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Tic22 Is an Essential Chaperone Required for Protein Import into the Apicoplast*

  • Stephanie Glaser
    Footnotes
    Affiliations
    Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, England
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  • Giel G. van Dooren
    Correspondence
    A QEII Fellow of the Australian Research Council. To whom correspondence may be addressed
    Footnotes
    Affiliations
    Research School of Biology, Australian National University, Canberra, ACT, 0200, Australia
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  • Swati Agrawal
    Footnotes
    Affiliations
    Department of Cellular Biology, University of Georgia, Athens, Georgia 30602
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  • Carrie F. Brooks
    Affiliations
    Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia 30602
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  • Geoffrey I. McFadden
    Footnotes
    Affiliations
    Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria, 3010, Australia
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  • Boris Striepen
    Correspondence
    A Georgia Research Alliance Distinguished Investigator. To whom correspondence may be addressed
    Affiliations
    Department of Cellular Biology, University of Georgia, Athens, Georgia 30602

    Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia 30602
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  • Matthew K. Higgins
    Correspondence
    To whom correspondence may be addressed
    Affiliations
    Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, England
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  • Author Footnotes
    * This work was funded by an Australian Research Council Discovery grant (to G. v. D.), a National Health and Medical Research Council Program Grant (to G. I. M.), a Medical Research Council grant (to M. K. H.), and a grant from the National Institutes of Health (AI 64671) (to B. S.).
    This article contains supplemental Figs. S1–S7.
    5 An ARC Federation Fellow.
    1 Both authors contributed equally to this work.
    2 Supported by a Medical Research Council Studentship.
    4 Supported by a predoctoral fellowship from the American Heart Association. Present address: Dept. of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48105.
Open AccessPublished:October 01, 2012DOI:https://doi.org/10.1074/jbc.M112.405100
      Most plastids proteins are post-translationally imported into organelles through multisubunit translocons. The TIC and TOC complexes perform this role in the two membranes of the plant chloroplast and in the inner two membranes of the apicoplasts of the apicomplexan parasites, Toxoplasma gondii and Plasmodium falciparum. Tic22 is a ubiquitous intermembrane translocon component that interacts with translocating proteins. Here, we demonstrate that T. gondii Tic22 is an apicoplast-localized protein, essential for parasite survival and protein import into the apicoplast stroma. The structure of Tic22 from P. falciparum reveals a fold conserved from cyanobacteria to plants, which displays a non-polar groove on each side of the molecule. We show that these grooves allow Tic22 to act as a chaperone. General chaperones are common components of protein translocation systems where they maintain cargo proteins in an unfolded conformation during transit. Such a chaperone had not been identified in the intermembrane space of plastids and we propose that Tic22 fulfills this role.

