.J.
      Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis.
      ). A model in which PAP acts as retrograde signal from chloroplast to nucleus during abiotic stress has been proposed (
      • Estavillo G.M.
      • Crisp P.A.
      • Pornsiriwong W.
      • Wirtz M.
      • Collinge D.
      • Carrie C.
      • Giraud E.
      • Whelan J.
      • David P.
      • Javot H.
      • Brearley C.
      • Hell R.
      • Marin E.
      • Pogson B.J.
      Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis.
      ) and corroborated by recent findings of redox regulation of SAL1 (
      • Chan K.X.
      • Mabbitt P.D.
      • Phua S.Y.
      • Mueller J.W.
      • Nisar N.
      • Gigolashvili T.
      • Stroeher E.
      • Grassl J.
      • Arlt W.
      • Estavillo G.M.
      • Jackson C.J.
      • Pogson B.J.
      Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase.
      ). Thus, oxidative stress leads to oxidation of a redox cysteine pair in SAL1 and strong inactivation of the enzyme. This in turn results in accumulation of PAP, its transport to the nucleus, and induction of expression of stress-response genes (
      • Chan K.X.
      • Mabbitt P.D.
      • Phua S.Y.
      • Mueller J.W.
      • Nisar N.
      • Gigolashvili T.
      • Stroeher E.
      • Grassl J.
      • Arlt W.
      • Estavillo G.M.
      • Jackson C.J.
      • Pogson B.J.
      Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase.
      ). Accordingly, PAP accumulation due to loss-of-function of SAL1 leads to stress tolerance, such as drought tolerance (
      • Chan K.X.
      • Wirtz M.
      • Phua S.Y.
      • Estavillo G.M.
      • Pogson B.J.
      Balancing metabolites in drought: the sulfur assimilation conundrum.
      ). In addition, the SAL1–PAP regulatory module has an intermediary role connecting hormonal signaling pathways, such as germination and stomatal closure (
      • Pornsiriwong W.
      • Estavillo G.M.
      • Chan K.X.
      • Tee E.E.
      • Ganguly D.
      • Crisp P.A.
      • Phua S.Y.
      • Zhao C.
      • Qiu J.
      • Park J.
      • Yong M.T.
      • Nisar N.
      • Yadav A.K.
      • Schwessinger B.
      • Rathjen J.
      • Cazzonelli C.I.
      • et al.
      A chloroplast retrograde signal, 3′-phosphoadenosine 5′-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination.
      ).
      It has to be noted that in Arabidopsis SAL1 is a member of a small gene family with seven members. SAL1 is, however, the only gene that has been found in the numerous genetic screens and that, when disrupted, causes the various phenotypes. Two additional isoforms, AHL and SAL2, were confirmed to function as PAP phosphatase (
      • Gil-Mascarell R.
      • López-Coronado J.M.
      • Bellés J.M.
      • Serrano R.
      • Rodríguez P.L.
      The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase.
      ), but only AHL is expressed at levels comparable with SAL1 (
      • Kim B.H.
      • von Arnim A.G.
      FIERY1 regulates light-mediated repression of cell elongation and flowering time via its 3′(2′),5′-bisphosphate nucleotidase activity.
      ). In contrast to SAL1, AHL does not seem to use inositol 1,4-bisphosphate as a substrate (
      • Gil-Mascarell R.
      • López-Coronado J.M.
      • Bellés J.M.
      • Serrano R.
      • Rodríguez P.L.
      The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase.
      ), and its overexpression complements the loss of SAL1 for at least some phenotypes (
      • Kim B.H.
      • von Arnim A.G.
      FIERY1 regulates light-mediated repression of cell elongation and flowering time via its 3′(2′),5′-bisphosphate nucleotidase activity.
      ). Although this is clear evidence for PAP being the causal metabolite for many phenotypes, the reason why in WT Arabidopsis AHL does not suffice to metabolize PAP remains to be elucidated. Another unsolved question is the physiological relevance of PAPS dephosphorylation.
      The alteration in glucosinolate synthesis is the first direct metabolic link of SAL1 with sulfation pathways (
      • Lee B.R.
