Advertisement

Skp1 Prolyl 4-Hydroxylase of Dictyostelium Mediates Glycosylation-independent and -dependent Responses to O2 without Affecting Skp1 Stability*

  • Dongmei Zhang
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, 73104

    Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, China
    Search for articles by this author
  • Hanke van der Wel
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, 73104
    Search for articles by this author
  • Jennifer M. Johnson
    Affiliations
    Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
    Search for articles by this author
  • Christopher M. West
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 975 NE 10th St., BRC 417, OUHSC, Oklahoma City, OK 73104. Tel.: 405-271-4147; Fax: 405-271-3910
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, 73104

    Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R01-GM037539 (to C. M. W.). This work was also supported by Grant HR10-181 from the Oklahoma Center for Advancement of Science and Technology.
    This article contains supplemental Table S1 and Fig. S1.
Open AccessPublished:November 29, 2011DOI:https://doi.org/10.1074/jbc.M111.314021
      Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O2 sensing in animals via modification of the transcriptional factor subunit HIFα, resulting in its polyubiquitination by the E3VHLubiquitin (Ub) ligase and degradation in the 26 S proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFα-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O2 sensor for development. Dictyostelium lacks HIFα, and P4H1 modifies a different protein, Skp1, an adaptor of the SCF class of E3-Ub ligases related to the E3VHLUb ligase that targets animal HIFα. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O2 requirement of P4H1-null cells, therefore revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Because the effect of the gnt1 deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Although Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub ligases.

      Introduction

      Cells of aerobic organisms sense O2 and use the information to modulate physiological and developmental processes. Many short and long term mechanisms of O2 sensing have evolved (
      • Ward J.P.
      Oxygen sensors in context.
      ,
      • Bien C.M.
      • Espenshade P.J.
      Sterol regulatory element-binding proteins in fungi: hypoxic transcription factors linked to pathogenesis.
      ,
      • Kaelin Jr., W.G.
      • Ratcliffe P.J.
      Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.
      ). Important for humans is the family of prolyl 4-hydroxylases (PHD 1–3)
      The abbreviations used are: PHD
      prolyl 4-hydroxylase domain protein
      αGlcNAcT
      α-N-acetylglucosaminyltransferase
      HIFα
      hypoxia-inducible factor-α
      Hyp
      (4R,2S)-hydroxyproline
      Ub
      ubiquitin
      SCF
      complex containing Skp1, cullin-1, and an FBP
      FBP
      F-box protein
      VBC
      complex containing von Hippel-Lindau protein, elongin B, and elongin C
      nt
      nucleotide(s)
      Bis-Tris
      2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol.
      that modify the O2-dependent degradation domain of hypoxia-inducible factor α (HIFα), a transcriptional factor subunit that positively controls the expression of many hypoxia-induced genes (
      • Kaelin Jr., W.G.
      • Ratcliffe P.J.
      Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.
      ). O2 serves as a substrate for PHD2 to hydroxylate HIFα, which leads to its recognition by the von Hippel-Lindau subunit of the E3VBCubiquitin (Ub) ligase and subsequent polyubiquitination. Polyubiquitinated HIFα is then degraded by the 26 S proteasome. HIFα accumulates in hypoxia because of low activity of PHD2.
      The social soil amoeba Dictyostelium is an important model organism for cell signaling and motility. Dictyostelium, which normally proliferates as solitary amoebae in the soil, undergoes starvation-induced development resulting in formation of a multicellular slug that migrates to the soil surface and culminates into a fruiting body (
      • Schindler J.
      • Sussman M.
      Ammonia determines the choice of morphogenetic pathways in Dictyostelium discoideum.
      ,
      • Newell P.C.
      • Telser A.
      • Sussman M.
      Alternative developmental pathways determined by environmental conditions in the cellular slime mold Dictyostelium discoideum.
      ,
      • Smith E.
      • Williams K.L.
      Evidence for tip control of the “slug/fruit” switch in slugs of Dictyostelium discoideum.
      ,
      • Singleton C.K.
      • Kirsten J.H.
      • Dinsmore C.J.
      Function of ammonium transporter A in the initiation of culmination of development in Dictyostelium discoideum.
      ). Culmination depends on a superphysiological level of O2 (
      • Sandonà D.
      • Gastaldello S.
      • Rizzuto R.
      • Bisson R.
      Expression of cytochrome c oxidase during growth and development of Dictyostelium.
      ), suggesting that O2 is an environmental factor that the slug uses to know that it is above ground, in a location suitable for culmination and subsequent dispersal of spores (
      • Bonner J.T.
      • Lamont D.S.
      Behavior of cellular slime molds in the soil.
      ).
      Dictyostelium expresses an HIFα-like prolyl 4-hydroxylase (P4H1) but lacks an HIFα-like gene based on sequence searches (
      • West C.M.
      • Wang Z.A.
      • van der Wel H.
      A cytoplasmic prolyl hydroxylation and glycosylation pathway modifies Skp1 and regulates O2-dependent development in Dictyostelium.
      ). P4H1 was initially discovered as the enzyme required for a novel form of cytoplasmic glycosylation of Skp1 (
      • van der Wel H.
      • Ercan A.
      • West C.M.
      The Skp1 prolyl hydroxylase from Dictyostelium is related to the hypoxia-inducible factor-α class of animal prolyl 4-hydroxylases.
      ). Subsequent to its 4(trans)-hydroxylation, Pro-143 is modified with a residue of GlcNAc by a reaction that can be catalyzed in vitro by Gnt1 (
      • van der Wel H.
      • Johnson J.M.
      • Xu Y.
      • Karunaratne C.V.
      • Wilson K.D.
      • Vohra Y.
      • Boons G.J.
      • Taylor C.M.
      • Bendiak B.
      • West C.M.
      Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in Dictyostelium.
      ), an αGlcNAcT purified based on this activity from cytosolic extracts of Dictyostelium (
      • Teng-Umnuay P
      • van der Wel H
      • West CM
      Identification of a UDP-GlcNAc:Skp1-hydroxyproline GlcNAc-transferase in the cytoplasm of Dictyostelium.
      ,
      • Van Der Wel H.
      • Morris H.R.
      • Panico M.
      • Paxton T.
      • Dell A.
      • Kaplan L.
      • West C.M.
      Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of Dictyostelium.
      ). Recombinant Gnt1 exhibits similar activity, but the suspected role of Gnt1 in cells has not been confirmed because of difficulty in disrupting the gnt1 locus. GlcNAc-O-Skp1 is subsequently acted on by four additional glycosyltransferase activities encoded by pgtA and agtA (
      • West C.M.
      • Wang Z.A.
      • van der Wel H.
      A cytoplasmic prolyl hydroxylation and glycosylation pathway modifies Skp1 and regulates O2-dependent development in Dictyostelium.
      ). Genetic manipulations have suggested that P4H1 behaves as an O2 sensor to control culmination (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ). Initial GlcNAc capping of Hyp-143 is sufficient for restoring a near normal O2 requirement (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ,
      • Wang Z.A.
      • Singh D.
      • van der Wel H.
      • West C.M.
      Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.
      ), and the addition of intermediate sugars by PgtA acts like a second chance mechanism to reverse the effect of hydroxylation until the terminal sugars are added by AgtA (
      • Wang Z.A.
      • van der Wel H.
      • Vohra Y.
      • Buskas T.
      • Boons G.J.
      • West C.M.
      Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
      ). Accumulating evidence suggests the importance of other forms of complex cytoplasmic and nuclear glycosylation in eukaryotes (
      • Funakoshi Y.
      • Suzuki T.
      Glycobiology in the cytosol: the bitter side of a sweet world.
      ,
      • Hart G.W.
      • West C.M.
      ,
      • Borst P.
      • Sabatini R.
      Base J: discovery, biosynthesis, and possible functions.
      ).
      Skp1 is best known as a subunit of the SCF class of E3-Ub ligases (
      • Willems A.R.
      • Schwab M.
      • Tyers M.
      A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin.
      ,
      • Deshaies R.J.
      • Joazeiro C.A.
      RING domain E3 ubiquitin ligases.
      ). E3SCFUb ligases are responsible for the polyubiquitination and degradation of potentially hundreds of proteins in the cell, including regulators of the cell cycle, transcription, and vesicle trafficking. This multifunctionality is due to the existence of >50 F-box proteins in humans and Dictyostelium
      Y. Xu and C. M. West, unpublished data.
      that may be associated with the SCF complex via the Skp1 adaptor. Reverse genetic studies indicate that Skp1 is also involved in O2 regulation (
      • Wang Z.A.
      • Singh D.
      • van der Wel H.
      • West C.M.
      Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.
      ). Furthermore, genetic interactions with the hydroxylation/glycosylation pathway, together with evidence that Skp1 is the only cellular substrate for these enzymes, suggest that Skp1 is a primary mediator of P4H1 signaling in the cell.
      P4H1 appears to be the Dictyostelium ortholog of human PHD2 based on biochemical and functional criteria (
      • West C.M.
      • Wang Z.A.
      • van der Wel H.
      A cytoplasmic prolyl hydroxylation and glycosylation pathway modifies Skp1 and regulates O2-dependent development in Dictyostelium.
      ,
      • van der Wel H.
      • Johnson J.M.
      • Xu Y.
      • Karunaratne C.V.
      • Wilson K.D.
      • Vohra Y.
      • Boons G.J.
      • Taylor C.M.
      • Bendiak B.
      • West C.M.
      Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in Dictyostelium.
      ), except that it modifies Skp1 rather than HIFα. With the successful disruption of the gnt1 locus presented here, the predicted role for Gnt1 in Skp1 glycosylation was confirmed and permitted comparison of the effects of prolyl hydroxylation of Skp1 and HIFα. Surprisingly, hydroxylated Skp1 appeared to be as stable as unmodified Skp1. Nevertheless, hydroxylation partially rescued the high O2 requirement of the P4H1 mutant, and Skp1 was the only substrate of Gnt1 that could be detected in gnt1-null cells, suggesting an alternative primordial mechanism of hydroxylation that nonetheless might still affect the stability of clients of Skp1-dependent Ub ligases.

