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Two Golgi-resident 3′-Phosphoadenosine 5′-Phosphosulfate Transporters Play Distinct Roles in Heparan Sulfate Modifications and Embryonic and Larval Development in Caenorhabditis elegans*

  • Katsufumi Dejima
    Footnotes
    Affiliations
    Department of Biology, Faculty of Sciences 33, Kyushu University, Fukuoka 812-8581, Japan

    Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan
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  • Daisuke Murata
    Affiliations
    Department of Biology, Faculty of Sciences 33, Kyushu University, Fukuoka 812-8581, Japan

    Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan
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  • Souhei Mizuguchi
    Footnotes
    Affiliations
    Department of Biology, Faculty of Sciences 33, Kyushu University, Fukuoka 812-8581, Japan

    Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan
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  • Kazuko H. Nomura
    Affiliations
    Department of Biology, Faculty of Sciences 33, Kyushu University, Fukuoka 812-8581, Japan

    Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan
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  • Tomomi Izumikawa
    Affiliations
    Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan

    Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan
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  • Hiroshi Kitagawa
    Affiliations
    Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan

    Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan
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  • Keiko Gengyo-Ando
    Footnotes
    Affiliations
    Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan

    Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo 162-8666, Japan
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  • Sawako Yoshina
    Affiliations
    Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan

    Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo 162-8666, Japan
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  • Tomomi Ichimiya
    Affiliations
    Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan
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  • Shoko Nishihara
    Affiliations
    Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan
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  • Shohei Mitani
    Affiliations
    Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan

    Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo 162-8666, Japan
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  • Kazuya Nomura
    Correspondence
    To whom correspondence should be addressed: Dept. of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan. Tel. and Fax: 81-92-642-4613
    Affiliations
    Department of Biology, Faculty of Sciences 33, Kyushu University, Fukuoka 812-8581, Japan

    Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan
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  • Author Footnotes
    * This work was supported by a grant-in-aid from the Japan Society for the Promotion of Science Fellows (to K. D.), a grant-in-aid for young scientists (B) (to S. M.), and Grant-in-Aid for Scientific Research B-21390025 (to H. K.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. This research was also supported by MEXT Grant-in-Aid for Scientific Research B-20370051 (to S. N.) and by the Core Research for Evolutional Science and Technology Program of the Japan Science and Technology Corp. (to K. N.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4 and Table S1.
    1 Present address: Dept. of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455.
    2 Present address: Dept. of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.
    3 Present address: Saitama University Brain Science Institute, Saitama 338–8570, Japan.
Open AccessPublished:June 06, 2010DOI:https://doi.org/10.1074/jbc.M109.088229
      Synthesis of extracellular sulfated molecules requires active 3′-phosphoadenosine 5′-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.