      • Huseby S.
      • Koprivova A.
      • Chételat A.
      • Wirtz M.
      • Mugford S.T.
      • Navid E.
      • Brearley C.
      • Saha S.
      • Mithen R.
      • Hell R.
      • Farmer E.E.
      • Kopriva S.
      Effects of fou8/fry1 mutation on sulfur metabolism: is decreased internal sulfate the trigger of sulfate starvation response?.
      ). In the fou8 allele of sal1 mutants, glucosinolate levels were lower than in WT Col-0 (
      • Lee B.R.
      • Huseby S.
      • Koprivova A.
      • Chételat A.
      • Wirtz M.
      • Mugford S.T.
      • Navid E.
      • Brearley C.
      • Saha S.
      • Mithen R.
      • Hell R.
      • Farmer E.E.
      • Kopriva S.
      Effects of fou8/fry1 mutation on sulfur metabolism: is decreased internal sulfate the trigger of sulfate starvation response?.
      ). This was caused by reduction in sulfation rate, as the mutants also accumulated the desulfo-glucosinolate precursors (
      • Lee B.R.
      • Huseby S.
      • Koprivova A.
      • Chételat A.
      • Wirtz M.
      • Mugford S.T.
      • Navid E.
      • Brearley C.
      • Saha S.
      • Mithen R.
      • Hell R.
      • Farmer E.E.
      • Kopriva S.
      Effects of fou8/fry1 mutation on sulfur metabolism: is decreased internal sulfate the trigger of sulfate starvation response?.
      ). The phenotype thus strongly resembled that of apk1 apk2 mutants with low provision of PAPS (
      • Mugford S.G.
      • Yoshimoto N.
      • Reichelt M.
      • Wirtz M.
      • Hill L.
      • Mugford S.T.
      • Nakazato Y.
      • Noji M.
      • Takahashi H.
      • Kramell R.
      • Gigolashvili T.
      • Flügge U.I.
      • Wasternack C.
      • Gershenzon J.
      • Hell R.
      • et al.
      Disruption of adenosine-5′-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites.
      ). Interestingly, combining the fou8 mutant with apk1 apk2 resulted in alleviation of many of the phenotypic alterations connected with loss of SAL1 function, strongly suggesting that PAP was the responsible metabolite (
      • Lee B.R.
      • Huseby S.
      • Koprivova A.
      • Chételat A.
      • Wirtz M.
      • Mugford S.T.
      • Navid E.
      • Brearley C.
      • Saha S.
      • Mithen R.
      • Hell R.
      • Farmer E.E.
      • Kopriva S.
      Effects of fou8/fry1 mutation on sulfur metabolism: is decreased internal sulfate the trigger of sulfate starvation response?.
      ). This observation forms a second direct link of SAL1 and sulfation pathways: the SAL1–PAP signaling depends on synthesis of PAPS and sulfation reactions, i.e. secondary sulfur metabolism (
      • Chan K.X.
      • Phua S.Y.
      • Breusegem F.V.
      Secondary sulfur metabolism in cellular signalling and oxidative stress responses.
      ). This is particularly important for plants, which do not synthesize glucosinolates or other major classes of sulfated secondary metabolites but still possess functional PAP signaling (
      • Manmathan H.
      • Shaner D.
      • Snelling J.
      • Tisserat N.
      • Lapitan N.
      Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance.
      ). Which sulfotransferase isoforms provide the majority of PAP for the stress signaling is, however, still unknown.

      Human BPNT1 and Golgi PAP phosphatases

      In humans, PAP is degraded at the sites of its production by a cytoplasmic and a Golgi PAP phosphatase. The human PAP phosphatase bisphosphate nucleotidase 1 (BPNT1) is a cytoplasmic enzyme (
      • Hudson B.H.
      • York J.D.
      Roles for nucleotide phosphatases in sulfate assimilation and skeletal disease.
      ), whereas the “Golgi PAP phosphatase” (gPAPP) is obviously located in the Golgi apparatus. For its side activity toward inositols, however, gPAPP is also known as inositol monophosphatase domain containing 1 (IMPAD1). The catalytic domain of this type II transmembrane protein is in the lumen of the Golgi (
      • Hudson B.H.