      EXPERIMENTAL PROCEDURES

      Growth and Development

      Dictyostelium discoideum cells (see Table 1 for list of strains) were typically grown axenically in HL-5 and cloned by growth on SM-agar plates containing Klebsiella aerogenes (
      • Fey P.
      • Kowal A.S.
      • Gaudet P.
      • Pilcher K.E.
      • Chisholm R.L.
      Protocols for growth and development of Dictyostelium discoideum.
      ). Dictyostelium purpureum (Dp1) was grown on K. aerogenes. Development was monitored after clearing bacteria, or axenically grown cells were centrifugally washed, resuspended in PDF buffer (33 mm NaH2PO4, 10.6 mm Na2HPO4, 20 mm KCl, 6 mm MgSO4, pH 5.8), and deposited on cellulose acetate filters (
      • Fey P.
      • Kowal A.S.
      • Gaudet P.
      • Pilcher K.E.
      • Chisholm R.L.
      Protocols for growth and development of Dictyostelium discoideum.
      ). O2 effects were analyzed by incubating the filters in chambers through which flowed a constant proportion of O2/N2 (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ). Development was monitored morphologically, and terminal differentiation was analyzed microscopically after incubation in Calcofluor White ST to render cellulosic cell walls fluorescent, which was visualized in the DAPI filter channel, as described (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ). Spore formation was quantitated by counting in a hemacytometer.
      TABLE 1Dictyostelium strains employed
      StrainParental strainDrug resistanceGenotypeGnt1 activity
      Summarized from Figs. 2 and 3. −, not detected; +, normal activity; (+++), conditional high activity in slug only; +/−, slightly less than normal.
      Ref.
      Ax3NC-4NonephyA+/gnt1+ (normal)+
      HW288Ax3Blasticidin SphyA+
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      HW260Ax3Blasticidin SpgtA+
      • Van Der Wel H.
      • Fisher S.Z.
      • West C.M.
      A bifunctional diglycosyltransferase forms the Fucα1,2Galβ1,3-disaccharide on Skp1 in the cytoplasm of Dictyostelium.
      HW261Ax3Blasticidin SpgtA+
      • Van Der Wel H.
      • Fisher S.Z.
      • West C.M.
      A bifunctional diglycosyltransferase forms the Fucα1,2Galβ1,3-disaccharide on Skp1 in the cytoplasm of Dictyostelium.
      HW420Ax3Blasticidin SagtA+
      • Ercan A.
      • Panico M.
      • Sutton-Smith M.
      • Dell A.
      • Morris H.R.
      • Matta K.L.
      • Gay D.F.
      • West C.M.
      Molecular characterization of a novel UDP-galactose:fucoside α3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.
      HW500Ax3Hygromycingnt1.1(H104D)This study
      HW501Ax3Hygromycingnt1.1(H104D)This study
      HW503Ax3Hygromycingnt1.3(H104D/Y253stop)This study
      HW504Ax3Hygromycingnt1.3(H104D/Y253stop)This study
      HW506HW500G418/hygromycinecmA::DpGnt1/gnt1.1(+++)This study
      HW508HW501G418/hygromycinecmA::DpGnt1/gnt1.3(+++)This study
      HW507HW500G418/hygromycinecmA::DpGnt1(D102A)[gnt1.2]/gnt1.1This study
      HW509HW501G418/hygromycinecmA::DpGnt1(D102A)[gnt1.2]/gnt1.3This study
      HW512HW288Hygromycin/blasticidin Sgnt1.1/phyAThis study
      HW513HW288Hygromycin/blasticidin Sgnt1.1/phyAThis study
      HW515HW288Hygromycin/blasticidin Sgnt1.3/phyAThis study
      HW516HW288Hygromycin/blasticidin Sgnt1.3/phyAThis study
      HW518HW420Hygromycin/blasticidin Sgnt1.0/agtA+/−This study
      a Summarized from FIGURE 2, FIGURE 3. −, not detected; +, normal activity; (+++), conditional high activity in slug only; +/−, slightly less than normal.
      Figure thumbnail gr3
      FIGURE 3Skp1 modification status in gnt1 mutant strains. A, summary of the Skp1 modification pathway. B, isoform-specific antibodies. Soluble (S100) extracts of growing cells of normal (Ax3 = wild type) and mutant strains were subjected to SDS-PAGE/Western blotting with pan-specific mAb 4E1, the previously described GlcNAc-O-Skp1-specific mAb 1C9, and two new isoform-selective pAbs UOK87 and UOK85. The series of mutant strains shown express incrementally modified Skp1 isoforms from left to right as indicated (Pro, unmodified; OH, hydroxylated; Gn, modified with GlcNAc; G, Gal-GlcNAc; F, Fuc-Gal-GlcNAc; or GG′, Gal-Gal-Fuc-Gal-GlcNAc). C, slug stage extracts of gnt1 mutant and overexpression strains were Western blotted using the indicated antibodies to assess the modification status of Skp1 and expression of DpGnt1. Note: The appearance of Skp1 as a doublet in these trials resulted from apparent proteolysis that occurs variably during preparation of slug cell extracts. D, MALDI-TOF-MS analysis of Skp1 isolated from gnt1.1 stationary stage cells. Skp1 was purified to homogeneity from a 30-liter culture of gnt1.1 cells and analyzed by MALDI-TOF-MS.
      Figure thumbnail gr2
      FIGURE 2Skp1-Hyp GlcNAcT activity in gnt1 mutant strains. Soluble (S100) extracts were prepared from slugs of normal and mutant strains and desalted, and ∼35 μg of protein was incubated with 2 μm recombinant HO-Skp1 in the presence of UDP-[3H]GlcNAc. Incorporation of 3H was measured using the SDS-PAGE assay, and the zero time blank value was subtracted. Error bars indicate ± S.E.

      Antisera

      Antiserum against DpGnt1 was induced by injection of New Zealand White female rabbits with a highly purified preparation of recombinant DdDpHis6Gnt1 (
      • van der Wel H.
      • Johnson J.M.
      • Xu Y.
      • Karunaratne C.V.
      • Wilson K.D.
      • Vohra Y.
      • Boons G.J.
      • Taylor C.M.
      • Bendiak B.
      • West C.M.
      Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in Dictyostelium.
      ) mixed with either Freund's complete (primary injection) or incomplete adjuvant. Antisera obtained after three booster injections reacted specifically at 1:100 dilution with DpGnt1 but not DdGnt1 in extracts using Western blotting. Similarly, antisera were raised against a synthetic Pro peptide that included Skp1 residues 137–149 (centered at Pro-143) that had been conjugated via its N terminus to keyhole limpet hemocyanin as described (
      • Wang Z.A.
      • van der Wel H.
      • Vohra Y.
      • Buskas T.
      • Boons G.J.
      • West C.M.
      Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
      ) and to the corresponding Hyp peptide. Final booster injections were conducted with longer peptides extending to residue 151. Antisera from selected animals exhibited desired Skp1 isoform specificity by Western blotting at titers of 1:5000 (see “Results”).