      Introduction

      Organogenesis, tissue morphogenesis, and cell growth require diverse types of extracellular sulfation. Sulfated molecules are crucial for the establishment of a hydrophilic extracellular environment and for intercellular signaling (
      • Kehoe J.W.
      • Bertozzi C.R.
      ,
      • Schwartz N.B.
      • Domowicz M.
      ,
      • Honke K.
      • Zhang Y.
      • Cheng X.
      • Kotani N.
      • Taniguchi N.
      ,
      • Zhang H.
      • Uchimura K.
      • Kadomatsu K.
      ,
      • Bishop J.R.
      • Schuksz M.
      • Esko J.D.
      ,
      • Sugahara K.
      • Mikami T.
      ,
      • Nadanaka S.
      • Kitagawa H.
      ,
      • Morita I.
      • Kizuka Y.
      • Kakuda S.
      • Oka S.
      ). In eukaryotes, Golgi-resident sulfotransferases transfer sulfates from an active sulfate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS),
      The abbreviations used are: PAPS
      3′-phosphoadenosine 5′-phosphosulfate
      DIC
      differential interference contrast
      DTC
      distal tip cell
      EGFP
      enhanced green fluorescent protein
      GFP
      green fluorescent protein
      GAG
      glycosaminoglycan
      HA
      hemagglutinin
      hPAPST
      human PAPS transporter
      HS
      heparan sulfate
      PAP
      3′-phosphoadenosine 5′-phosphate
      Sqv
      squashed vulva
      NS
      2-N-sulfate
      2S
      2-O-sulfate
      6S
      6-O-sulfate.
      to membrane-associated and secreted molecules like glycosaminoglycans (GAGs) (
      • Robbins P.W.
      • Lipmann F.
      ,
      • Klaassen C.D.
      • Boles J.W.
      ,
      • Strott C.A.
      ). The sulfation reaction yields 3′-phosphoadenosine 5′-phosphate (PAP) as a byproduct. Biochemical and cytological studies have revealed that PAPS is synthesized by PAPS synthase, a bifunctional enzyme found in the nucleus and/or cytosol but not in the Golgi apparatus (
      • Robbins P.W.
      • Lipmann F.
      ,
      • Zaruba M.E.
      • Schwartz N.B.
      • Tennekoon G.I.
      ,
      • Besset S.
      • Vincourt J.B.
      • Amalric F.
      • Girard J.P.
      ,
      • Dejima K.
      • Seko A.
      • Yamashita K.
      • Gengyo-Ando K.
      • Mitani S.
      • Izumikawa T.
      • Kitagawa H.
      • Sugahara K.
      • Mizuguchi S.
      • Nomura K.
      ). PAPS transporters transport PAPS from the cytosol into the Golgi lumen (
      • Schwarz J.K.
      • Capasso J.M.
      • Hirschberg C.B.
      ). Recent reports have demonstrated that PAPS/PAP concentrations in the Golgi apparatus are important for biosynthetic regulation of sulfated molecules, including heparan sulfate (HS) (
      • Carlsson P.
      • Presto J.
      • Spillmann D.
      • Lindahl U.
      • Kjellén L.
      ,
      • Sohaskey M.L.
      • Yu J.
      • Diaz M.A.
      • Plaas A.H.
      • Harland R.M.
      ,
      • Frederick J.P.
      • Tafari A.T.
      • Wu S.M.
      • Megosh L.C.
      • Chiou S.T.
      • Irving R.P.
      • York J.D.
      ,
      • Dick G.
      • Grøndahl F.
      • Prydz K.
      ). Thus, understanding how PAPS metabolism is regulated by PAPS transporters in vivo will provide important insight into the mechanisms underlying developmental control of extracellular sulfation.
      PAPS transporter activity was first demonstrated in rat liver Golgi-derived vesicles (
      • Schwarz J.K.
      • Capasso J.M.
      • Hirschberg C.B.
      ). The characterization of purified proteins involved in PAPS transport activity suggests that they act through an antiport mechanism (
      • Zaruba M.E.
      • Schwartz N.B.
      • Tennekoon G.I.
      ,
      • Capasso J.M.
      • Hirschberg C.B.
      ,
      • Mandon E.C.
      • Milla M.E.
      • Kempner E.
      • Hirschberg C.B.
      ,
      • Ozeran J.D.
      • Westley J.
      • Schwartz N.B.
      ,
      • Ozeran J.D.
      • Westley J.
      • Schwartz N.B.