      • York J.D.
      Roles for nucleotide phosphatases in sulfate assimilation and skeletal disease.
      ). Its main substrate is PAP from Golgi-residing sulfotransferases (
      • Frederick J.P.
      • Tafari A.T.
      • Wu S.M.
      • Megosh L.C.
      • Chiou S.T.
      • Irving R.P.
      • York J.D.
      A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation.
      ).
      Mice with an inactivated gPAPP/IMPAD1 gene show neonatal lethality, abnormalities in the lung, and bone and cartilage malformation (
      • Frederick J.P.
      • Tafari A.T.
      • Wu S.M.
      • Megosh L.C.
      • Chiou S.T.
      • Irving R.P.
      • York J.D.
      A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation.
      ). This may be due to under-sulfated chondroitin and perturbed formation of heparan sulfate due to the inhibition of the corresponding sulfotransferases by the accumulated PAP (
      • Frederick J.P.
      • Tafari A.T.
      • Wu S.M.
      • Megosh L.C.
      • Chiou S.T.
      • Irving R.P.
      • York J.D.
      A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation.
      ). The human gPAPP/IMPAD1 gene lies in the genomic region 8p11-p12 that is frequently amplified in breast cancer (
      • Parris T.Z.
      • Kovács A.
      • Hajizadeh S.
      • Nemes S.
      • Semaan M.
      • Levin M.
      • Karlsson P.
      • Helou K.
      Frequent MYC coamplification and DNA hypomethylation of multiple genes on 8q in 8p11-p12-amplified breast carcinomas.
      ); however, a functional role in tumorigenesis remains to be established. Patients with truncation mutations in gPAPP/IMPAD1 are characterized by short stature, joint dislocations, brachydactyly, and cleft palate (
      • Nizon M.
      • Alanay Y.
      • Tuysuz B.
      • Kiper P.O.
      • Genevieve D.
      • Sillence D.
      • Huber C.
      • Munnich A.
      • Cormier-Daire V.
      IMPAD1 mutations in two Catel-Manzke like patients.
      ,
      • Vissers L.E.
      • Lausch E.
      • Unger S.
      • Campos-Xavier A.B.
      • Gilissen C.
      • Rossi A.
      • Del Rosario M.
      • Venselaar H.
      • Knoll U.
      • Nampoothiri S.
      • Nair M.
      • Spranger J.
      • Brunner H.G.
      • Bonafé L.
      • Veltman J.A.
      • et al.
      Chondrodysplasia and abnormal joint development associated with mutations in IMPAD1, encoding the Golgi-resident nucleotide phosphatase, gPAPP.
      ). Some patients also had a homozygous missense mutation D77N (
      • Vissers L.E.
      • Lausch E.
      • Unger S.
      • Campos-Xavier A.B.
      • Gilissen C.
      • Rossi A.
      • Del Rosario M.
      • Venselaar H.
      • Knoll U.
      • Nampoothiri S.
      • Nair M.
      • Spranger J.
      • Brunner H.G.
      • Bonafé L.
      • Veltman J.A.
      • et al.
      Chondrodysplasia and abnormal joint development associated with mutations in IMPAD1, encoding the Golgi-resident nucleotide phosphatase, gPAPP.
      ). This mutation was recently introduced into mice, and the phenotype of homozygous gPAPP/Impad1 knockin animals overlaps with the lethal phenotype described previously in Impad1 knockout mice (
      • Capulli M.
      • Costantini R.
      • Sonntag S.
      • Maurizi A.
      • Paganini C.
      • Monti L.
      • Forlino A.
      • Shmerling D.
      • Teti A.
      • Rossi A.
      Testing the Cre-mediated genetic switch for the generation of conditional knock-in mice.
      ). The gPAPP phosphatase is only found in animals; hence, it may have co-evolved with the many Golgi sulfotransferases as a critical modulator of glycosaminoglycan and proteoglycan sulfation.
      With an inhibition constant Ki of 157 μm, the only other human PAP phosphatase BPNT1 is an exceptionally lithium-sensitive enzyme (
      • Spiegelberg B.D.