      SDS-PAGE and Western Blotting

      Samples were diluted in concentrated SDS sample buffer to a final concentration of 20 mm dithiothreitol and applied to an Invitrogen NuPAGE® Novex 4–12% Bis-Tris midi gel using the MES buffer system. Proteins were transferred to a 0.2-μm pore diameter nitrocellulose membrane using an Invitrogen iBlot® dry blotting system. Blots were probed with antisera diluted in 5% nonfat dry milk prepared in Tris-buffered saline (50 mm Tris-HCl, 100 mm NaCl, pH 7.4), followed by Alexa Fluor 680-conjugated goat anti-mouse IgG (LI-COR Biosciences) also diluted 1:10,000 in the milk solution, and imaged at 700 nm in a LI-COR Biosciences Odyssey infrared scanner. Apparent Mr values were estimated using the Benchmark prestained protein ladder from Invitrogen.

      αGlcNAc-transferase Reactions

      αGlcNAcT activity was assayed as described previously (
      • Teng-Umnuay P
      • van der Wel H
      • West CM
      Identification of a UDP-GlcNAc:Skp1-hydroxyproline GlcNAc-transferase in the cytoplasm of Dictyostelium.
      ). Briefly, soluble (S100) extracts were prepared from the slug or growth stage, desalted, and incubated with 2 μm recombinant HO-Skp1 (prehydroxylated in vitro) in the presence of UDP-[3H]GlcNAc for 2 h. Incorporation of 3H was measured using the SDS-PAGE assay, and the zero time blank value was subtracted. For screening αGlcNAcT acceptor substrates, the reactions were amended with highly purified recombinant DpGnt1, and exogenous Skp1 was not added.

      Construction of gnt1 Replacement Vectors

      Three replacement DNA fragments were designed to disrupt gnt1 by homologous recombination via a double-crossover mechanism. The DNAs were constructed using standard PCR and cloning procedures. At most steps, DNAs were purified by electrophoresis on a 1% (w/v) SeaKem GTG-agarose (BioWhittaker) gel, extracted using a freeze-squeeze method, and EtOH-precipitated. The 710-nt upstream targeting DNA was amplified from pTYB1GnT51 or pTYB1GnT51(H104D) (
      • Van Der Wel H.
      • Morris H.R.
      • Panico M.
      • Paxton T.
      • Dell A.
      • Kaplan L.
      • West C.M.
      Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of Dictyostelium.
      ) using primers GnD and Gn6 (see Fig. 1A, supplemental Table S1, and supplemental Fig. S1) and cloned into pCR4-TOPO (Invitrogen) to yield pTOPO+1 and pTOPO+1(H104D). To replace a homopolymeric stretch of poly(N), 310-nt of Gnt1 3′-coding DNA was amplified from D. purpureum genomic DNA using GnG and Gn4 (see Fig. 1B), cloned into pCR4-TOPO to yield pTOPO+2. Amplification fidelity was confirmed by direct sequencing. The 710-nt targeting DNAs were excised using SpeI and StuI and ligated to corresponding sites of pTOPO+2 to yield pTOPO+1+2 and pCR4-TOPO+1(H104D)+2. A 1304-nt downstream targeting DNA was amplified from D. discoideum genomic DNA using primers GnH and Gn2 and cloned into pCR4-TOPO to yield pTOPO+4. The hygromycin resistance cassette from pHygT(plus)/pG7 (a gift from M. Nelson, M. Fukuzawa, and J. G. Williams) was excised using BamHI and cloned in reverse orientation into the BglII site of pTOPO+4, yielding pTOPO+4(hygr)+5. The (710+310)-DNA was excised from the pTOPO+1+2 plasmids with SpeI and BamHI and cloned into similarly digested pTOPO+4(hygr)+5 to yield pTOPO+1(±H104D)+2+4(hygr)+5 (see Fig. 1D). The Y253stop mutation (see Fig. 1D) was introduced into the H104D DNA by site-directed mutagenesis using oligonucleotides GnF and Gn7.
      Figure thumbnail gr1
      FIGURE 1Modifications of gnt1 locus. A, genomic locus of gnt1 from D. discoideum. Positions of oligonucleotides used for cloning and sequencing (see and ) are shown. B, corresponding coding region of gnt1 from D. purpureum. C, hybrid DdDpGnt1.b (gnt1.0) resulting from gene replacement strategy shown in panel D. D, gene replacement constructs designed for homologous recombination based on a double-crossover mechanism, after selection based on resistance to hygromycin. Left, 5′-targeting DNA including most of exon-2 of Dd-gnt1; right, 3′-targeting DNA including the coding region of the downstream gene. Replacement DNA-1 contains the inactivating H104D substitution (termed gnt1.1); replacement DNA-2 contains, in addition, Y253stop (gnt1.3). E and F, complementation constructs consisting of the coding regions of DpGnt1 or mutant DpGnt1(D102A) (gnt1.2), positioned downstream of the ecmA promoter and adjacent to a G418 resistance cassette; transformants are expected to overexpress Gnt1 in prestalk cells from chromosomally integrated multicopy tandem arrays. G, PCR analysis of gene replacement strains. PCR reactions conducted on genomic DNA from strains transformed with DNA-1 or -2, using the indicated primer pairs, one from outside and the other from within the disruption DNA. Std, DNA ladder standard.
      The replacement DNAs were excised with EcoRI and SpeI, briefly trimmed with Bal31 exonuclease, and electroporated into D. discoideum strain Ax3 or HW288 (phyA, where phyA is the Dictyostelium gene encoding P4H1) as described previously (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ). Transformants were initially selected at 35 μg/ml hygromycin and then cloned on bacteria plates (
      • van der Wel H.
      • Ercan A.
      • West C.M.
      The Skp1 prolyl hydroxylase from Dictyostelium is related to the hypoxia-inducible factor-α class of animal prolyl 4-hydroxylases.
      ). Oligonucleotides used for PCR trials to confirm the expected recombination events are shown in supplemental Table S1 and Fig. 1A. Strains are listed in Table 1.

      Construction of gnt1 Overexpression Strains

      Native DpGnt1 cDNA (1035 nt) was PCR-amplified from pDEST527-DdDpGnt1.a (chimera a) (
      • van der Wel H.
      • Johnson J.M.
      • Xu Y.
      • Karunaratne C.V.
      • Wilson K.D.
      • Vohra Y.
      • Boons G.J.
      • Taylor C.M.
      • Bendiak B.
      • West C.M.
      Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in Dictyostelium.
      ) using GnB and Gn3 (see Fig. 1E, supplemental Table S1, and supplemental Fig. S1) and cloned into pCR4-TOPO to yield pTOPO-DpGnt1. DpGnt1 cDNA was excised using KpnI and BamHI and ligated into similarly digested pVSE (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ) to yield pVSE-DpGnt1 (see Fig. 1E). Site-directed mutagenesis using primers GnE and Gn8 was employed to generate pVSE-DpGnt1(D102A) (see Fig. 1F). These vectors were designed to overexpress native or enzymatically inactive DpGnt1 under control of the ecmA promoter in prestalk cells. After electroporation of plasmids into D. discoideum, G418-resistant cells were initially selected at 20 μg/ml G418 and cloned on bacteria plates.

      Skp1 Metabolic Labeling and Immunoprecipitation

      To estimate the rate of total protein synthesis, logarithmically growing cells were centrifuged and resuspended at 107/ml in 1 ml of HL-5 medium containing 71 μCi/ml [35S]Met/Cys (carrier-free Trans-label; Amersham Biosciences), 0.1 mm Met, and 0.1 mm Cys and shaken at 22 °C for 40 min. Cells were recovered by centrifugation at 1000 × g for 1 min, resuspended in cold PDF buffer, centrifuged again, and frozen at −80 °C. Incorporation of 35S into protein was determined by trichloroacetic acid precipitation as described (
      • Sassi S.
      • Sweetinburgh M.
      • Erogul J.
      • Zhang P.
      • Teng-Umnuay P.
      • West C.M.
      Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium.
      ). To determine incorporation into Skp1 of growing cells, cells were incubated as above in the absence of added unlabeled Met and Cys for 1 h. Incorporation into Skp1 of 15-h developing cells (slugs) was performed by transferring a filter containing the cells into a Petri dish on which had been deposited five 20-μl aliquots of 0.71 mCi/ml [35S]Met/Cys in PDF and incubating for 1 h. For pulse-chase studies, 1-h labeled cells in HL-5 were centrifuged and resuspended in fresh HL-5 containing unlabeled 0.1 mm Met and 0.1 mm Cys. At the indicated times, cells were pelleted and stored at −80 °C, subsequently solubilized in 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 8 m urea, and protease inhibitors (1 mm PMSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin), and centrifuged at 16,000 × g for 30 min at 4 °C. The supernatant was diluted 10-fold in IP buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, and protease inhibitors, precleared with 10 μg of mouse IgG and 25 μl of a 25% slurry of UltraLink protein G beads (Thermo Scientific), and immunoprecipitated by the sequential addition of 5 μg of mAb 4E1 (1 h on ice) and 25 μl of the UltraLink protein G bead suspension. After incubation for 2 h at 4 °C with gentle mixing, beads were recovered by centrifugation and washed in IP buffer. Pellets were subjected to SDS-PAGE. The washed gel was incubated in 1 m sodium sulfosalicylate for 30 min, dried on Whatman 3MM paper, and autoradiographed using BioMax MR film (Kodak) and a Fisher Biotech L-Plus intensifying screen. The films were scanned and densitometrically analyzed using ImageJ software. A semilogarithmic transformation of the data was fitted by linear regression in Microsoft Excel, based on minimizing the sum of the squares of the error terms.