      ). Recently, two human PAPS transporter genes were cloned and named PAPST1 (Slc35b2) (
      • Kamiyama S.
      • Suda T.
      • Ueda R.
      • Suzuki M.
      • Okubo R.
      • Kikuchi N.
      • Chiba Y.
      • Goto S.
      • Toyoda H.
      • Saigo K.
      • Watanabe M.
      • Narimatsu H.
      • Jigami Y.
      • Nishihara S.
      ) and PAPST2 (Slc35b3) (
      • Kamiyama S.
      • Sasaki N.
      • Goda E.
      • Ui-Tei K.
      • Saigo K.
      • Narimatsu H.
      • Jigami Y.
      • Kannagi R.
      • Irimura T.
      • Nishihara S.
      ,
      • Goda E.
      • Kamiyama S.
      • Uno T.
      • Yoshida H.
      • Ueyama M.
      • Kinoshita-Toyoda A.
      • Toyoda H.
      • Ueda R.
      • Nishihara S.
      ). The Drosophila slalom gene, which encodes PAPST1, was identified as a segment polarity gene (
      • Lüders F.
      • Segawa H.
      • Stein D.
      • Selva E.M.
      • Perrimon N.
      • Turco S.J.
      • Häcker U.
      ,
      • Ali R.A.
      • Mellenthin K.
      • Fahmy K.
      • Da Rocha S.
      • Baumgartner S.
      ). In zebrafish, mutations in pinscher, the gene encoding PAPST1, caused defects in skeletal development and axon sorting (
      • Clément A.
      • Wiweger M.
      • von der Hardt S.
      • Rusch M.A.
      • Selleck S.B.
      • Chien C.B.
      • Roehl H.H.
      ). Although the gene encoding PAPST2 showed genetic interactions with the genes that encode HS modification enzymes in the fruit fly, its physiological roles are largely unknown (
      • Goda E.
      • Kamiyama S.
      • Uno T.
      • Yoshida H.
      • Ueyama M.
      • Kinoshita-Toyoda A.
      • Toyoda H.
      • Ueda R.
      • Nishihara S.
      ).
      The nematode Caenorhabditis elegans is a model organism that is well suited for developmental genetics because of its simple and well organized organs, including those of the reproductive, digestive, nervous, and epithelial tissue systems. The C. elegans genome contains orthologs of all the known enzymes involved in PAPS metabolism, including PAPS synthase (pps-1, T14G10.1), PAPS reductase (R53.1), Golgi-resident PAP phosphatase (Y6B3B.5), sulfate transporters (sulp-2, F14D12.5; sulp-4, K12G11.1) (
      • Sherman T.
      • Chernova M.N.
      • Clark J.S.
      • Jiang L.
      • Alper S.L.
      • Nehrke K.
      ), and PAPS transporters (pst-1, M03F8.2; pst-2, F54E7.1). In addition, C. elegans expresses genes that are involved in the sulfation of tyrosine and HS but not chondroitin (
      • Bülow H.E.
      • Hobert O.
      ,
      • Mizuguchi S.
      • Dejima D.
      • Nomura K.
      ,
      • Yamada S.
      • Van Die I.
      • Van den Eijnden D.H.
      • Yokota A.
      • Kitagawa H.
      • Sugahara K.
      ,
      • Toyoda H.
      • Kinoshita-Toyoda A.
      • Selleck S.B.
      ,
      • Nabetani T.
      • Miyazaki K.
      • Tabuse Y.
      • Tsugita A.
      ). This represents an advantage of using C. elegans over vertebrate species to investigate the role of PAPS metabolism in development. In vertebrates, chondroitin sulfate is required for chondrogenesis (
      • Schwartz N.B.
      • Domowicz M.
      ,
      • Sohaskey M.L.
      • Yu J.
      • Diaz M.A.
      • Plaas A.H.
      • Harland R.M.
      ,
      • Frederick J.P.
      • Tafari A.T.
      • Wu S.M.
      • Megosh L.C.
      • Chiou S.T.
      • Irving R.P.
      • York J.D.
      ,
      • Clément A.
      • Wiweger M.
      • von der Hardt S.
      • Rusch M.A.
      • Selleck S.B.
      • Chien C.B.
      • Roehl H.H.
      ), and defects in chondroitin sulfation obscure the importance of other sulfated molecules. Previous studies have demonstrated that extracellular sulfated molecules play pivotal roles in nervous system development, gonadal cell migration, cuticle formation, and embryogenesis in C. elegans (
      • Kim T.H.
      • Hwang S.B.
      • Jeong P.Y.
      • Lee J.
      • Cho J.W.
      ,