      Introduction

      Endosymbiosis has been a major driver of eukaryotic evolution. Plastids, such as the chloroplasts of plants, evolved through the endosymbiotic acquisition of a cyanobacterium by a eukaryotic cell. Plastids are critical to the survival of cells in which they are found, and have central roles in carbon fixation and synthesis of biomolecules such as tetrapyrroles, amino acids, fatty acids, and isoprenoids. After the initial endosymbiosis that led to the evolution of primary plastids in plants and red algae, plastids spread through multiple eukaryotic phyla through a number of secondary endosymbioses. Here, a plastid-bearing eukaryote became incorporated into a heterotrophic eukaryote, bestowing the many biochemical advantages of a plastid. Plastids derived in this manner are found in organisms as diverse as brown algae, dinoflagellates, and a group of intracellular parasites called the Apicomplexa. Apicomplexa include Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, the cause of toxoplasmosis. They contain a plastid known as the apicoplast (
      • McFadden G.I.
      • van Dooren G.G.
      Evolution: red algal genome affirms a common origin of all plastids.
      ). Although highly reduced in function compared with the chloroplasts of plants, apicoplasts are critical for parasite survival as they contain essential enzymatic pathways for the synthesis of fatty acids, heme, iron-sulfur clusters, and isoprenoids (
      • Ralph S.A.
      • van Dooren G.G.
      • Waller R.F.
      • Crawford M.J.
      • Fraunholz M.J.
      • Foth B.J.
      • Tonkin C.J.
      • Roos D.S.
      • McFadden G.I.
      Tropical infectious diseases: Metabolic maps and functions of the Plasmodium falciparum apicoplast.
      ).
      Most proteins that function in plastids are encoded by nuclear genes and are post-translationally trafficked by multi-subunit translocons across the two or more membranes that surround the organelle (
      • Soll J.
      • Schleiff E.
      Protein import into chloroplasts.
      ). In plant chloroplasts, this is mediated by the Translocons of the Inner and Outer Chloroplast membranes (the TIC
      The abbreviations used are: TIC
      translocons of the inner chloroplast membranes
      TOC
      translocons of the outer chloroplast membranes
      ATc
      anhydrotetracycline.
      and TOC complexes). The TOC complex recognizes motifs in plastid-targeted proteins and feeds them through the pore formed by the β-barrel of Toc75 (
      • Soll J.
      • Schleiff E.
      Protein import into chloroplasts.
      ). Reconstitution of just Toc75 and Toc159 into liposomes generates a functional import apparatus, suggesting that the TOC complex can act independently of any other components to drive proteins across the outer membrane (
      • Schleiff E.
      • Jelic M.
      • Soll J.
      A GTP-driven motor moves proteins across the outer envelope of chloroplasts.
      ). In vivo, some proteins are completely translocated through the TOC complex before engaging the inner membrane TIC complex (
      • Scott S.V.
      • Theg S.M.
      A new chloroplast protein import intermediate reveals distinct translocation machineries in the two envelope membranes: energetics and mechanistic implications.
      ), while proteins destined for the intermembrane space use the TOC complex alone to reach their final destination (
      • Vojta L.
      • Soll J.
      • Bölter B.
      Protein transport in chloroplasts, targeting to the intermembrane space.
      ). More commonly, proteins are translocated simultaneously across both TIC and TOC complexes at contact sites (
      • Schnell D.J.
      • Blobel G.
      Identification of intermediates in the pathway of protein import into chloroplasts and their localization to envelope contact sites.
      ), engaging with the TIC machinery before completing their passage through Toc75 (
      • Schleiff E.
      • Jelic M.
      • Soll J.
      A GTP-driven motor moves proteins across the outer envelope of chloroplasts.
      ).
      The TIC complex of plants is thought to consist of at least seven subunits, although their functions and association with one another are not well understood. Both Tic110 and Tic20 have been proposed to serve as pores, although they may be part of different TIC complexes (
      • Kikuchi S.
      • Oishi M.
      • Hirabayashi Y.
      • Lee D.W.
      • Hwang I.
      • Nakai M.
      A 1-megadalton translocation complex containing Tic20 and Tic21 mediates chloroplast protein import at the inner envelope membrane.
      ,
      • Kovács-Bogdán E.
      • Benz J.P.
      • Soll J.
      • Bölter B.
      Tic20 forms a channel independent of Tic110 in chloroplasts.
      ). The TIC complex of plants also contains subunits that likely function in redox regulation, a subunit (Tic40) that recruits the chaperone ClpC to the stromal (inside) side of the TIC complex to mediate protein translocation, and a subunit of unknown function called Tic22 (
      • Chou M.L.
      • Chu C.C.
      • Chen L.J.
      • Akita M.
      • Li H.M.
      Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts.
      ,
      • Kovacs-Bogdan E.
      • Soll J.
      • Bolter B.
      Protein import into chloroplasts: the Tic complex and its regulation.
      ,
      • Kouranov A.
      • Chen X.
      • Fuks B.
      • Schnell D.J.
      Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane.
      ).
      Tic22 was originally identified through association with plastid-targeted proteins in cross-linking studies (
      • Kouranov A.
      • Chen X.
      • Fuks B.
      • Schnell D.J.
      Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane.
      ,
      • Kouranov A.
      • Schnell D.J.
      Analysis of the interactions of preproteins with the import machinery over the course of protein import into chloroplasts.
      ). It is a 22 kDa protein that localizes to the chloroplast intermembrane space. Although Tic22 is soluble, it is found in inner membrane preparations and a small percentage co-fractionates with the integral inner membrane component Tic20 (
      • Kouranov A.
      • Chen X.
      • Fuks B.
      • Schnell D.J.
      Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane.
      ). Tic22 also interacts with Toc64, and cofractionates with the TOC complex (
      • Kouranov A.
      • Chen X.
      • Fuks B.
      • Schnell D.J.
      Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane.
      ,
      • Becker T.
      • Hritz J.
      • Vogel M.
      • Caliebe A.
      • Bukau B.
      • Soll J.
      • Schleiff E.
      Toc12, a novel subunit of the intermembrane space preprotein translocon of chloroplasts.
      ). Whether these interactions occur simultaneously or transiently is uncertain, and roles for Tic22 in either bridging the TIC and TOC complexes, or in shuttling translocated proteins between the two have been proposed (
      • Soll J.
      • Schleiff E.
      Protein import into chloroplasts.
      ). A recent study of a cyanobacterial Tic22 homologue proposes a role of Tic22 in bacterial outer membrane biogenesis, perhaps functioning as a chaperone (
      • Tripp J.
      • Hahn A.
      • Koenig P.
      • Flinner N.
      • Bublak D.
      • Brouwer E.M.
      • Ertel F.
      • Mirus O.
      • Sinning I.
      • Tews I.
      • Schleiff E.
      Structure and conservation of the periplasmic targeting factor Tic22 from plants and cyanobacteria.
      ).
      Apicoplasts are bound by four membranes, the inner two of which are homologous to the two membranes that surround plant chloroplasts. As in primary plastids, each apicoplast membrane appears to contain a translocon that mediates protein import. Apicomplexan genomes contain homologues of subunits of both the TIC and TOC complexes (
      • McFadden G.I.
      • van Dooren G.G.
      Evolution: red algal genome affirms a common origin of all plastids.
      ,
      • Bullmann L.
      • Haarmann R.
      • Mirus O.
      • Bredemeier R.
      • Hempel F.
      • Maier U.G.
      • Schleiff E.
      Filling the gap, evolutionarily conserved Omp85 in plastids of chromalveolates.
      ,
      • Kalanon M.
      • Tonkin C.J.
      • McFadden G.I.
      Characterization of two putative protein translocation components in the apicoplast of Plasmodium falciparum.
      ,
      • van Dooren G.G.
      • Tomova C.
      • Agrawal S.
      • Humbel B.M.
      • Striepen B.
      Toxoplasma gondii Tic20 is essential for apicoplast protein import.
      ). The apicomplexan homologue of Tic20 localizes to the inner apicoplast membrane and is essential for parasite survival and for protein import (
      • van Dooren G.G.
      • Tomova C.
      • Agrawal S.
      • Humbel B.M.
      • Striepen B.
      Toxoplasma gondii Tic20 is essential for apicoplast protein import.
      ). Apicomplexan genomes also harbor a plastid-targeted Tic22 homologue and an, as yet, uncharacterized homologue of Toc75 (
      • Bullmann L.
      • Haarmann R.
      • Mirus O.
      • Bredemeier R.
      • Hempel F.
      • Maier U.G.
      • Schleiff E.
      Filling the gap, evolutionarily conserved Omp85 in plastids of chromalveolates.
      ,
      • Kalanon M.
      • Tonkin C.J.
      • McFadden G.I.
      Characterization of two putative protein translocation components in the apicoplast of Plasmodium falciparum.
      ). The presence of these homologues suggest that protein translocation across the inner two membranes of apicoplasts is similar to that in plants. Furthermore, conservation of these TIC and TOC homologues across vast evolutionary distances suggests that they most likely represent “core” components of the plastid import machinery.
      Although Tic22 is ubiquitous in plastids of plants, algae and Apicomplexa parasites, its function and importance during protein import are unknown. In this study, we combine genetics and structural biology to define the function of Tic22. We use targeted genome manipulation in Toxoplasma to demonstrate a requirement for Tic22 in apicoplast protein import. We then establish the structure of Tic22 in the related parasite P. falciparum and show that Tic22 from both Toxoplasma and Plasmodium act as chaperone proteins.

      EXPERIMENTAL PROCEDURES

      T. gondii parasites were cultured in human foreskin fibroblasts with Dulbecco's modified Eagles medium, supplemented with 1% fetal bovine serum and antibiotics. Where relevant, we added anhydrotetracycline to the growth medium at a final concentration of 0.5 μg/ml. Parasite manipulations and mutant generation was performed as previously described (
      • van Dooren G.G.
      • Tomova C.
      • Agrawal S.
      • Humbel B.M.
      • Striepen B.
      Toxoplasma gondii Tic20 is essential for apicoplast protein import.
      ). Details of TgTic22 cloning and vector construction and parasite genetic modifications are described in the supplemental information.
      Growth assays were performed using a SpectraMax M2e microplate reader (Molecular Devices) as previously described (
      • van Dooren G.G.
      • Tomova C.
      • Agrawal S.
      • Humbel B.M.
      • Striepen B.
      Toxoplasma gondii Tic20 is essential for apicoplast protein import.
      ). For measuring apicoplast biogenesis in the TgTic22 mutant, we introduced an apicoplast-targeted red fluorescent protein (
      • van Dooren G.G.
      • Tomova C.
      • Agrawal S.
      • Humbel B.M.
      • Striepen B.
      Toxoplasma gondii Tic20 is essential for apicoplast protein import.
      ). We counted 100 four-cell vacuoles at each time point and measured parasites for the presence or absence of an apicoplast. Pulse-chase data were analyzed by autoradiography and PhosphorImaging (GE Healthcare). To quantify the level of import in the pulse-chase assays for TgCpn60 and FNR-mDHFR-cmyc, we calculated the ratio of the amount of mature protein in the chase lane to the amount of precursor in the pulse lane. To quantify the amount of TgPDH-E2-LA, we compared the amount of TgPDH-E2-LA to the amount of lower mito-E2-LA band in the chase lanes. To enable comparison between different import assays, we normalized the data, setting the no ATc value to 100%. Band intensities were quantified using either ImageJ or PhosphorImaging software.
      Western blotting, pulse-chase analyses, immunoprecipitations, immunofluorescence assays, sodium carbonate extractions, and TX-114 partitioning were all performed as previously described (
      • van Dooren G.G.
      • Tomova C.
      • Agrawal S.
      • Humbel B.M.
      • Striepen B.
      Toxoplasma gondii Tic20 is essential for apicoplast protein import.
      ,
      • Agrawal S.
      • van Dooren G.G.
      • Beatty W.L.
      • Striepen B.
      Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins.
      ). We used the following antibodies for Western blotting, immunoprecipitations, and immunofluorescence assays: rabbit anti-TgACP, rat anti-HA (Roche), mouse anti-c-Myc (Roche and Santa Cruz Biotechnology), mouse anti-TgGRA8 (a kind gift from Gary Ward, University of Vermont), rabbit anti-TgCpn60, rabbit anti-MIC5 (a kind gift from Vern Carruthers, University of Michigan), and rabbit anti-lipoic acid (Calbiochem). Secondary antibodies used for immunofluorescence assays were goat anti-rabbit Alexa Fluor 546 (1:500), goat anti-mouse Alexa Fluor 546 (1:500), goat anti-mouse Alexa Fluor 647 (1:200), and goat anti-rat Alexa Fluor 488 (1:200; Invitrogen). Parasites were imaged using a DeltaVision set-up on an inverted Olympus IX71 microscope fitted with an Olympus objective lens (UPlanSApo 100x/1.40 Oil), with images recorded using a Photometrics CoolSNAP HQ or HQ2 camera. Images were deconvolved and adjusted for contrast.
      PfTic22 was expressed, purified, and crystallized as described (
      • Glaser S.
      • Higgins M.K.
      Overproduction, purification and crystallization of PfTic22, a component of the import apparatus from the apicoplast of.
      ) with a selenomethionine derivative used to derive phase information. TgTic22 was also expressed in Escherichia coli and purified. Insulin aggregation assays were performed by mixing insulin and Tic22 proteins at defined ratios, inducing insulin aggregation by the addition of DTT and following light scattering at 360 nm. Detailed procedures are included in SI “Experimental Procedures.”