      RESULTS

      Disruption of gnt1

      Native and recombinant Gnt1 exhibits UDP-GlcNAc:Skp1-Hyp αGlcNAc-transferase activity in vitro. However, efforts to confirm this role in vivo have been thwarted by the inability to disrupt gnt1 using either of two separate linear targeting DNAs expected to homologously recombine via a double-crossover mechanism.
      L. Kaplan, Z. A. Wang, H. van der Wel, and C. M. West, unpublished data.
      In the third attempt reported here, the strategy was modified to express an enzymatically inactive protein in case the Gnt1 protein itself is required for viability. In addition, a new 5′-targeting DNA consisting of most of exon 2 of the gnt1 coding sequence and a new 3′-DNA consisting of the downstream intergenic region and the coding sequence of the neighboring gene (Fig. 1C) were selected to guide targeting. A reverse-oriented hygromycin resistance cassette was positioned between with its transcription terminator expected to function for both gnt1 and hygr. In addition, the C-terminal 143 amino acids of Gnt1, which include a non-conserved degenerate 79-amino acid domain highly enriched in Asn residues (polyN in Fig. 1A and supplemental Fig. S1) that interfere with cloning and expression in Escherichia coli (
      • Van Der Wel H.
      • Morris H.R.
      • Panico M.
      • Paxton T.
      • Dell A.
      • Kaplan L.
      • West C.M.
      Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of Dictyostelium.
      ), were replaced with the corresponding 71-amino acid region of Gnt1 from the related species D. purpureum (
      • Sucgang R.
      • Kuo A.
      • Tian X.
      • Salerno W.
      • Parikh A.
      • Feasley C.L.
      • Dalin E.
      • Tu H.
      • Huang E.
      • Barry K.
      • Lindquist E.
      • Shapiro H.
      • Bruce D.
      • Schmutz J.
      • Salamov A.
      • Fey P.
      • Gaudet P.
      • Anjard C.
      • Babu M.M.
      • Basu S.
      • Bushmanova Y.
      • van der Wel H.
      • Katoh-Kurasawa M.
      • Dinh C.
      • Coutinho P.M.
      • Saito T.
      • Elias M.
      • Schaap P.
      • Kay R.R.
      • Henrissat B.
      • Eichinger L.
      • Rivero F.
      • Putnam N.H.
      • West C.M.
      • Loomis W.F.
      • Chisholm R.L.
      • Shaulsky G.
      • Strassmann J.E.
      • Queller D.C.
      • Kuspa A.
      • Grigoriev I.V.
      Comparative genomics of the social amoebae Dictyostelium discoideum and Dictyostelium purpureum.
      ) that lacks the degenerate region (Fig. 1B). The GlcNAcT activity of DpGnt1 was recently shown to be indistinguishable from that of DdGnt1 in vitro (
      • van der Wel H.
      • Johnson J.M.
      • Xu Y.
      • Karunaratne C.V.
      • Wilson K.D.
      • Vohra Y.
      • Boons G.J.
      • Taylor C.M.
      • Bendiak B.
      • West C.M.
      Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in Dictyostelium.
      ), and this chimera (referred to as DdDpGnt1.b, or gnt1.0) was active in vivo (see below). The gnt1-coding sequence (within the 5′-DNA) was modified by a point mutation (H104D) expected, based on analysis of recombinant Gnt1 (
      • Van Der Wel H.
      • Morris H.R.
      • Panico M.
      • Paxton T.
      • Dell A.
      • Kaplan L.
      • West C.M.
      Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of Dictyostelium.
      ), to inactivate the enzyme activity without affecting protein expression. In a second generation strategy (see below), the same disruption DNA was further modified by the introduction of a stop codon at the junction of the Dd and Dp coding regions (codon 253) of the chimera (Fig. 1D) that, based on previous studies (
      • Van Der Wel H.
      • Morris H.R.
      • Panico M.
      • Paxton T.
      • Dell A.
      • Kaplan L.
      • West C.M.
      Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of Dictyostelium.
      ), resulted in an enzymatically inactive truncated protein when expressed in E. coli.
      Electroporation of replacement DNA-1 (Fig. 1D) into normal strain Ax3 cells led to the appearance of stable hygromycin-resistant clones in which the gnt1 locus was successfully replaced as expected, based on the size and sequence of PCR products amplified from mutant clone DNA (Fig. 1G, and data not shown). Similar results were obtained using replacement DNA-2. The crossover positions for both disruption DNAs occurred at the 5′-side of the point mutations, thereby incorporating the mutations into the genomic locus as desired and yielding gnt1.1 and gnt1.3, respectively (Table 1).
      To analyze genetic interactions with P4H1, which catalyzes formation of a substrate for Gnt1, the gnt1 replacement DNAs were introduced into the previously described P4H1-null strain HW288 (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ). Clones were isolated with the same insertions as above (Fig. 1G) and confirmed to have incorporated the desired point mutations. Replacement DNA-1 was also introduced into the agtA-null strain HW420. Although this DNA integrated in identical fashion based on PCR analysis (Fig. 1G), sequencing revealed the presence of the original H104 codon, indicating that the crossover point for inserting the upstream DNA occurred downstream of H104 position, resulting in expression of the DdDpGnt1.b chimera (gnt1.0) expected to be active. Varied crossover positions also occurred in our previous replacement of the Skp1A gene (
      • Wang Z.A.
      • Singh D.
      • van der Wel H.
      • West C.M.
      Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.
      ).
      To confirm an effect on Gnt1, soluble extracts from vegetative cells were assayed for Gnt1 activity using HO-Skp1 that had been prepared from E. coli transfected with a dual expression plasmid encoding both Skp1 and P4H1 (encoded by phyA).
      M. O. Sheikh and C. M. West, unpublished data.
      Extracts from gnt1.1, gnt1.3, and gnt1.1/phyA cells exhibited negligible activity (<1%) when compared with parental Ax3 and phyA cells, as expected (Fig. 2). Extracts of gnt1.0/agtA cells had near normal Gnt1 activity, consistent with failure to incorporate the H104D mutation because of its downstream crossover point, confirming that the DdDpGnt1.b chimera protein (gnt1.0 allele) was expressed and active.
      These replacements of gnt1 were predicted to block modification of Skp1 past the hydroxylation step (see the pathway schematic in Fig. 3A). This was first assessed by analyzing the mobility of Skp1 on SDS-PAGE gels. As shown in Fig. 3B, Western blotting with the pan-specific mAb 4E1 showed that Skp1 from gnt1.1 cells migrated more similarly to that of phyA and pgtA cells than to Skp1 from Ax3 cells, which is modified by five sugars. This was further analyzed using newly raised rabbit antisera from animals immunized with synthetic peptides corresponding to the Pro-143 region of Skp1 and conjugated to keyhole limpet hemocyanin (KLH) (
      • Wang Z.A.
      • Singh D.
      • van der Wel H.
      • West C.M.
      Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.
      ,
      • Wang Z.A.
      • van der Wel H.
      • Vohra Y.
      • Buskas T.
      • Boons G.J.
      • West C.M.
      Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
      ). As shown in Fig. 3B, UOK87, induced by the Pro peptide, bound strongly to Skp1 from phyA cells and only weakly to the other modification isoforms that accumulate in available pathway mutants, including gnt1.1. In comparison, UOK85, induced by the Hyp peptide, reacted only with Skp1 from gnt1.1 cells and none of the other pathway mutants. mAb 1C9, previously shown to selectively bind GlcNAc-O-Skp1 (
      • Wang Z.A.
      • van der Wel H.
      • Vohra Y.
      • Buskas T.
      • Boons G.J.
      • West C.M.
      Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
      ), failed to react with Skp1 from gnt1.1. Similar results were obtained for gnt1.3 cells (Fig. 3C, and data not shown). To confirm that the majority of Skp1 accumulates as HO-Skp1, Skp1 was purified from stationary stage gnt1.1 cells and analyzed by MALDI-TOF-MS (Fig. 3D). The expected m/z for the singly charged hydroxylated protein, taking into consideration known processing events and alkylation, was 18,932 and 18,916 for Skp1A and Skp1B, respectively. The observed m/z of 18,932 indicates the absence of glycosylation. Analysis of peptides generated by endo Lys-C digestion revealed that the most abundant isoform consisted of the Hyp peptide (data not shown). Very minor levels of the fully glycosylated peptide were detected, which might represent contamination from previous purifications on the chromatography columns used or low level processing of Skp1 by residual activity. Because gnt1 is the only Gnt1-like gene in D. discoideum, Gnt1 appears to be the sole Skp1:Hyp αGlcNAcT in cells.