      • Xiong J.P.
      • Smith J.J.
      • Gu R.F.
      • York J.D.
      Cloning and characterization of a mammalian lithium-sensitive bisphosphate 3′-nucleotidase inhibited by inositol 1,4-bisphosphate.
      ). Hence, there was speculation whether BPNT1 is the actual target for lithium as a treatment for bipolar disorder. At least in C. elegans, lithium causes BPNT1-mediated selective toxicity to specific neurons and leads to behavior changes (
      • Meisel J.D.
      • Kim D.H.
      Inhibition of lithium-sensitive phosphatase BPNT-1 causes selective neuronal dysfunction in C. elegans.
      ). A study in rats, however, questioned the role of PAP phosphatases for the therapeutic effect of lithium, as there was no PAP accumulation detected in the brain after prolonged lithium exposure (
      • Shaltiel G.
      • Deutsch J.
      • Rapoport S.I.
      • Basselin M.
      • Belmaker R.H.
      • Agam G.
      Is phosphoadenosine phosphate phosphatase a target of lithium's therapeutic effect?.
      ). Additionally, the knockout of Bpnt1 in mice leads to an early aging phenotype (
      • Hudson B.H.
      • Frederick J.P.
      • Drake L.Y.
      • Megosh L.C.
      • Irving R.P.
      • York J.D.
      Role for cytoplasmic nucleotide hydrolysis in hepatic function and protein synthesis.
      ). Bpnt1−/− mice do not show a skeletal phenotype, but develop liver pathologies, hypoalbuminemia, hepatocellular damage, and deadly whole-body edema by just 7 weeks of age (
      • Hudson B.H.
      • Frederick J.P.
      • Drake L.Y.
      • Megosh L.C.
      • Irving R.P.
      • York J.D.
      Role for cytoplasmic nucleotide hydrolysis in hepatic function and protein synthesis.
      ). PAP accumulation is thought to interfere with RNA processing leading to defective ribosomes (
      • Hudson B.H.
      • Frederick J.P.
      • Drake L.Y.
      • Megosh L.C.
      • Irving R.P.
      • York J.D.
      Role for cytoplasmic nucleotide hydrolysis in hepatic function and protein synthesis.
      ). A recent re-evaluation of the same mouse model linked the toxic accumulation of a BPNT1 substrate, such as PAP, directly or indirectly to changes in HIF-2α levels and iron homeostasis (
      • Hudson B.H.
      • Hale A.T.
      • Irving R.P.
      • Li S.
      • York J.D.
      Modulation of intestinal sulfur assimilation metabolism regulates iron homeostasis.
      ). Looking at the tissue distribution of the BPNT1 and enriched levels of PAP, Hudson and York (
      • Hudson B.H.
      • York J.D.
      Tissue-specific regulation of 3′-nucleotide hydrolysis and nucleolar architecture.
      ) reported a mismatch between broad expression of BPNT1, but measurable PAP accumulation only in liver, duodenum, and kidneys.
      Interesting questions about how BPNT1 is regulated in a tissue-specific manner, whether redox regulation plays a role, and what involvement this PAP phosphatase has in further regulatory pathways remain to be answered. In worms at least, a genetic interaction of BPNT1 and the exoribonuclease XRN2 in polycistronic gene regulation has recently been reported (
      • Miki T.S.
      • Carl S.H.
      • Stadler M.B.
      • Grosshans H.
      XRN2 Autoregulation and control of polycistronic gene expression in Caenorhabditis elegans.
      ).

      Subcellular localization and transporters in sulfation pathways

      The products of sulfation pathways represent intracellular metabolites as well as external proteins/peptides and carbohydrates. Therefore, the sulfotransferase enzymes have to be located in at least two compartments of the cytosol and Golgi apparatus. However, animal sulfate activation occurs in the cytoplasm and the nucleus as PAPS synthases shuttle between these compartments (
      • Schröder E.
      • Gebel L.
      • Eremeev A.A.
      • Morgner J.
      • Grum D.
      • Knauer S.K.
