Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Moonlighting proteins Hal3 and Vhs3 form a heteromeric PPCDC with Ykl088w in yeast CoA biosynthesis

Nature Chemical Biology volume 5, pages 920–928 (2009)Cite this article

Abstract

Unlike most other organisms, the essential five-step coenzyme A biosynthetic pathway has not been fully resolved in yeast. Specifically, the genes encoding the phosphopantothenoylcysteine decarboxylase (PPCDC) activity still remain unidentified. Sequence homology analyses suggest three candidates—Ykl088w, Hal3 and Vhs3—as putative PPCDC enzymes in Saccharomyces cerevisiae. Notably, Hal3 and Vhs3 have been characterized as negative regulatory subunits of the Ppz1 protein phosphatase. Here we show that YKL088w does not encode a third Ppz1 regulatory subunit, and that the essential roles of Ykl088w and the Hal3 and Vhs3 pair are complementary, cannot be interchanged and can be attributed to PPCDC-related functions. We demonstrate that while known eukaryotic PPCDCs are homotrimers, the active yeast enzyme is a heterotrimer that consists of Ykl088w and Hal3/Vhs3 monomers that separately provides two essential catalytic residues. Our results unveil Hal3 and Vhs3 as moonlighting proteins involved in both CoA biosynthesis and protein phosphatase regulation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Comparison of known and putative PPCDC proteins and the reaction they catalyze.
Figure 2: Functional characterization of YKL088w as a putative regulatory subunit of Ppz1.
Figure 3: In vitro activity analysis of putative yeast PPCDCs allows the proposal of a model for functional S. cerevisiae PPCDC.
Figure 4: The yeast PPCDC is a trimer consisting of Hal3, Vhs3 and Ykl088w.
Figure 5: Replacement of the sequence of Ykl088w's motif II with the sequence of the same motif from Hal3 results in a functional PPCDC protein.
Figure 6: The divergence of PPCDC monomers in yeasts.

Similar content being viewed by others

References

  1. Begley, T.P., Kinsland, C. & Strauss, E. The biosynthesis of coenzyme A in bacteria. Vitam. Horm. 61, 157–171 (2001).

    Article  CAS  Google Scholar 

  2. Mercer, A.C. & Burkart, M.D. The ubiquitous carrier protein–a window to metabolite biosynthesis. Nat. Prod. Rep. 24, 750–773 (2007).

    Article  CAS  Google Scholar 

  3. Daugherty, M. et al. Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics. J. Biol. Chem. 277, 21431–21439 (2002).

    Article  CAS  Google Scholar 

  4. Kupke, T., Hernandez-Acosta, P. & Culianez-Macia, F.A. 4′-phosphopantetheine and coenzyme A biosynthesis in plants. J. Biol. Chem. 278, 38229–38237 (2003).

    Article  CAS  Google Scholar 

  5. Ye, Y., Osterman, A., Overbeek, R. & Godzik, A. Automatic detection of subsystem/pathway variants in genome analysis. Bioinformatics 21 Suppl 1: i478–i486 (2005).

    Article  CAS  Google Scholar 

  6. Osterman, A. & Overbeek, R. Missing genes in metabolic pathways: a comparative genomics approach. Curr. Opin. Chem. Biol. 7, 238–251 (2003).

    Article  CAS  Google Scholar 

  7. Kupke, T., Hernandez-Acosta, P., Steinbacher, S. & Culianez-Macia, F.A. Arabidopsis thaliana flavoprotein AtHAL3a catalyzes the decarboxylation of 4′-Phosphopantothenoylcysteine to 4′-phosphopantetheine, a key step in coenzyme A biosynthesis. J. Biol. Chem. 276, 19190–19196 (2001).

    Article  CAS  Google Scholar 

  8. Strauss, E., Kinsland, C., Ge, Y., McLafferty, F.W. & Begley, T.P. Phosphopantothenoylcysteine synthetase from Escherichia coli. Identification and characterization of the last unidentified coenzyme A biosynthetic enzyme in bacteria. J. Biol. Chem. 276, 13513–13516 (2001).

    Article  CAS  Google Scholar 

  9. Hernandez-Acosta, P., Schmid, D.G., Jung, G., Culianez-Macia, F.A. & Kupke, T. Molecular characterization of the Arabidopsis thaliana flavoprotein AtHAL3a reveals the general reaction mechanism of 4′-phosphopantothenoylcysteine decarboxylases. J. Biol. Chem. 277, 20490–20498 (2002).