      Complementation of gnt1

      To verify that the absence of Gnt1 activity could be attributed to the disruption of gnt1, gnt1.1 and gnt1.3 cells were electroporated with pVSE-derived expression plasmids, which direct expression of DpGnt1 under control of the prestalk cell-specific promoter ecmA. Cells were analyzed for expression of DdDpGnt1 using a new antiserum raised against recombinant DpHis6Gnt1 expressed in and purified from E. coli. A reactive band at the expected Mr position for DpGnt1 was detected in slug cells (Fig. 3C), but not vegetative cells (data not shown), as expected according to the strict slug specificity of expression of the ecmA promoter. No binding was detected in parental D. discoideum cells, indicating species specificity of the antiserum. The expressed protein was enzymatically active as extracts of mutant slugs exhibited about 50-fold increase in Gnt1-like activity relative to normal Ax3 cells (Fig. 2). Finally, the great majority of Skp1 was modified in the gnt1.1 and gnt1.3 slugs expressing DpGnt1, based on loss of the UOK85 epitope and the decreased mobility by SDS-PAGE (Fig. 3C). However, only partial rescue of Skp1 modification was expected as the ecmA promoter is active in prestalk cells. The implication that Skp1 of prespore cells, the major cell type of the slug, is also modified suggests possible cell type conversion, which was previously noted when P4H1 was disrupted or overexpressed (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ). Glycosylation rescue depended on the enzymatic activity of Gnt1 because overexpression of inactive mutant DpGnt1(D102A) (gnt1.2) did not lead to detectable Skp1 glycosylation (Fig. 3C).

      Gnt1 Contributes to Orderly Morphogenesis

      Clonal growth of gnt1.1 and gnt1.3 cells on bacterial agar plates was normal except that plaque sizes were slightly smaller than those of phyA cells and slightly larger than those of Ax3 cells.
      D. Zhang, H. van der Wel, and C. M. West, unpublished data.
      Proliferation rates were similar when cells were grown in suspension in HL-5 axenic medium, indicating that the difference was specific to bacterial phagocytosis or digestion. A greater difference was evident after the gnt1 mutant cells developed to form fruiting bodies after exhausting the bacteria. The majority of fruiting bodies formed by gnt1.1 and gnt1.3 cells were deformed with their stalks exhibiting extensive varicosities and frequent bends, in contrast to the predominantly straight and slender profile of the normal stalk (Fig. 4A and data not shown). Microscopic observation in the presence of Calcofluor White ST, a fluorescence brightener whose affinity to cellulose reveals the organization of stalk cell walls and the cellulosic stalk tube proper that envelopes the stalk cells, revealed that mutant stalks lacked the uniform diameter of normal stalks and contained regions of local swelling filled with stalk cells (data not shown). These defects were corrected by overexpression of DpGnt1 under control of the ecmA promoter, which directs expression in prestalk and stalk cells (Fig. 4A). Correction depended on the catalytic activity of DpGnt1 because clones overexpressing the enzymatically inactive D102A mutant remained abnormal. Similar differences were observed in independent gnt1 mutant and Gnt1-complemented clones (data not shown). The morphology of fruiting bodies was more normal when axenically grown cells were developed on filters so, as for proliferation, the effects of gnt1 disruption on stalk formation depended on the environmental context.
      Figure thumbnail gr4
      FIGURE 4O2 dependence of culmination of gnt1 mutant cells. A, gnt1 mutant strains, in parallel with normal (Ax3) and complemented strains, were allowed to culminate following consumption of bacteria on SM-agar and photographed from above. Typical fruiting bodies, consisting of a spherical droplet of spores perched on an upright, linear stalk, are circled in yellow. B, axenically grown gnt1 mutant strains, in parallel with Ax3 and phyA cells, were induced to develop for 42 h on filters at 10 or 15% O2. C, spores were recovered from trials like those shown in panel B and counted in a hemacytometer. Spore numbers were normalized to the maximal number formed at 40 or 21% O2, which varied ±20%. Sporulation corresponded to culmination in these strains. Data are merged from two large trials and are representative of observations in seven independent trials. D, strains in which Gnt1 alleles were overexpressed under control of the ecmA promoter were analyzed as in panel C. E, average O2 values (± S.E.) required for 50% sporulation from the seven trials are plotted.

      Prolyl Hydroxylation Alone Affects the O2 Requirement for Culmination

      Previous studies showed that P4H1-null cells exhibited a substantially elevated requirement for O2, ∼21%, to culminate, compared with the ≤10% required for the parental Ax3 strain (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ). We also reported that disruption of pgtA, such that Skp1 cannot be processed past the GlcNAc-O-Skp1 isoform, was delayed in development but appeared to have a near normal O2 requirement to culminate. The availability of gnt1 mutant cells made it possible to determine whether hydroxylation per se can rescue or whether GlcNAc capping is also required. Based on morphological analyses (Fig. 4B), gnt1.1 and gnt1.3 cells required ∼15% O2 to culminate, i.e. less than for P4H1-null cells but more than for wild-type (Ax3) cells, which required ≤10% O2 in this trial. At 10–12% O2, cells remained organized as slugs that oscillated between a horizontal migratory phase and a stationary state in which the slug rears up into a semivertical position, based on time-lapse microscopy.3 At a low but variable frequency, the vertical hypoxic slugs propelled themselves upward on their slime sheaths to form delicate aerial slugs that do not terminally differentiate (data not shown), as originally described for slugger mutants in normoxia (
      • Newell P.C.
      • Ross F.M.
      Genetic analysis of the slug stage of Dictyostelium discoideum.
      ) and normal cells in hypoxia (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ).
      Quantitation of the number of spores produced, which depends on normal culmination, yielded a similar finding. Some mutant terminal differentiation occurred at 12% O2, but 15% was required to generate normal spore numbers (Fig. 4C), and the O2 level required for 50% culmination (∼13%) was ∼3% higher than for strain Ax3 in the example shown. Spore counting showed that pgtA cells also required higher O2 than did Ax3 to sporulate, with an O2 level required for 50% culmination indistinguishable from that of gnt1 mutant cells. Because we did not previously observe a difference between Ax3 and pgtA (
      • Wang Z.A.
      • van der Wel H.
      • Vohra Y.
      • Buskas T.
      • Boons G.J.
      • West C.M.
      Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
      ), this comparison was repeated six additional times, always with a difference between the two strains. As reported previously (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ), the absolute O2 threshold varies between trials presumably because of other variables such as light level, although the relative order of O2 thresholds between mutants is invariant. Higher resolution was achieved in the present experimental series because of a broader threshold range separating phyA and Ax3 cells and the closer O2 level increments tested. Plotting the average O2 level required for 50% maximal sporulation over all experiments (Fig. 4E) confirmed that hydroxylation alone caused a substantial decrease in the O2 threshold from the unmodified state (i.e. phyA) that was indistinguishable from that of the mono-GlcNAcylated state (pgtA) and confirmed rescue by overexpression of Gnt1 but not mutant Gnt1.2. As reported previously (
      • Wang Z.A.
      • van der Wel H.
      • Vohra Y.
      • Buskas T.
      • Boons G.J.
      • West C.M.
      Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
      ), further glycosylation past the GlcNAc addition step to the trisaccharide state (agtA) reversed the effect (Fig. 4, C and E) before the addition of the terminal αGal residues reduced the O2 threshold to the wild-type level (Ax3). Thus P4H1 contributes both glycosylation-independent and glycosylation-dependent functions to establishing the O2 threshold.
      To test whether the reduced O2 threshold (relative to P4H1 cells) was specific to loss of αGlcNAcT activity, the gnt1.1 and gnt1.3 strains that had been complemented by overexpression of the DpGnt1 under the ecmA promoter were analyzed. As shown in Fig. 4, D and E, these strains exhibited an Ax3-like O2 dependence. Rescue depended on the αGlcNAcT activity as similar overexpression (Fig. 3C) of mutant DpGnt1(D102A) (Gnt1.2), which carries an inactivating mutation similar to that in Gnt1.3, did not affect the O2 requirement for culmination and sporulation. Overexpression of active Gnt1 did not reduce the O2 threshold to below the wild-type level, and this was confirmed by testing the overexpression of either active or inactive Gnt1 in the wild-type background (Fig. 4D). Therefore Gnt1 overexpression did not exert a dominant negative effect and, furthermore, Gnt1 did not appear to be rate-limiting for signaling because, unlike for P4H1 (
      • West C.M.
      • van der Wel H.
      • Wang Z.A.
      Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
      ), overexpression did not drive the O2 threshold to a lower level.