      • Bayer P.
      • Mueller J.W.
      Human PAPS synthase isoforms are dynamically regulated enzymes with access to nucleus and cytoplasm.
      ). Conserved nuclear localization and export signals govern this subcellular distribution, including a nuclear localization signal at the very N terminus of the APK domain as well as an atypical nuclear export signal at the APK dimer interface (
      • Schröder E.
      • Gebel L.
      • Eremeev A.A.
      • Morgner J.
      • Grum D.
      • Knauer S.K.
      • Bayer P.
      • Mueller J.W.
      Human PAPS synthase isoforms are dynamically regulated enzymes with access to nucleus and cytoplasm.
      ).
      Human soluble SULT enzymes are mainly cytoplasmic (
      • Coughtrie M.W.H.
      Function and organization of the human cytosolic sulfotransferase (SULT) family.
      ); however, they are sometimes also present in the nucleus (
      • He D.
      • Falany C.N.
      Characterization of proline-serine-rich carboxyl terminus in human sulfotransferase 2B1b: immunogenicity, subcellular localization, kinetic properties, and phosphorylation.
      ). They receive the necessary PAPS co-factor from PAPS synthases via diffusion through the bulk medium; however, transient protein interactions might facilitate this process (
      • Mueller J.W.
      • Idkowiak J.
      • Gesteira T.F.
      • Vallet C.
      • Hardman R.
      • van den Boom J.
      • Dhir V.
      • Knauer S.K.
      • Rosta E.
      • Arlt W.
      Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.
      ). Because of their very high expression in liver and some other tissues, cytoplasmic sulfotransferases may outnumber PAPS synthases and the co-factor itself (
      • Mueller J.W.
      • Idkowiak J.
      • Gesteira T.F.
      • Vallet C.
      • Hardman R.
      • van den Boom J.
      • Dhir V.
      • Knauer S.K.
      • Rosta E.
      • Arlt W.
      Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.
      ,
      • Riches Z.
      • Stanley E.L.
      • Bloomer J.C.
      • Coughtrie M.W.
      Quantitative evaluation of the expression and activity of five major sulfotransferases (SULTs) in human tissues: the SULT “pie”.
      ). Therefore, interactions between sulfotransferases and PAPS synthases may be a mechanism to overcome the substrate limitation and add an additional level of control (
      • Mueller J.W.
      • Idkowiak J.
      • Gesteira T.F.
      • Vallet C.
      • Hardman R.
      • van den Boom J.
      • Dhir V.
      • Knauer S.K.
      • Rosta E.
      • Arlt W.
      Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.
      ).
      The many Golgi sulfotransferases rely on the import of PAPS by the human PAPS transporters PAPST1 and PAPST2, also referred to as SLC35B2 and SLC35B3, respectively (
      • Kamiyama S.
      • Sasaki N.
      • Goda E.
      • Ui-Tei K.
      • Saigo K.
      • Narimatsu H.
      • Jigami Y.
      • Kannagi R.
      • Irimura T.
      • Nishihara S.
      Molecular cloning and characterization of a novel 3′-phosphoadenosine 5′-phosphosulfate transporter, PAPST2.
      ,
      • Kamiyama S.
      • Suda T.
      • Ueda R.
      • Suzuki M.
      • Okubo R.
      • Kikuchi N.
      • Chiba Y.
      • Goto S.
      • Toyoda H.
      • Saigo K.
      • Watanabe M.
      • Narimatsu H.
      • Jigami Y.
      • Nishihara S.
      Molecular cloning and identification of 3′-phosphoadenosine 5′-phosphosulfate transporter.
      ). Both transporters belong to the group of nucleotide–sugar transporters but are specific for PAPS and only share 24% amino acid identity with each other (
      • Kamiyama S.
      • Suda T.
      • Ueda R.
      • Suzuki M.
      • Okubo R.
      • Kikuchi N.
      • Chiba Y.
      • Goto S.
      • Toyoda H.
      • Saigo K.
      • Watanabe M.
      • Narimatsu H.
      • Jigami Y.
      • Nishihara S.
      Molecular cloning and identification of 3′-phosphoadenosine 5′-phosphosulfate transporter.