    Article  CAS  Google Scholar 

  10. Steinbacher, S. et al. Crystal structure of the plant PPC decarboxylase AtHAL3a complexed with an ene-thiol reaction intermediate. J. Mol. Biol. 327, 193–202 (2003).

    Article  CAS  Google Scholar 

  11. Strauss, E., Zhai, H., Brand, L.A., McLafferty, F.W. & Begley, T.P. Mechanistic studies on phosphopantothenoylcysteine decarboxylase: trapping of an enethiolate intermediate with a mechanism-based inactivating agent. Biochemistry 43, 15520–15533 (2004).

    Article  CAS  Google Scholar 

  12. Ferrando, A., Kron, S.J., Rios, G., Fink, G.R. & Serrano, R. Regulation of cation transport in Saccharomyces cerevisiae by the salt tolerance gene HAL3. Mol. Cell. Biol. 15, 5470–5481 (1995).

    Article  CAS  Google Scholar 

  13. Di Como, C.J., Bose, R. & Arndt, K.T. Overexpression of SIS2, which contains an extremely acidic region, increases the expression of SWI4, CLN1 and CLN2 in sit4 mutants. Genetics 139, 95–107 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Ariño, J. Novel protein phosphatases in yeast. Eur. J. Biochem. 269, 1072–1077 (2002).

    Article  Google Scholar 

  15. de Nadal, E. et al. The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase. Proc. Natl. Acad. Sci. USA 95, 7357–7362 (1998).

    Article  CAS  Google Scholar 

  16. Posas, F., Camps, M. & Ariño, J. The PPZ protein phosphatases are important determinants of salt tolerance in yeast cells. J. Biol. Chem. 270, 13036–13041 (1995).

    Article  CAS  Google Scholar 

  17. Yenush, L., Mulet, J.M., Ariño, J. & Serrano, R. The Ppz protein phosphatases are key regulators of K+ and pH homeostasis: implications for salt tolerance, cell wall integrity and cell cycle progression. EMBO J. 21, 920–929 (2002).

    Article  CAS  Google Scholar 

  18. Ruiz, A., Yenush, L. & Ariño, J. Regulation of ENA1 Na(+)-ATPase gene expression by the Ppz1 protein phosphatase is mediated by the calcineurin pathway. Eukaryot. Cell 2, 937–948 (2003).

    Article  CAS  Google Scholar 

  19. Lee, K.S., Hines, L.K. & Levin, D.E. A pair of functionally redundant yeast genes (PPZ1 and PPZ2) encoding type 1-related protein phosphatases function within the PKC1-mediated pathway. Mol. Cell. Biol. 13, 5843–5853 (1993).

    Article  CAS  Google Scholar 

  20. Merchan, S., Bernal, D., Serrano, R. & Yenush, L. Response of the Saccharomyces cerevisiae Mpk1 mitogen-activated protein kinase pathway to increases in internal turgor pressure caused by loss of Ppz protein phosphatases. Eukaryot. Cell 3, 100–107 (2004).

    Article  CAS  Google Scholar 

  21. Clotet, J., Gari, E., Aldea, M. & Ariño, J. The yeast ser/thr phosphatases sit4 and ppz1 play opposite roles in regulation of the cell cycle. Mol. Cell. Biol. 19, 2408–2415 (1999).

    Article  CAS  Google Scholar 

  22. Munoz, I., Simon, E., Casals, N., Clotet, J. & Ariño, J. Identification of multicopy suppressors of cell cycle arrest at the G1-S transition in Saccharomyces cerevisiae. Yeast 20, 157–169 (2003).

    Article  CAS  Google Scholar 

  23. Ruiz, A. et al. Functional characterization of the Saccharomyces cerevisiae VHS3 gene: a regulatory subunit of the Ppz1 protein phosphatase with novel, phosphatase-unrelated functions. J. Biol. Chem. 279, 34421–34430 (2004).

    Article  CAS  Google Scholar 

  24. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002).

    Article  CAS  Google Scholar 

  25. Espinosa-Ruiz, A., Belles, J.M., Serrano, R. & Culianez-Macia, F.A. Arabidopsis thaliana AtHAL3: a flavoprotein related to salt and osmotic tolerance and plant growth. Plant J. 20, 529–539 (1999).