      Skp1 Is the Only Detected Acceptor Substrate of Gnt1 in gnt1.1 Cells

      Because the known function of Gnt1 is to enzymatically modify the product of the P4H1 reaction, we predicted that there would be no consequence of gnt1 mutations in a P4H1 background. This was tested by analyzing the gnt1.1/phyA and gnt1.3/phyA double mutants described above. Both mutants exhibited O2 requirements similar to that of phyA cells (Fig. 4C), indicating that Gnt1 only modifies Hyp products of the P4H1 reaction in cells. A previous study showed that the action of PgtA, whose only detectable substrate in cells is Skp1, can reverse the contribution of P4H1 to O2 signaling (
      • Wang Z.A.
      • van der Wel H.
      • Vohra Y.
      • Buskas T.
      • Boons G.J.
      • West C.M.
      Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
      ), suggesting that Gnt1 and P4H1 also signal via Skp1. To explore this model further, the in vivo specificity of Gnt1 was examined by screening an extract of gnt1.1 cells for potential acceptors expected to accumulate in the absence of the enzyme. After the addition of recombinant DpGnt1 and UDP[3H]GlcNAc to extracts of gnt1.1 and Ax3 and allowing the labeling reaction to proceed, the reaction product was displayed on an SDS-PAGE gel, which was analyzed for radioactivity. 3H incorporation was observed only in gnt1.1 cells, and the great majority occurred at the Mr position of Skp1 (Fig. 5A). The minimal incorporation seen at higher Mr positions was also observed in the absence of added Gnt1 (panel B) and thus appeared to be nonspecific. Increased incorporation into fractions 14, 20, and 21, which was <0.7% of the incorporation into the Skp1 band, was not reproducible in separate trials with independent gnt1 mutant clones or a phyA/gnt1 double mutant strain extract supplemented with Gnt1 or Gnt1 + P4H1 (data not shown). The near negligible labeling of Skp1 in parental Ax3 cells supplemented with DpGnt1 (Fig. 5B) indicated that the hydroxylated isoform did not accumulate in cells under these conditions. The even lower level of incorporation into higher Mr positions of Ax3 extracts probably reflects less contamination from vesicular glycosyltransferases and glycoprotein acceptors in this preparation. These results suggest that Skp1 is the only substrate that accumulates in the absence of Gnt1 and thus is potentially a unique Gnt1 acceptor substrate.
      Figure thumbnail gr5
      FIGURE 5Gnt1 substrate detection by in vitro complementation. A, soluble extracts from growing gnt1.1 and normal (Ax3) cells were incubated with UDP-[3H]GlcNAc ± recombinant DpGnt1. The quenched reaction was subjected to SDS-PAGE, and the apparent Mr distribution of 3H incorporation was determined by liquid scintillation counting of sequential gel slices. B, replot of the same data using a different ordinate scale.

      Analysis of Skp1 Half-life in gnt1.1 Cells

      The Western blot analyses of Fig. 3 show that the steady-state level of Skp1 in growing cells was not affected by hydroxylation in the absence of capping by GlcNAc. This finding differs from that observed for HIFα, whose levels sharply decrease following the hydroxylation events that led to its rapid polyubiquitination. To verify that Skp1 turnover was not affected, growing cells were metabolically labeled with [35S]Cys/Met and incorporation of radiolabel into total protein, as determined by trichloroacetic acid precipitation, or Skp1, as determined by immunoprecipitation and SDS-PAGE, was measured. Western blot analysis showed that >90% of Skp1 was recovered by immunoprecipitation (data not shown), and the autoradiogram shown in Fig. 6E shows the selectivity of the method. A comparison of P4H1-null and gnt1.1 cells showed that similar levels of radiolabel were incorporated into total protein (Fig. 6A) and Skp1 after 1 h of labeling (Fig. 6B). Similar labeling of Skp1 also occurred in slug cells (Fig. 6C) suggesting that, because Skp1 levels do not vary during development, turnover rates are also similar at this stage. The similar rate of Skp1 synthesis indicates that its turnover rate is also similar among the strains because Skp1 levels appeared to be constant. This was examined further after replacing the medium of 1-h labeled cells with medium containing unlabeled Cys/Met. A semilogarithmic plot of the level of radioactivity remaining in Skp1 as a function of the chase time revealed an approximately linear decrease (Fig. 6E) consistent with simple first-order breakdown. Skp1 was long lived in both strains with a calculated half-life of 15–18 h (Fig. 6D). A second trial also revealed indistinguishable turnover kinetics between the two strains, with an apparent shorter half-life of 10–12 h (data not shown). The strain comparison indicates that the pool of Skp1 that accumulates over the course of a 1-h labeling period decays with similar kinetics whether it is hydroxylated or not.
      Figure thumbnail gr6
      FIGURE 6Effect of prolyl hydroxylation on Skp1 stability. phyA (P4H1) and gnt1.1 cells were compared in 21% O2. A, proliferating cells were labeled with low specific activity [35S]Met/Cys for 1 h and incorporation into total protein determined by trichloroacetic acid precipitation. Average values ± S.E. are shown. B, proliferating cells were labeled with high specific activity [35S]Met/Cys for 1 h, and incorporation into Skp1 was determined by immunoprecipitation and SDS-PAGE. dpm were determined by scintillation counting and are presented as the percentage of incorporation into total protein. C, a comparison of labeling of slug stage Skp1, normalized to input cell number. D, half-lives of proliferating cell Skp1 were calculated from data shown in panel E. E, results from a pulse-chase study of 1-h labeled proliferating cells are plotted as a function of time. The line was fitted from a linear regression analysis and used to calculated Skp1 half-lives in growing cells. The autoradiogram at the left shows the selectivity of the immunoprecipitation method. Less incorporation was observed into the phyA strain in the example shown, but the fraction of label relative to total incorporation was similar between the two strains.

      DISCUSSION

      Target Specificity of Gnt1

      Gnt1 is a polypeptide αGlcNAcT that belongs to the lineage of enzymes responsible for initiation of mucin-type O-glycans in eukaryotes. In protists, the initial sugar is typically an αGlcNAc linked to Thr or Ser (
      • Ercan A.
      • West C.M.
      Kinetic analysis of a Golgi UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-α-glucosaminyltransferase from Dictyostelium.
      ,
      • Heise N.
      • Singh D.
      • van der Wel H.
      • Sassi S.O.
      • Johnson J.M.
      • Feasley C.L.
      • Koeller C.M.
      • Previato J.O.
      • Mendonça-Previato L.
      • West C.M.
      Molecular analysis of a UDP-GlcNAc:polypeptide α-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi.
      ), whereas in animals, the initial sugar is almost always its 4-epimer αGalNAc linked to either of the same hydroxyamino acids (
      • Ten Hagen K.G.
      • Fritz T.A.
      • Tabak L.A.
      All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases.
      ). A phylogenetic analysis of these sequences suggests that an ancient gene duplication allowed divergent evolution of the version of this enzyme that populates the Golgi apparatus to O-glycosylate cell surface and secretory proteins (
      • West C.M.
      • Van Der Wel H.
      • Sassi S.
      • Gaucher E.A.
      Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways.
      ). The product of the other gene retained its apparently primordial location in the cytoplasm where, at least in Dictyostelium and probably in Toxoplasma gondii (
      • West C.M.
      • van der Wel H.
      • Blader I.J.
      Detection of cytoplasmic glycosylation associated with hydroxyproline.
      ), it catalyzes the initiation of O-glycosylation of Hyp on Skp1. Here we confirm that this enzyme is solely responsible for Skp1 O-glycosylation at Hyp-143 and present evidence that, in contrast to other enzymes of this gene family, its catalytic activity is devoted to only a single protein target.
      The interpretation that Skp1 is the only protein acceptor substrate for Gnt1 derives from both biochemical and functional data. When recombinant Gnt1 was added back to extracts of gnt1.1 cells, expected to accumulate any Gnt1 substrates, the only protein labeled by the radioactive donor substrate above background levels (<0.7%) was Skp1 (Fig. 5). This finding is consistent with the results of previous similar experiments in which AgtA, PgtA, or P4H1/Gnt1 was added back to the corresponding enzyme mutant (
      • Wang Z.A.
      • Singh D.
      • van der Wel H.
      • West C.M.
      Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.
      ,
      • Wang Z.A.
      • van der Wel H.
      • Vohra Y.
      • Buskas T.
      • Boons G.J.
      • West C.M.
      Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
      ,
      • Ercan A.
      • Panico M.
      • Sutton-Smith M.
      • Dell A.
      • Morris H.R.
      • Matta K.L.
      • Gay D.F.
      • West C.M.
      Molecular characterization of a novel UDP-galactose:fucoside α3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.
      ). In addition, the effect of Gnt1 on the O2 requirement for culmination depended on P4H1 (Fig. 4C), which most likely indicates that Gnt1 acts on the product of P4H1. Thus Gnt1 is confirmed to modify Hyp during signaling and, because further Gnt1-dependent glycosylation reverses the P4H1 effect (
      • Wang Z.A.
      • van der Wel H.
      • Vohra Y.
      • Buskas T.
      • Boons G.J.
      • West C.M.
      Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
      ), P4H1 apparently signals via Skp1. Consistent with this model, genetic manipulations of Skp1 levels have effects on the O2 dependence of culmination in a manner that is affected by the presence of the target Pro-143 (
      • Wang Z.A.
      • Singh D.
      • van der Wel H.
      • West C.M.
      Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.
      ). Specificity for Skp1 may involve the previously described bipartite recognition mechanisms surmised for P4H1 and Gnt1 from characterizations of the in vitro reactions using purified proteins (
      • van der Wel H.
      • Johnson J.M.
      • Xu Y.
      • Karunaratne C.V.
      • Wilson K.D.
      • Vohra Y.
      • Boons G.J.
      • Taylor C.M.
      • Bendiak B.
      • West C.M.
      Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in Dictyostelium.
      ).