      DISCUSSION

      In this study we use genetic and structural approaches to define the function of Tic22, a molecule with a predicted role in protein import in both plant chloroplasts and apicomplexan apicoplasts. We demonstrate that, as with its plant homologues, TgTic22 is likely a soluble protein that localizes to the organellar periphery. Four membranes surround the apicoplast, and analogy with chloroplasts predicts that Tic22 may localize to the space between the inner two membranes. TgTic22 exists as both pre-processed and N-terminally processed isoforms, as is also the case for Tic22 homologues in plants and P. falciparum, with the N-terminal extension likely functioning as a plastid-targeting domain (
      • Kouranov A.
      • Chen X.
      • Fuks B.
      • Schnell D.J.
      Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane.
      ,
      • Kalanon M.
      • Tonkin C.J.
      • McFadden G.I.
      Characterization of two putative protein translocation components in the apicoplast of Plasmodium falciparum.
      ).
      Tic22 has been postulated to play a role in protein import into plastids based on its association with precursor proteins and components of the TIC and TOC complexes (
      • Kouranov A.
      • Chen X.
      • Fuks B.
      • Schnell D.J.
      Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane.
      ,
      • Becker T.
      • Hritz J.
      • Vogel M.
      • Caliebe A.
      • Bukau B.
      • Soll J.
      • Schleiff E.
      Toc12, a novel subunit of the intermembrane space preprotein translocon of chloroplasts.
      ). Here, we utilize the robust genetics of T. gondii to demonstrate for the first time for any organism that cells deficient in Tic22 are impaired in plastid protein import. Loss of TgTic22 corresponds with a considerable reduction in apicoplast protein import, at a time when cell growth, apicoplast biogenesis and endosomal protein targeting are all unaffected. This supports a direct role for TgTic22 in apicoplast protein import. In contrast to cyanobacterial Tic22, which is proposed to play a specific role in bacterial outer membrane protein insertion (
      • Tripp J.
      • Hahn A.
      • Koenig P.
      • Flinner N.
      • Bublak D.
      • Brouwer E.M.
      • Ertel F.
      • Mirus O.
      • Sinning I.
      • Tews I.
      • Schleiff E.
      Structure and conservation of the periplasmic targeting factor Tic22 from plants and cyanobacteria.
      ), Tic22 has a more ubiquitous role in the apicoplast, with its disruption affecting import of all tested proteins into the stroma. Examining the role of TgTic22 in biogenesis of TgToc75 will now be of particular interest to see if this function is conserved from its cyanobacterial counterpart.
      As with other apicoplast import mutants we have generated in T. gondii (e.g. TgTic20 and TgDer1), loss of apicoplast import is promptly followed by major defects in apicoplast biogenesis (
      • van Dooren G.G.
      • Tomova C.
      • Agrawal S.
      • Humbel B.M.
      • Striepen B.
      Toxoplasma gondii Tic20 is essential for apicoplast protein import.
      ,
      • Glaser S.
      • Higgins M.K.
      Overproduction, purification and crystallization of PfTic22, a component of the import apparatus from the apicoplast of.
      ), consistent with the importance of apicoplast-localized proteins for organellar maintenance. The loss of apicoplasts is the most likely reason for the subsequent inhibition of parasite growth in TgTic22-less parasites.
      The structure of PfTic22 reveals a fold with hydrophobic grooves on both sides of the molecule. The same has recently been observed for a periplasmic Tic22 homologue from a cyanobacteria suggesting it to be an ancient fold conserved from cyanoabacteria to plants (
      • Tripp J.
      • Hahn A.
      • Koenig P.
      • Flinner N.
      • Bublak D.
      • Brouwer E.M.
      • Ertel F.
      • Mirus O.
      • Sinning I.
      • Tews I.
      • Schleiff E.
      Structure and conservation of the periplasmic targeting factor Tic22 from plants and cyanobacteria.
      ), but has been observed in no other protein. We demonstrate here that the hydrophobic grooves are critical to allow Tic22 to function as a nonspecific, ATP-independent chaperone, preventing aggregation of a model protein. Although we have only tested apicoplast Tic22s for chaperone function, these grooves are also present in plant Tic22 and most likely have the same properties.
      Holdases are ATP-independent chaperones that prevent aggregation of their binding partners and are important components of many protein import or secretion systems. Bacteria use a cytosolic chaperone, SecB, that stabilizes unfolded proteins in a suitable state for insertion into the SecYEG translocon. Gram-negative bacteria also contain chaperones in the periplasm, with both SurA and Skp interacting with outer membrane proteins to keep them unfolded and primed for membrane insertion (
      • Danese P.N.
      • Silhavy T.J.
      Targeting and assembly of periplasmic and outer-membrane proteins in Escherichia coli.