    Article  CAS  Google Scholar 

  26. Albert, A. et al. The X-ray structure of the FMN-binding protein AtHal3 provides the structural basis for the activity of a regulatory subunit involved in signal transduction. Structure 8, 961–969 (2000).

    Article  CAS  Google Scholar 

  27. Strauss, E. & Begley, T.P. Mechanistic studies on phosphopantothenoylcysteine decarboxylase. J. Am. Chem. Soc. 123, 6449–6450 (2001).

    Article  CAS  Google Scholar 

  28. Strauss, E. & Begley, T.P. Stereochemical studies on phosphopantothenoylcysteine decarboxylase from Escherichia coli. Bioorg. Med. Chem. Lett. 13, 339–342 (2003).

    Article  CAS  Google Scholar 

  29. Kupke, T. & Schwarz, W. 4′-phosphopantetheine biosynthesis in Archaea. J. Biol. Chem. 281, 5435–5444 (2006).

    Article  CAS  Google Scholar 

  30. de Nadal, E., Fadden, R.P., Ruiz, A., Haystead, T. & Ariño, J. A role for the Ppz Ser/Thr protein phosphatases in the regulation of translation elongation factor 1Balpha. J. Biol. Chem. 276, 14829–14834 (2001).

    Article  CAS  Google Scholar 

  31. Costigan, C., Kolodrubetz, D. & Snyder, M. NHP6A and NHP6B, which encode HMG1-like proteins, are candidates for downstream components of the yeast SLT2 mitogen-activated protein kinase pathway. Mol. Cell. Biol. 14, 2391–2403 (1994).

    Article  CAS  Google Scholar 

  32. Gavin, A.C. et al. Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631–636 (2006).

    Article  CAS  Google Scholar 

  33. Brown, M.T., Goetsch, L. & Hartwell, L.H. MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae. J. Cell Biol. 123, 387–403 (1993).

    Article  CAS  Google Scholar 

  34. Kupke, T. et al. Molecular characterization of lantibiotic-synthesizing enzyme EpiD reveals a function for bacterial Dfp proteins in coenzyme A biosynthesis. J. Biol. Chem. 275, 31838–31846 (2000).

    Article  CAS  Google Scholar 

  35. Hazbun, T.R. et al. Assigning function to yeast proteins by integration of technologies. Mol. Cell 12, 1353–1365 (2003).

    Article  CAS  Google Scholar 

  36. Krogan, N.J. et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440, 637–643 (2006).

    Article  CAS  Google Scholar 

  37. Collins, S.R. et al. Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae. Mol. Cell. Proteomics 6, 439–450 (2007).

    Article  CAS  Google Scholar 

  38. Spitzer, E.D. & Weiss, B. dfp Gene of Escherichia coli K-12, a locus affecting DNA synthesis, codes for a flavoprotein. J. Bacteriol. 164, 994–1003 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Spitzer, E.D., Jimenez-Billini, H.E. & Weiss, B. beta-Alanine auxotrophy associated with dfp, a locus affecting DNA synthesis in Escherichia coli. J. Bacteriol. 170, 872–876 (1988).

    Article  CAS  Google Scholar 

  40. Dujon, B. Yeasts illustrate the molecular mechanisms of eukaryotic genome evolution. Trends Genet. 22, 375–387 (2006).

    Article  CAS  Google Scholar 

  41. Munoz, I. et al. Functional characterization of the yeast Ppz1 phosphatase inhibitory subunit Hal3: a mutagenesis study. J. Biol. Chem. 279, 42619–42627 (2004).

    Article  CAS  Google Scholar 

  42. Bucovaz, E.T., Macleod, R.M., Morrison, J.C. & Whybrew, W.D. The coenzyme A-synthesizing protein complex and its proposed role in CoA biosynthesis in bakers' yeast. Biochimie 79, 787–798 (1997).

    Article  CAS  Google Scholar 

  43. Bucovaz, E.T. et al. Coenzyme A-synthesizing protein complex of Saccharomyces cerevisiae. Mol. Cell. Biochem. 30, 7–26 (1980).

    Article  CAS  Google Scholar 

  44. Olzhausen, J., Schubbe, S. & Schuller, H.J. Genetic analysis of coenzyme A biosynthesis in the yeast Saccharomyces cerevisiae: identification of a conditional mutation in the pantothenate kinase gene CAB1. Curr. Genet. 55, 163–173 (2009).