      ). The PAPST1 homologue from Drosophila is essential for viability of the flies (
      • Kamiyama S.
      • Suda T.
      • Ueda R.
      • Suzuki M.
      • Okubo R.
      • Kikuchi N.
      • Chiba Y.
      • Goto S.
      • Toyoda H.
      • Saigo K.
      • Watanabe M.
      • Narimatsu H.
      • Jigami Y.
      • Nishihara S.
      Molecular cloning and identification of 3′-phosphoadenosine 5′-phosphosulfate transporter.
      ). In humans, the two transporters are expressed in different tissues and may impact different subsets of sulfation pathways (
      • Dick G.
      • Akslen-Hoel L.K.
      • Grøndahl F.
      • Kjos I.
      • Maccarana M.
      • Prydz K.
      PAPST1 regulates sulfation of heparan sulfate proteoglycans in epithelial MDCK II cells.
      ).
      A complementary mechanism to having enzymes in multiple compartments is that the substrates and/or sulfated products themselves can traffic around the cell. Many low-molecular-weight compounds such as steroids are believed to be membrane-permeable. Notably, however, a recent study challenges the dogma of freely membrane-permeable steroids (and maybe also other smaller compounds). Okamoto et al. (
      • Okamoto N.
      • Viswanatha R.
      • Bittar R.
      • Li Z.
      • Haga-Yamanaka S.
      • Perrimon N.
      • Yamanaka N.
      A membrane transporter is required for steroid hormone uptake in Drosophila.
      ) have reported that in Drosophila, steroid hormones require a protein transporter for passing through cellular membranes. Moreover, once steroids become sulfated, they are trapped within the cell (
      • Mueller J.W.
      • Gilligan L.C.
      • Idkowiak J.
      • Arlt W.
      • Foster P.A.
      The regulation of steroid action by sulfation and desulfation.
      ). Release into the circulation and uptake into other cells depends on organic anion transporters, the OATPs (
      • Nigam S.K.
      • Bush K.T.
      • Martovetsky G.
      • Ahn S.Y.
      • Liu H.C.
      • Richard E.
      • Bhatnagar V.
      • Wu W.
      The organic anion transporter (OAT) family: a systems biology perspective.
      ). These individual transporters thus represent an additional layer of regulation (
      • Hartmann K.
      • Bennien J.
      • Wapelhorst B.
      • Bakhaus K.
      • Schumacher V.
      • Kliesch S.
      • Weidner W.
      • Bergmann M.
      • Geyer J.
      • Fietz D.
      Current insights into the sulfatase pathway in human testis and cultured Sertoli cells.
      ).
      Plants also require transporters for function of sulfation pathways. Although all the PAPS-dependent sulfotransferases are located outside of plastids, the majority of the APS kinase activity is located within the chloroplast (
      • Mugford S.G.
      • Yoshimoto N.
      • Reichelt M.
      • Wirtz M.
      • Hill L.
      • Mugford S.T.
      • Nakazato Y.
      • Noji M.
      • Takahashi H.
      • Kramell R.
      • Gigolashvili T.
      • Flügge U.I.
      • Wasternack C.
      • Gershenzon J.
      • Hell R.
      • et al.
      Disruption of adenosine-5′-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites.
      ). Hence, plant cytosolic sulfation pathways are dependent on export of PAPS from plastids and those in the Golgi additionally on import of cytosolic PAPS. The first plant PAPS transporter was identified through co-expression with genes for glucosinolate synthesis and transport assays in liposomes (
      • Gigolashvili T.
      • Geier M.
      • Ashykhmina N.
      • Frerigmann H.
      • Wulfert S.
      • Krueger S.
      • Mugford S.G.
      • Kopriva S.
      • Haferkamp I.
      • Flügge U.I.
      The Arabidopsis thylakoid ADP/ATP carrier TAAC has an additional role in supplying plastidic phosphoadenosine 5′-phosphosulfate to the cytosol.