      Berninsone, P. M., (December, 18, 2006) in WormBook (C. elegans Research Community, ed) doi/10.1895/wormbook.1.125.1, http://www.wormbook.org

      ,
      • Gumienny T.L.
      • MacNeil L.T.
      • Wang H.
      • de Bono M.
      • Wrana J.L.
      • Padgett R.W.
      ,
      • Franks D.M.
      • Izumikawa T.
      • Kitagawa H.
      • Sugahara K.
      • Okkema P.G.
      ,
      • Kitagawa H.
      • Izumikawa T.
      • Mizuguchi S.
      • Dejima K.
      • Nomura K.H.
      • Egusa N.
      • Taniguchi F.
      • Tamura J.
      • Gengyo-Ando K.
      • Mitani S.
      • Nomura K.
      • Sugahara K.
      ,
      • Bülow H.E.
      • Tjoe N.
      • Townley R.A.
      • Didiano D.
      • van Kuppevelt T.H.
      • Hobert O.
      ). Most recently, pst-1 alleles were shown to be required for viability and neuronal development (
      • Bhattacharya R.
      • Townley R.A.
      • Berry K.L.
      • Bülow H.E.
      ).
      In this study, we aimed to investigate the roles of PAPS transporters in development and morphogenesis. To that end, we isolated C. elegans deletion mutants of the PAPS transporters and analyzed the defects in development and morphogenesis.