      Roles of P4H1 and Gnt1 in O2 Signaling

      The ground state O2 level required for culmination in the absence of Skp1 modification is established by the phyA strain and is ∼21% O2 (Fig. 4E). Hydroxylation reduces the threshold to 12–13%, as established by the gnt1 mutant strains, and this value is not affected further by the addition of GlcNAc alone, as determined in the pgtA strain. Thus hydroxylation alone has a substantial effect on O2 sensitivity. Although GlcNAc addition has little further effect, discrete steps in subsequent elongation of the sugar chain (see Fig. 3A for pathway) differentially modulate the O2 requirement (
      • West C.M.
      • Wang Z.A.
      • van der Wel H.
      A cytoplasmic prolyl hydroxylation and glycosylation pathway modifies Skp1 and regulates O2-dependent development in Dictyostelium.
      ). Therefore hydroxylation renders both glycosylation-independent and glycosylation-dependent effects on cell physiology. Because the effect of catalytically inactive Gnt1 was indistinguishable from that of truncated Gnt1 and because overexpression of inactive or active Gnt1 had no dominant negative effect, the role of Gnt1 may be to simply provide a platform for further glycosylation that affects development.

      Effects of P4H1 and GlcNAc on Skp1

      The comparison of Skp1 and Skp1-OH, from phyA and gnt1.1 or gnt1.3 cells, respectively, revealed no evidence for differences in level (Fig. 3), rate of synthesis, or stability (Fig. 6). A previous study showed a similar result for Skp1 from phyA, agtA, and normal cells using a cycloheximide treatment method (
      • Wang Z.A.
      • Singh D.
      • van der Wel H.
      • West C.M.
      Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.
      ). This contrasts with the effect of hydroxylation by the animal ortholog of P4H1, which leads to rapid degradation of HIFα based on recognition by the VHL subunit of the E3VBCUb ligase and subsequent polyubiquitination (
      • Kaelin Jr., W.G.
      • Ratcliffe P.J.
      Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.
      ). The difference is consistent with the absence of recognizable subunits of the VBC complex, except for elongin C, in the Dictyostelium genome.
      C. M. West, unpublished studies.
      Future studies are required to evaluate the possibility of indirect effects on the half-life of other proteins via the E3SCFUb ligase of which Skp1, evolutionarily related to elongin C of the E3VBCUb ligase, is a subunit.
      The mechanisms of action of the modifications toward Skp1 function are unclear. Because the gnt1 mutations also affect stalk morphogenesis in normoxia (Fig. 4A), Gnt1 action extends beyond the culmination checkpoint per se. Distinct developmental phenotypes appear in other pathway mutants (
      • Wang Z.A.
      • Singh D.
      • van der Wel H.
      • West C.M.
      Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.
      ), reinforcing the concept that global Skp1 functions, perhaps hydroxylation-dependent but not necessarily O2-regulated, are affected by Skp1 modification. Skp1 is an adaptor in the multisubunit SCF complex family, with potentially 50 members based on the number of predicted FBP genes,3 some of which are active in Dictyostelium development (
      • Mohanty S.
      • Lee S.
      • Yadava N.
      • Dealy M.J.
      • Johnson R.S.
      • Firtel R.A.
      Regulated protein degradation controls PKA function and cell type differentiation in Dictyostelium.
      ,
      • Wang B.
      • Kuspa A.
      CulB, a putative ubiquitin ligase subunit, regulates prestalk cell differentiation and morphogenesis in Dictyostelium spp. Eukaryot.
      ,
      • Nelson M.K.
      • Clark A.
      • Abe T.
      • Nomura A.
      • Yadava N.
      • Funair C.J.
      • Jermyn K.A.
      • Mohanty S.
      • Firtel R.A.
      • Williams J.G.
      An F-Box/WD40 repeat-containing protein important for Dictyostelium cell type proportioning, slug behavior, and culmination.
      ,
      • Ennis H.L.
      • Dao D.N.
      • Pukatzki S.U.
      • Kessin R.H.
      Dictyostelium amoebae lacking an F-box protein form spores rather than stalk in chimeras with wild type.
      ). Studies from other organisms indicate that in addition to interacting with F-box proteins and cullins and the modification enzymes discussed here, Skp1 can homodimerize (
      • Tan A.
      • Henzl M.T.
      Conformational stabilities of guinea pig OCP1 and OCP2.
      ) and interact with other proteins such as Sgt1 and Siah-interacting protein (SIP). If the modifications influence local Skp1 conformation, as suggested by related studies in HIFα (
      • Loenarz C.
      • Mecinovi J.
      • Chowdhury R.
      • McNeill L.A.
      • Flashman E.
      • Schofield C.J.
      Evidence for a stereoelectronic effect in human oxygen sensing.
      ), then disturbances of the kinetics or equilibria of any of these interactions may influence Skp1 activities. An alternative “extrinsic” model in which accessory proteins that recognize discrete modification states is not excluded by these considerations. Conservation of P4H1- and Gnt1-like genes in numerous other protists, including pathogens such as Acanthamoeba, Phytophthora spp., and the agent for human toxoplasmosis, T. gondii (
      • West C.M.
      • Wang Z.A.
      • van der Wel H.
      A cytoplasmic prolyl hydroxylation and glycosylation pathway modifies Skp1 and regulates O2-dependent development in Dictyostelium.
      ,
      • West C.M.
      • Van Der Wel H.
      • Sassi S.
      • Gaucher E.A.
      Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways.
      ), suggests that mechanisms operating in Dictyostelium will be relevant for O2 and metabolic sensing in a broad spectrum of unicellular organisms.