      ) and belongs to the ADP/ATP carriers of the mitochondrial carrier family. AtPAPST1 also transports PAP, which has to be imported to plastids for degradation by SAL1; therefore, the transporter most probably serves as a PAP/PAPS antiporter (
      • Gigolashvili T.
      • Geier M.
      • Ashykhmina N.
      • Frerigmann H.
      • Wulfert S.
      • Krueger S.
      • Mugford S.G.
      • Kopriva S.
      • Haferkamp I.
      • Flügge U.I.
      The Arabidopsis thylakoid ADP/ATP carrier TAAC has an additional role in supplying plastidic phosphoadenosine 5′-phosphosulfate to the cytosol.
      ). The loss-of-function mutant papst1 accumulates desulfo-glucosinolate precursors and shows decreased glucosinolate levels similar to but to a lower extent than apk1 apk2, suggesting the existence of a second plastidic PAPS transporter. Indeed, AtPAPST2 was recently identified in Arabidopsis as a transporter located dually in membranes of chloroplasts and mitochondria (
      • Ashykhmina N.
      • Lorenz M.
      • Frerigmann H.
      • Koprivova A.
      • Hofsetz E.
      • Stührwohldt N.
      • Flügge U.I.
      • Haferkamp I.
      • Kopriva S.
      • Gigolashvili T.
      PAPST2 plays critical roles in removing the stress signaling molecule 3′-phosphoadenosine 5′-phosphate from the cytosol and its subsequent degradation in plastids and mitochondria.
      ). The AtPAPST2 gene is not co-expressed with glucosinolate genes, and its loss had only a minor effect on glucosinolate accumulation (
      • Ashykhmina N.
      • Lorenz M.
      • Frerigmann H.
      • Koprivova A.
      • Hofsetz E.
      • Stührwohldt N.
      • Flügge U.I.
      • Haferkamp I.
      • Kopriva S.
      • Gigolashvili T.
      PAPST2 plays critical roles in removing the stress signaling molecule 3′-phosphoadenosine 5′-phosphate from the cytosol and its subsequent degradation in plastids and mitochondria.
      ). Localization links AtPAPST2 to SAL1, which is also present in plastids and mitochondria. Thus, it seems that AtPAPST1 has a major role in exporting PAPS from chloroplast to cytosol for sulfation reactions and AtPAPST2 in importing PAP into the organelles for degradation by SAL1 (
      • Ashykhmina N.
      • Lorenz M.
      • Frerigmann H.
      • Koprivova A.
      • Hofsetz E.
      • Stührwohldt N.
      • Flügge U.I.
      • Haferkamp I.
      • Kopriva S.
      • Gigolashvili T.
      PAPST2 plays critical roles in removing the stress signaling molecule 3′-phosphoadenosine 5′-phosphate from the cytosol and its subsequent degradation in plastids and mitochondria.
      ). It also seems that the two transporters, AtPAPST1 and AtPAPST2, are not sufficient to explain all phenotypes connected to movement of PAPS and PAP between cytosol and the organelles, particularly the accumulation of glucosinolates and their desulfo-precursors. This metabolic phenotype can be expected to be found in mutants of the additional transporter gene(s) and enable their identification.

      Natural genetic variation

      The enzymes connected to sulfation pathways show a large variation between the different lineages and taxa. Many of them are found in several isoforms, further expanding their variation. However, the individual gene/enzyme isoforms also show variability within a single species, in different accessions and populations or even in individuals. Rare genetic mutations have been extremely informative in the study of many components of the sulfation pathways (
      • Herrmann J.
      • Ravilious G.E.
      • McKinney S.E.
      • Westfall C.S.
      • Lee S.G.
      • Baraniecka P.
      • Giovannetti M.
      • Kopriva S.
      • Krishnan H.B.
      • Jez J.M.
      Structure and mechanism of soybean ATP sulfurylase and the committed step in plant sulfur assimilation.
      ,
      • Oostdijk W.
      • Idkowiak J.
      • Mueller J.W.
      • House P.J.
      • Taylor A.E.
      • O’Reilly M.W.
      • Hughes B.A.
      • de Vries M.C.
      • Kant S.G.
      • Santen G.W.
      • Verkerk A.J.
      • Uitterlinden A.G.