      DISCUSSION

      This study showed that, in C. elegans, the PAPS transporter pst-1 gene, but not the pst-2 gene, is essential for diverse aspects of epithelial development, somatic gonadal cell migration, and viability. We observed embryonic defects in pst-1 knock-out worms similar to those observed in embryos deficient in pps-1 (
      • Dejima K.
      • Seko A.
      • Yamashita K.
      • Gengyo-Ando K.
      • Mitani S.
      • Izumikawa T.
      • Kitagawa H.
      • Sugahara K.
      • Mizuguchi S.
      • Nomura K.
      ) or rib-1/rib-2, which lacked HS-synthesizing enzymes (
      • Kitagawa H.
      • Izumikawa T.
      • Mizuguchi S.
      • Dejima K.
      • Nomura K.H.
      • Egusa N.
      • Taniguchi F.
      • Tamura J.
      • Gengyo-Ando K.
      • Mitani S.
      • Nomura K.
      • Sugahara K.
      ,
      • Morio H.
      • Honda Y.
      • Toyoda H.
      • Nakajima M.
      • Kurosawa H.
      • Shirasawa T.
      ). During larval development, the pst-1 mutant showed defective cuticle formation similar to that observed in larvae depleted of pps-1 or tyrosylprotein sulfotransferase-A (tpst-1) genes (
      • Dejima K.
      • Seko A.
      • Yamashita K.
      • Gengyo-Ando K.
      • Mitani S.
      • Izumikawa T.
      • Kitagawa H.
      • Sugahara K.
      • Mizuguchi S.
      • Nomura K.
      ,
      • Kim T.H.
      • Hwang S.B.
      • Jeong P.Y.
      • Lee J.
      • Cho J.W.
      ). Although disaccharide analysis revealed that both pst-1 and pst-2 were involved in HS sulfation, none of the defects caused by the pst-1 mutation could be restored by the heterogeneous expression of hPAPST2 or PST-2. Moreover, HS sulfation patterns isolated from pst-1 mutant animals were clearly different from those isolated from pst-2 mutant animals. Furthermore, pst-1 and pst-2 displayed different expression patterns. These observations indicated that hPAPST1/PST-1 has distinct characteristics from hPAPST2/PST-2 in vivo. Our data also suggested that subcellular localization of the PST-1·EGFP protein was slightly different from that of the PST-2·EGFP protein. Thus, each PAPS transporter may reside in different intracellular compartments; this would allow differential sulfation reactions within a single cell. Intriguingly, differential “Golgi units” are proposed to regulate different glycosylation reactions in Drosophila cells (
      • Yano H.
      • Yamamoto-Hino M.
      • Abe M.
      • Kuwahara R.
      • Haraguchi S.
      • Kusaka I.
      • Awano W.
      • Kinoshita-Toyoda A.
      • Toyoda H.
      • Goto S.
      ).
      Analysis using the yeast heterologous system clearly suggested that PST-1 and PST-2 are PAPS-specific transporters. Although PST-2 showed weaker transport activity compared with PST-1, the transporter activity in our assay was statistically significant, and PST-2 showed no transport activity for nucleotide sugars. These results together with the reduced sulfation in the pst-2 null allele HS disaccharide analysis strongly indicate that PST-2 is also a PAPS transporter. It is intriguing that the total amount of HS disaccharide units was decreased in pst-2 mutant but not in the pst-1 null allele. Because sulfation takes place simultaneously with elongation of the GAG chain (
      • Silbert J.E.
      • Sugumaran G.
      ), depletion of PAPS in the intracellular compartment containing PST-2 may specifically influence the synthesis of GAG chains and reduce the heparan sulfate content measured in pst-2 null mutant animals.
      The pst-1, pst-2 double mutant showed that PST-1 and PST-2 had synergistic effects in cuticle formation. Precise cuticle formation requires both epithelial cells and pharyngeal gland cells (
      • Nelson F.K.
      • Albert P.S.
      • Riddle D.L.
      ). Epithelial cells synthesize the cuticle, and pharyngeal gland cells are thought to secrete a surface coating that covers the cuticle. Our results showed that pst-2 was expressed in pharyngeal gland cells, and thus in these cells, PST-2 may be involved in the secretion or synthesis of components of the cuticle surface coat. Another nucleotide sugar transporter, SRF-3, transports UDP-GlcNAc and UDP-Gal into Golgi apparatus-enriched vesicles from the cytosol. SRF-3 is expressed in pharyngeal gland cells and seam cells. It is involved in the biosynthesis of glycoconjugates for the outer surface and the cuticle (
      • Höflich J.
      • Berninsone P.
      • Göbel C.
      • Gravato-Nobre M.J.
      • Libby B.J.
      • Darby C.
      • Politz S.M.
      • Hodgkin J.
      • Hirschberg C.B.
      • Baumeister R.
      ,
      • Cipollo J.F.
      • Awad A.M.
      • Costello C.E.
      • Hirschberg C.B.
      ). Considering the expression patterns of PST-2, it is possible that PST-2 may cooperate with SRF-3 in this process. We also found that PST-2 was strongly expressed in the intestine, consistent with the tissue distribution of hPAPST2 transcripts (
      • Kamiyama S.
      • Sasaki N.
      • Goda E.
      • Ui-Tei K.
      • Saigo K.
      • Narimatsu H.
      • Jigami Y.
      • Kannagi R.
      • Irimura T.
      • Nishihara S.
      ). This implies that PST-2 and hPAPST2 may play a common role in the intestine; for example, they may participate in host-pathogen interactions.
      Vulval morphogenesis, including invagination and final cell positioning along the anterior-posterior axis, was affected in pst-1 mutant animals. Our data suggest that pst-1 is required for the precise expression of lin-11::gfp. Similar findings have been reported for worms that expressed mutant lin-17, a gene that encodes the Frizzled Wnt receptor (
      • Gupta B.P.
      • Sternberg P.W.
      ,
      • Sternberg P.W.
      • Horvitz H.R.
      ,
      • Sawa H.
      • Lobel L.
      • Horvitz H.R.
      ). HS proteoglycans are essential for Wnt signaling, both in vertebrates and invertebrates (
      • Lin X.
      ), and thus the sulfation of HS proteoglycans could modulate Wnt/Frizzled signaling or lin-11 transcriptional regulation in vulval cells.
      Immunostaining experiments have indicated that HS is present in vulval cells and around the vulva (Ref.
      • Minniti A.N.
      • Labarca M.
      • Hurtado C.
      • Brandan E.
      and our unpublished data). Consistent with this finding, abnormal vulval morphogenesis is thought to result from the loss of rib-2 (a glucosaminyltransferase) function (
      • Bender A.M.
      • Kirienko N.V.
      • Olson S.K.
      • Esko J.D.
      • Fay D.S.
      ). Other glycosyltransferase genes (sqv-6, sqv-2, sqv-8, and sqv-3) involved in establishing proteoglycan linkages are required for synthesis of both HS and chondroitin. Mutations of sqv genes cause the Sqv phenotype, which was also observed in the pst-1 mutant. As ample evidence indicates that chondroitin is not sulfated in this organism (
      • Yamada S.
      • Van Die I.
      • Van den Eijnden D.H.
      • Yokota A.
      • Kitagawa H.
      • Sugahara K.
      ,
      • Toyoda H.
      • Kinoshita-Toyoda A.
      • Selleck S.B.
      ,
      • Nabetani T.
      • Miyazaki K.
      • Tabuse Y.
      • Tsugita A.
      ), the Sqv phenotype in pst-1 mutant would be due to undersulfation of HS rather than chondroitin. However, animals with mutations in the sqv genes do not exhibit abnormal anterior-posterior cell positioning or small ectopic invaginations (
      • Herman T.
      • Hartwieg E.
      • Horvitz H.R.
      ), despite their requirement for HS synthesis. This apparent discrepancy might be ascribed to the different half-lives of the different gene products studied (mRNA or proteins) and/or to different metabolic functions of the enzymes studied (PAPS transport versus GAG linkage). Further study of these genes in the vulval development will provide useful information concerning the fundamental machinery of GAG synthesis and metabolism, as well as the regulation of extracellular signaling by GAGs.
      Mutation of pst-1 also resulted in abnormal EMS cell division and post-embryonic seam cell development, which are regulated by Wnt signaling (