      Supplementary Material

      REFERENCES

        • Ward J.P.
        Oxygen sensors in context.
        Biochim. Biophys. Acta. 2008; 1777: 1-14
        • Bien C.M.
        • Espenshade P.J.
        Sterol regulatory element-binding proteins in fungi: hypoxic transcription factors linked to pathogenesis.
        Eukaryot. Cell. 2010; 9: 352-359
        • Kaelin Jr., W.G.
        • Ratcliffe P.J.
        Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.
        Mol. Cell. 2008; 30: 393-402
        • Schindler J.
        • Sussman M.
        Ammonia determines the choice of morphogenetic pathways in Dictyostelium discoideum.
        J. Mol. Biol. 1977; 116: 161-169
        • Newell P.C.
        • Telser A.
        • Sussman M.
        Alternative developmental pathways determined by environmental conditions in the cellular slime mold Dictyostelium discoideum.
        J. Bacteriol. 1969; 100: 763-768
        • Smith E.
        • Williams K.L.
        Evidence for tip control of the “slug/fruit” switch in slugs of Dictyostelium discoideum.
        J. Embryol. Exp. Morphol. 1980; 57: 233-240
        • Singleton C.K.
        • Kirsten J.H.
        • Dinsmore C.J.
        Function of ammonium transporter A in the initiation of culmination of development in Dictyostelium discoideum.
        Eukaryot. Cell. 2006; 5: 991-996
        • Sandonà D.
        • Gastaldello S.
        • Rizzuto R.
        • Bisson R.
        Expression of cytochrome c oxidase during growth and development of Dictyostelium.
        J. Biol. Chem. 1995; 270: 5587-5593
        • Bonner J.T.
        • Lamont D.S.
        Behavior of cellular slime molds in the soil.
        Mycologia. 2005; 97: 178-184
        • West C.M.
        • Wang Z.A.
        • van der Wel H.
        A cytoplasmic prolyl hydroxylation and glycosylation pathway modifies Skp1 and regulates O2-dependent development in Dictyostelium.
        Biochim. Biophys. Acta. 2010; 1800: 160-171
        • van der Wel H.
        • Ercan A.
        • West C.M.
        The Skp1 prolyl hydroxylase from Dictyostelium is related to the hypoxia-inducible factor-α class of animal prolyl 4-hydroxylases.
        J. Biol. Chem. 2005; 280: 14645-14655
        • van der Wel H.
        • Johnson J.M.
        • Xu Y.
        • Karunaratne C.V.
        • Wilson K.D.
        • Vohra Y.
        • Boons G.J.
        • Taylor C.M.
        • Bendiak B.
        • West C.M.
        Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in Dictyostelium.
        Biochemistry. 2011; 50: 1700-1713
        • Teng-Umnuay P
        • van der Wel H
        • West CM
        Identification of a UDP-GlcNAc:Skp1-hydroxyproline GlcNAc-transferase in the cytoplasm of Dictyostelium.
        J. Biol. Chem. 1999; 274: 36392-36402
        • Van Der Wel H.
        • Morris H.R.
        • Panico M.
        • Paxton T.
        • Dell A.
        • Kaplan L.
        • West C.M.
        Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of Dictyostelium.
        J. Biol. Chem. 2002; 277: 46328-46337
        • West C.M.
        • van der Wel H.
        • Wang Z.A.
        Prolyl 4-hydroxylase-1 mediates O2 signaling during development of Dictyostelium.
        Development. 2007; 134: 3349-3358
        • Wang Z.A.
        • Singh D.
        • van der Wel H.
        • West C.M.
        Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.
        Dev. Biol. 2011; 349: 283-295
        • Wang Z.A.
        • van der Wel H.
        • Vohra Y.
        • Buskas T.
        • Boons G.J.
        • West C.M.
        Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
        J. Biol. Chem. 2009; 284: 28896-28904
        • Funakoshi Y.
        • Suzuki T.
        Glycobiology in the cytosol: the bitter side of a sweet world.
        Biochim. Biophys. Acta. 2009; 1790: 81-94
        • Hart G.W.
        • West C.M.
        Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. Essentials of Glycobiology. 2nd Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2009: 249-262
        • Borst P.
        • Sabatini R.
        Base J: discovery, biosynthesis, and possible functions.
        Annu. Rev. Microbiol. 2008; 62: 235-251
        • Willems A.R.
        • Schwab M.
        • Tyers M.
        A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin.
        Biochim. Biophys. Acta. 2004; 1695: 133-170
        • Deshaies R.J.
        • Joazeiro C.A.
        RING domain E3 ubiquitin ligases.
        Annu. Rev. Biochem. 2009; 78: 399-434
        • Fey P.
        • Kowal A.S.
        • Gaudet P.
        • Pilcher K.E.
        • Chisholm R.L.
        Protocols for growth and development of Dictyostelium discoideum.
        Nat. Protoc. 2007; 2: 1307-1316
        • Sassi S.
        • Sweetinburgh M.
        • Erogul J.
        • Zhang P.
        • Teng-Umnuay P.
        • West C.M.
        Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium.
        Glycobiology. 2001; 11: 283-295
        • Sucgang R.
        • Kuo A.
        • Tian X.
        • Salerno W.
        • Parikh A.
        • Feasley C.L.
        • Dalin E.
        • Tu H.
        • Huang E.
        • Barry K.
        • Lindquist E.
        • Shapiro H.
        • Bruce D.
        • Schmutz J.
        • Salamov A.
        • Fey P.
        • Gaudet P.
        • Anjard C.
        • Babu M.M.
        • Basu S.
        • Bushmanova Y.
        • van der Wel H.
        • Katoh-Kurasawa M.
        • Dinh C.
        • Coutinho P.M.
        • Saito T.
        • Elias M.
        • Schaap P.
        • Kay R.R.
        • Henrissat B.
        • Eichinger L.
        • Rivero F.
        • Putnam N.H.
        • West C.M.
        • Loomis W.F.
        • Chisholm R.L.
        • Shaulsky G.
        • Strassmann J.E.
        • Queller D.C.
        • Kuspa A.
        • Grigoriev I.V.
        Comparative genomics of the social amoebae Dictyostelium discoideum and Dictyostelium purpureum.
        Genome Biol. 2011; 12: R20
        • Newell P.C.
        • Ross F.M.
        Genetic analysis of the slug stage of Dictyostelium discoideum.
        J. Gen. Microbiol. 1982; 128: 1639-1652
        • Ercan A.
        • West C.M.
        Kinetic analysis of a Golgi UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-α-glucosaminyltransferase from Dictyostelium.
        Glycobiology. 2005; 15: 489-500
        • Heise N.
        • Singh D.
        • van der Wel H.
        • Sassi S.O.
        • Johnson J.M.
        • Feasley C.L.
        • Koeller C.M.
        • Previato J.O.
        • Mendonça-Previato L.
        • West C.M.
        Molecular analysis of a UDP-GlcNAc:polypeptide α-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi.
        Glycobiology. 2009; 19: 918-933
        • Ten Hagen K.G.
        • Fritz T.A.
        • Tabak L.A.
        All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases.
        Glycobiology. 2003; 13: 1R-16R
        • West C.M.
        • Van Der Wel H.
        • Sassi S.
        • Gaucher E.A.
        Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways.
        Biochim. Biophys. Acta. 2004; 1673: 29-44
        • West C.M.
        • van der Wel H.
        • Blader I.J.
        Detection of cytoplasmic glycosylation associated with hydroxyproline.
        Methods Enzymology. 2006; 417: 385-400
        • Ercan A.
        • Panico M.
        • Sutton-Smith M.
        • Dell A.
        • Morris H.R.
        • Matta K.L.
        • Gay D.F.
        • West C.M.
        Molecular characterization of a novel UDP-galactose:fucoside α3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.
        J. Biol. Chem. 2006; 281: 12713-12721
        • Mohanty S.
        • Lee S.
        • Yadava N.
        • Dealy M.J.
        • Johnson R.S.
        • Firtel R.A.
        Regulated protein degradation controls PKA function and cell type differentiation in Dictyostelium.
        Genes Dev. 2001; 15: 1435-1448
        • Wang B.
        • Kuspa A.
        CulB, a putative ubiquitin ligase subunit, regulates prestalk cell differentiation and morphogenesis in Dictyostelium spp. Eukaryot.
        Cell. 2002; 1: 126-136
        • Nelson M.K.
        • Clark A.
        • Abe T.
        • Nomura A.
        • Yadava N.
        • Funair C.J.
        • Jermyn K.A.
        • Mohanty S.
        • Firtel R.A.
        • Williams J.G.
        An F-Box/WD40 repeat-containing protein important for Dictyostelium cell type proportioning, slug behavior, and culmination.
        Dev. Biol. 2000; 224: 42-59
        • Ennis H.L.
        • Dao D.N.
        • Pukatzki S.U.
        • Kessin R.H.
        Dictyostelium amoebae lacking an F-box protein form spores rather than stalk in chimeras with wild type.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 3292-3297
        • Tan A.
        • Henzl M.T.
        Conformational stabilities of guinea pig OCP1 and OCP2.
        Biophys. Chem. 2009; 144: 108-118
        • Loenarz C.
        • Mecinovi J.
        • Chowdhury R.
        • McNeill L.A.
        • Flashman E.
        • Schofield C.J.
        Evidence for a stereoelectronic effect in human oxygen sensing.
        Angew Chem. Int. Ed. Engl. 2009; 48: 1784-1787
        • Van Der Wel H.
        • Fisher S.Z.
        • West C.M.
        A bifunctional diglycosyltransferase forms the Fucα1,2Galβ1,3-disaccharide on Skp1 in the cytoplasm of Dictyostelium.
        J. Biol. Chem. 2002; 277: 46527-46534