Abstract
GPI2 encodes for one of the six accessory subunits of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) complex that catalyzes the first step of GPI biosynthesis in S. cerevisiae and C. albicans. It has been previously reported in S. cerevisiae that this subunit physically interacts with and negatively modulates Ras signaling. On the other hand, studies from our lab have shown that the homologous subunit in C. albicans is a positive modulator of Ras signaling. Are the functions of this subunit therefore strictly species dependent? We present here functional complementation studies on GPI2 from S. cerevisiae and C. albicans that were carried out to address this issue. Expression of CaGPI2 in a ScGPI2 conditional lethal mutant could not restore its growth defects. Likewise, ScGPI2 overexpression in a CaGPI2 heterozygous mutant could not restore its deficient GPI-GnT activity or reverse defects in its cell wall integrity and could only poorly restore filamentation. However, interestingly, ScGPI2 could restore lanosterol demethylase (CaERG11) levels and reverse azole resistance of the CaGPI2 heterozygote. It appeared to do this by regulating levels of another GPI-GnT subunit, CaGPI19, which we have previously shown to be involved in cross-talk with CaERG11. Thus, the effect of CaGPI2 on sterol biosynthesis in C. albicans is independent of its interaction with the GPI-GnT complex and Ras signaling pathways. In addition, the interaction of Gpi2 with other subunits of the GPI-GnT complex as well as with Ras signaling appears to have evolved differently in the two organisms.
Similar content being viewed by others
References
Orlean, P., Menon, A.K.: Thematic review series: lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids. J. Lipid Res. 48, 993–1011 (2007)
Eisenhaber, B., Maurer-Stroh, S., Novatchkova, M., Schneider, G., Eisenhaber, F.: Enzymes and auxiliary factors for GPI lipid anchor biosynthesis and post-translational transfer to proteins. Bioessays. 25, 367–385 (2003)
Yadav, B., Bhatnagar, S., Ahmad, M.F., Jain, P., Pratyusha, V.A., Kumar, P., Komath, S.S.: First Step of Glycosylphosphatidylinositol (GPI) Biosynthesis Cross-talks with Ergosterol Biosynthesis and Ras Signaling in Candida albicans. J. Biol. Chem. 289, 3365–3382 (2014)
Victoria, G.S., Kumar, P., Komath, S.S.: The Candida albicans homologue of PIG-P, CaGpi19p: gene dosage and role in growth and filamentation. Microbiol. Read. Engl. 156, 3041–3051 (2010)
Victoria, G.S., Yadav, B., Hauhnar, L., Jain, P., Bhatnagar, S., Komath, S.S.: Mutual co-regulation between GPI-N-acetylglucosaminyltransferase and ergosterol biosynthesis in Candida albicans. Biochem. J. 443, 619–625 (2012)
Sobering, A.K., Watanabe, R., Romeo, M.J., Yan, B.C., Specht, C.A., Orlean, P., Riezman, H., Levin, D.E.: Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER. Cell 117, 637–648 (2004)
Newman, H.A., Romeo, M.J., Lewis, S.E., Yan, B.C., Orlean, P., Levin, D.E.: Gpi19, the Saccharomyces cerevisiae homologue of mammalian PIG-P, is a subunit of the initial enzyme for glycosylphosphatidylinositol anchor biosynthesis. Eukaryot. Cell. 4, 1801–1807 (2005)
Ito, H., Fukuda, Y., Murata, K., Kimura, A.: Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168 (1983)
Figler, R.A., Omote, H., Nakamoto, R.K., Al-Shawi, M.K.: Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein. Arch. Biochem. Biophys. 376, 34–46 (2000)
Fonzi, W.A., Irwin, M.Y.: Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728 (1993)
Ketel, C., Wang, H.S.W., McClellan, M., Bouchonville, K., Selmecki, A., Lahav, T., Gerami-Nejad, M., Berman, J.: Neocentromeres form efficiently at multiple possible loci in Candida albicans. PLoS Genet. 5, e1000400 (2009)
Nolan, T., Hands, R.E., Bustin, S.A.: Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1, 1559–1582 (2006)
Leidich, S.D., Kostova, Z., Latek, R.R., Costello, L.C., Drapp, D.A., Gray, W., Fassler, J.S., Orlean, P.: Temperature-sensitive yeast GPI anchoring mutants gpi2 and gpi3 are defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol. Cloning of the GPI2 gene. J. Biol. Chem. 270, 13029–13035 (1995)
Richard, M., Ibata-Ombetta, S., Dromer, F., Bordon-Pallier, F., Jouault, T., Gaillardin, C.: Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages. Mol. Microbiol. 44, 841–853 (2002)
Harcus, D., Nantel, A., Marcil, A., Rigby, T., Whiteway, M.: Transcription profiling of cyclic AMP signaling in Candida albicans. Mol. Biol. Cell 15, 4490–4499 (2004)
Miller, J.P., Lo, R.S., Ben-Hur, A., Desmarais, C., Stagljar, I., Noble, W.S., Fields, S.: Large-scale identification of yeast integral membrane protein interactions. Proc. Natl. Acad. Sci. 102, 12123–12128 (2005)
Acknowledgments
This work was supported by funding from Department of Biotechnology (DBT) to SSK. Partial funding by DBT-BUILDER programme to SSK is also acknowledged. We thank Dr. Marwan Al Shawi for YEpHIS vector and Prof. Alistair JP Brown for pACT1-GFP vector. SLS and BY were supported by Junior and Senior Research fellowships from ICMR and CSIR while Anshuman received support from the DBT project to SSK. BY would also like to thank DST-PURSE for a short-term fellowship. Microscopy and RT-PCR and facilities were used at the Central Instrumentation Facility, School of Life Sciences, Jawaharlal Nehru University.
Author information
Authors and Affiliations
Corresponding author
Additional information
Anshuman Yadav and Sneh Lata Singh Equal contributing authors
Rights and permissions
About this article
Cite this article
Yadav, A., Singh, S.L., Yadav, B. et al. Saccharomyces cerevisiae Gpi2, an accessory subunit of the enzyme catalyzing the first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis, selectively complements some of the functions of its homolog in Candida albicans . Glycoconj J 31, 497–507 (2014). https://doi.org/10.1007/s10719-014-9536-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10719-014-9536-8