      Eisenmann, D. M., (June 25, 2005) in WormBook (C. elegans Research Community, ed) doi/10.1895/wormbook.1.7.1, http://www.wormbook.org

      ). This gives rise to the intriguing possibility that sulfation of extracellular molecules could be involved in regulation of proteins associated with Wnt signaling in diverse processes in C. elegans.
      The lethality observed in the pst-1 mutants isolated in this study occurred at a different embryonic stage than that observed in the pps-1 null mutant (tm1109) that we isolated previously (
      • Dejima K.
      • Seko A.
      • Yamashita K.
      • Gengyo-Ando K.
      • Mitani S.
      • Izumikawa T.
      • Kitagawa H.
      • Sugahara K.
      • Mizuguchi S.
      • Nomura K.
      ). The animals with pst-1 mutations survived through the embryonic stage of the second generation; in contrast, animals with pps-1 mutations died in L2/L3 of the first generation. This difference in timing can be explained by several possibilities. (i) Cytosolic sulfation is essential for L2/L3 growth. To date, no null mutation of ssu-1, which encodes the only known C. elegans ortholog of cytosolic sulfotransferase, has been isolated (
      • Carroll B.T.
      • Dubyak G.R.
      • Sedensky M.M.
      • Morgan P.G.
      ,
      • Hattori K.
      • Inoue M.
      • Inoue T.
      • Arai H.
      • Tamura H.O.
      ,
      • Sönnichsen B.
      • Koski L.B.
      • Walsh A.
      • Marschall P.
      • Neumann B.
      • Brehm M.
      • Alleaume A.M.
      • Artelt J.
      • Bettencourt P.
      • Cassin E.
      • Hewitson M.
      • Holz C.
      • Khan M.
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      • Martin C.
      • Nitzsche B.
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      • Heinkel R.
      • Röder M.
      • Finell J.
      • Häntsch H.
      • Jones S.J.
      • Jones M.
      • Piano F.
      • Gunsalus K.C.
      • Oegema K.
      • Gönczy P.
      • Coulson A.
      • Hyman A.A.
      • Echeverri C.J.
      ). However, deficiencies in ssu-1, by RNA interference or reduction-of-function mutations, did not result in a lethal phenotype. (ii) Extracellular sulfation is essential for L2/L3 growth, and PST-1 may not be the only Golgi-resident PAPS transporter, or there may be a novel mechanism for providing PAPS to Golgi-resident sulfotransferases. (iii) Neither cytosolic nor extracellular sulfation is required for L2/L3 growth, and PPS-1 has a function distinct from PAPS synthesis in vivo. Understanding how lethality is caused by pps-1 and pst-1 deficiencies will shed light on the mechanisms that underlie the regulation of PAPS metabolism and sulfation in C. elegans development.
      While this manuscript was in preparation, Bülow and colleagues (
      • Bhattacharya R.
      • Townley R.A.
      • Berry K.L.
      • Bülow H.E.
      ) published a paper concentrating on the analysis of pst-1 and pst-2 functions in the nervous system of C. elegans. They show that pst-1 is essential in nervous system development and other functions, and our results are complementary to their results indicating the essentiality of PAPS transporters in the organism.

      Acknowledgments

      We thank Dr. K. Fukushima and Prof. K. Yamashita (Tokyo Institute of Technology, Yokohama, Japan) for providing the mammalian total cDNA. We also thank the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health, NCRR, for the worms and E. coli strains. We also thank Adam Kleinschmit for useful comments.

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