      • Wit J.M.
      • Losekoot M.
      • Arlt W.
      PAPSS2 deficiency causes androgen excess via impaired DHEA sulfation–in vitro and in vivo studies in a family harboring two novel PAPSS2 mutations.
      ,
      • Ahmad M.
      • Faiyaz Ul Haque M.
      • Ahmad W.
      • Abbas H.
      • Haque S.
      • Krakow D.
      • Rimoin D.L.
      • Lachman R.S.
      • Cohn D.H.
      Distinct, autosomal recessive form of spondyloepimetaphyseal dysplasia segregating in an inbred Pakistani kindred.
      ). Because of vastly increased sequencing capacities, such genetic variation is now studied on a population scale, both in plants and in humans.

      Human genetic variation and clinical outcomes

      Genetic defects in the gene for human PAPSS1 have not been reported so far. Gene defects in human PAPSS2, however, have been known to cause various forms of bone and cartilage malformation, due to an under-sulfation of the extracellular matrix (
      • Oostdijk W.
      • Idkowiak J.
      • Mueller J.W.
      • House P.J.
      • Taylor A.E.
      • O’Reilly M.W.
      • Hughes B.A.
      • de Vries M.C.
      • Kant S.G.
      • Santen G.W.
      • Verkerk A.J.
      • Uitterlinden A.G.
      • Wit J.M.
      • Losekoot M.
      • Arlt W.
      PAPSS2 deficiency causes androgen excess via impaired DHEA sulfation–in vitro and in vivo studies in a family harboring two novel PAPSS2 mutations.
      ,
      • Ahmad M.
      • Faiyaz Ul Haque M.
      • Ahmad W.
      • Abbas H.
      • Haque S.
      • Krakow D.
      • Rimoin D.L.
      • Lachman R.S.
      • Cohn D.H.
      Distinct, autosomal recessive form of spondyloepimetaphyseal dysplasia segregating in an inbred Pakistani kindred.
      ). A steroid sulfation defect was reported for the first time in a girl with the compound–heterozygous mutations T48R/R329* in PAPSS2 (
      • Noordam C.
      • Dhir V.
      • McNelis J.C.
      • Schlereth F.
      • Hanley N.A.
      • Krone N.
      • Smeitink J.A.
      • Smeets R.
      • Sweep F.C.
      • Claahsen-van der Grinten H.L.
      • Arlt W.
      Inactivating PAPSS2 mutations in a patient with premature pubarche.
      ). A subsequent study with two brothers carrying the compound–heterozygous mutations G270D and the frameshift mutation W462Cfs*3, resulting in an early termination codon, in PAPSS2 confirmed disrupted sulfation of DHEA, the most abundant steroid in the human circulation (
      • Mueller J.W.
      • Gilligan L.C.
      • Idkowiak J.
      • Arlt W.
      • Foster P.A.
      The regulation of steroid action by sulfation and desulfation.
      ), and increased androgen activation (
      • Oostdijk W.
      • Idkowiak J.
      • Mueller J.W.
      • House P.J.
      • Taylor A.E.
      • O’Reilly M.W.
      • Hughes B.A.
      • de Vries M.C.
      • Kant S.G.
      • Santen G.W.
      • Verkerk A.J.
      • Uitterlinden A.G.
      • Wit J.M.
      • Losekoot M.
      • Arlt W.
      PAPSS2 deficiency causes androgen excess via impaired DHEA sulfation–in vitro and in vivo studies in a family harboring two novel PAPSS2 mutations.
      ). These studies of individual patients (
      • Oostdijk W.
      • Idkowiak J.
      • Mueller J.W.
      • House P.J.
      • Taylor A.E.
      • O’Reilly M.W.
      • Hughes B.A.
      • de Vries M.C.
      • Kant S.G.
      • Santen G.W.
      • Verkerk A.J.
      • Uitterlinden A.G.
      • Wit J.M.
      • Losekoot M.
      • Arlt W.
      PAPSS2 deficiency causes androgen excess via impaired DHEA sulfation–in vitro and in vivo studies in a family harboring two novel PAPSS2 mutations.