      ). Similarly the mitochondrial intermembrane space contains Tim9-Tim10 and Tim8-Tim13 chaperone complexes. These tiny TIM complexes associate with a subset of imported proteins as they emerge from the outer membrane TOM complex, and deliver them to an inner membrane translocon (
      • Chacinska A.
      • Koehler C.M.
      • Milenkovic D.
      • Lithgow T.
      • Pfanner N.
      Importing mitochondrial proteins: machineries and mechanisms.
      ).
      While a number of ATP-dependent chaperones play a role in trafficking through the TOC and TIC complex, including a putative intermembrane space Hsp70, no holdase has yet been discovered in this system. Our data demonstrate that Tic22 prevents protein aggregation in an ATP-independent, concentration-dependent manner. We therefore propose that Tic22 acts as such a chaperone in the intermembrane spaces of plant, algal, and apicomplexan plastids, maintaining proteins in an unfolded state during translocation between the inner and outer membranes.
      Structural studies of holdases show that diverse features are used for function, but in each case surface exposed non-polar patches on the chaperone are available to interact with aromatic or non-polar residues from target proteins to reduce the driving force for folding and aggregation. In the case of SecB, two long hydrophobic grooves perform this function (
      • Xu Z.
      • Knafels J.D.
      • Yoshino K.
      Crystal structure of the bacterial protein export chaperone secB.
      ), while Skp and the complex of Tim9 and Tim10 adopt jelly-fish like structures with the inside surfaces of the tentacles providing a hydrophobic environment in which unfolded proteins can be held (
      • Walton T.A.
      • Sousa M.C.
      Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation.
      ,
      • Webb C.T.
      • Gorman M.A.
      • Lazarou M.
      • Ryan M.T.
      • Gulbis J.M.
      Crystal structure of the mitochondrial chaperone TIM9.10 reveals a six-bladed α-propeller.
      ). Dynamic re-association can also be involved, with SurA adopting a variety of quaternary structures, forming different hydrophobic channels to allow interaction with different unfolded protein components (
      • Xu X.
      • Wang S.
      • Hu Y.X.
      • McKay D.B.
      The periplasmic bacterial molecular chaperone SurA adapts its structure to bind peptides in different conformations to assert a sequence preference for aromatic residues.
      ). In the case of Tic22, exposed hydrophobic grooves are likely to bind to exposed hydrophobic residues on unfolded target proteins, maintaining them in solution. Although Tic22 is a monomer in the crystals, it shows a propensity to form dimers and higher order oligomers when studied in solution by analytical ultracentrifugation, with the dimer as the predominant species for PfTic22 (supplemental Fig. S7). Whether or not this oligomerization contributes to chaperone function, as seen in SurA (
      • Xu X.
      • Wang S.
      • Hu Y.X.
      • McKay D.B.
      The periplasmic bacterial molecular chaperone SurA adapts its structure to bind peptides in different conformations to assert a sequence preference for aromatic residues.
      ), is uncertain.
      Whether Tic22 is required only for the import of a subset of proteins or more universally is also uncertain. SecB is used ubiquitously in delivery of unfolded proteins to the SecYEG translocon (
      • Danese P.N.
      • Silhavy T.J.
      Targeting and assembly of periplasmic and outer-membrane proteins in Escherichia coli.
      ), but the use of chaperones in the mitochondrial intermembrane space is limited to a subset of inner membrane proteins (
      • Chacinska A.
      • Koehler C.M.
      • Milenkovic D.
      • Lithgow T.
      • Pfanner N.
      Importing mitochondrial proteins: machineries and mechanisms.
      ). Cyanobacterial Tic22 appears to play a role in outer membrane biogenesis (
      • Tripp J.
      • Hahn A.
      • Koenig P.
      • Flinner N.
      • Bublak D.
      • Brouwer E.M.
      • Ertel F.
      • Mirus O.
      • Sinning I.
      • Tews I.
      • Schleiff E.
      Structure and conservation of the periplasmic targeting factor Tic22 from plants and cyanobacteria.
      ), and plastid Tic22 may have a similar function. However, TgTic22 ablation disrupts import of all stromal-targeted proteins tested to date, arguing for a more universal role in plastids. This suggests that a bacterial chaperone with a role in outer membrane biogenesis had been adopted in plastids as a chaperone used to maintain proteins in a translocation-competent state during transit across the inner membrane space. Regardless of whether it is ubiquitously used or required for more specific import pathways, the chaperone Tic22 is clearly critical for both plastid protein import and cell survival.

      Acknowledgments

      We thank Gary Ward (University of Vermont) and Vern Carruthers (University of Michigan) for sharing antibodies, and Jack de Puit (Campbell High School, ACT, Australia) for assistance with microscopy.

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