    Article  CAS  Google Scholar 

  45. Gancedo, C. & Flores, C.L. Moonlighting proteins in yeasts. Microbiol. Mol. Biol. Rev. 72, 197–210 (2008).

    Article  CAS  Google Scholar 

  46. Adams, A., Gottschling, D.E., Kaiser, C.A. & Stearns, T. Methods in Yeast Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1997).

  47. Treco, D.A. & Winston, F. Growth and manipulation of yeast. in Current Protocols in Molecular Biology (eds. Ausubel, F.M. et al.) 13.2.10–13.2.12 (John Wiley & Sons, New York, 1998).

    Google Scholar 

  48. Garcia-Gimeno, M.A., Munoz, I., Ariño, J. & Sanz, P. Molecular characterization of Ypi1, a novel Saccharomyces cerevisiae type 1 protein phosphatase inhibitor. J. Biol. Chem. 278, 47744–47752 (2003).

    Article  CAS  Google Scholar 

  49. Meyer, C.R., Rustin, P. & Wedding, R.T. A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity. Plant Physiol. 86, 325–328 (1988).

    Article  CAS  Google Scholar 

  50. Choi, J.H., Jung, H.Y., Kim, H.S. & Cho, H.G. PhyloDraw: a phylogenetic tree drawing system. Bioinformatics 16, 1056–1058 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Thanks are given to B. Weiss (Emory University School of Medicine) for E. coli strain BW369, to A. Osterman (Burnham Institute for Medical Research) for the human PPCDC cDNA and to R. Serrano (Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas) and P. Sanz (Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas) for diverse constructs and plasmids. We thank H. Ceulemans, E. Simón, A. Barceló and A. Vivó for their contribution in different phases of the project. The excellent technical assistance of A. Vilalta, M.J. Álvarez and M. Robledo is acknowledged. This work was supported by grants BFU2005-06388-C4-04-BMC and BFU2008-04188-C03-01 to J.A. (Ministerio de Educación y Ciencia, Spain and Fondo Europeo de Desarrollo Regional) and grant FA2007041600013 to E.S. (National Research Foundation, South Africa). J.A. is recipient of an “Ajut de Suport a les Activitats dels Grups de Recerca” (2009SGR-1091, Generalitat de Catalunya). A.G. was recipient of a fellowship from the Spanish Ministry of Education and Science. J.A. and E.S. are recipients of a Spain/South Africa Research Cooperation grant (HS2007-0022) from the National Research Foundation (South Africa) and the Ministerio de Educación y Ciencia (Spain). J.A. dedicates this paper to the memory of his father.

Author information

Author notes

  1. Amparo Ruiz, Ivan Muñoz & Raquel Serrano

    Present address: Present addresses: Department of Genetics and Development and Department of Microbiology and Immunology, Columbia University, New York, New York, USA (A.R.); Medical Research Council Protein Phosphorylation Unit, Sir James Black Centre, University of Dundee, Dundee, Scotland, UK (I.M.); Department of Biological Sciences, Stanford University, Stanford, California, USA (R.S.).,

  2. Amparo Ruiz and Asier González: These authors contributed equally to this work.

Authors and Affiliations

  1. Departament de Bioquimica i Biologia Molecular, Universitat Autònoma de Barcelona,, Barcelona, Spain

    Amparo Ruiz, Asier González, Ivan Muñoz, Raquel Serrano & Joaquín Ariño

  2. Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain

    Asier González & Joaquín Ariño

  3. Department of Biochemistry, Stellenbosch University, Stellenbosch, South Africa

    J Albert Abrie & Erick Strauss

Authors
  1. Amparo Ruiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Asier González
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ivan Muñoz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Raquel Serrano
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J Albert Abrie
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Erick Strauss
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Joaquín Ariño
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.R. performed genetic, phenotypic and biochemical experiments. A.G. performed genetic experiments, prepared recombinant proteins and carried out binding and cross-linking experiments and in vitro activity measurements. I.M. and R.S. performed genetic experiments. J.A.A. prepared recombinant proteins and performed in vitro activity measurements. E.S. and J.A. directed the research and co-wrote the paper.

Corresponding author

Correspondence to Joaquín Ariño.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–3 and Supplementary Methods (PDF 799 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ruiz, A., González, A., Muñoz, I. et al. Moonlighting proteins Hal3 and Vhs3 form a heteromeric PPCDC with Ykl088w in yeast CoA biosynthesis. Nat Chem Biol 5, 920–928 (2009). https://doi.org/10.1038/nchembio.243

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.243

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing