1887

Abstract

accumulates large amounts of the polyols glycerol and -arabitol when the cells are exposed to physiological conditions relevant to stress and virulence in animals. Intracellular concentrations of glycerol are determined by rates of glycerol production and catabolism and of glycerol uptake and efflux through the plasma membrane. We and others have studied glycerol production in , but glycerol uptake by has not been studied. In the present study, we found that [C]glycerol uptake by SC5314 was (i) accumulative; (ii) dependent on proton-motive force; (iii) unaffected by carbon source; and (iv) unaffected by large molar excesses of -arabitol or other polyols. The respective and values were 2.1 mM and 460 μmol h (g dry wt) in glucose medium and 2.6 mM and 268 μmol h (g dry wt) in glycerol medium. To identify the glycerol uptake protein(s), we cloned the homologues of the genes and , both of which are known to be involved in glycerol transport. When multicopy plasmids encoding , and were introduced into the corresponding null mutants, the transformants all acquired the ability to grow on minimal glycerol medium; however, only null mutants transformed with actively took up extracellular [C]glycerol. When both chromosomal alleles of were deleted from BWP17, the resulting null mutants grew poorly on minimal glycerol medium, and their ability to transport [C]glycerol into the cell was markedly reduced. In contrast, deletion of both chromosomal alleles of or of had no significant effects on [C]glycerol uptake or the ability to grow on minimal glycerol medium. Northern blot analysis indicated that was expressed in both glucose and glycerol media, conditions under which we detected wild-type active glycerol uptake. Furthermore, was highly expressed in salt-stressed cells; however, the null mutant was no more sensitive to salt stress than wild-type controls. We also detected high levels of expression in glycerol-grown cells, even though deletion of this gene did not influence glycerol uptake activity in glycerol-grown cells. We conclude from the results above that a plasma-membrane H symporter encoded by actively transports glycerol into cells.

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2009-05-01
2020-01-26
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References

  1. Alonso-Monge R., Navarro-Garcia F., Molero G., Diez-Orejas R., Gustin M., Pla J., Sanchez M., Nombela C.. 1999; Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans . J Bacteriol181:3058–3068
    [Google Scholar]
  2. Alonso-Monge R., Navarro-García F., Roman E., Negredo A. I., Eisman B. C., Nombela C., Pla J.. 2003; The Hog1 MAP kinase is essential in the oxidative stress response and chlamydospore formation in Candida albicans . Eukaryot Cell2:351–361
    [Google Scholar]
  3. Amberg D. C., Burke D. J., Strathern J. N.. 2005; Methods in Yeast Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
  4. Ansell R., Granath K., Hohmann S., Thevelein J. M., Adler L.. 1997; The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaption and redox regulation. EMBO J16:2179–2187
    [Google Scholar]
  5. Bernard E. M., Christiansen L. J., Tsang S. F., Kiehn T. E., Armstrong D.. 1981; Rate of arabinitol production by pathogenic yeast species. J Clin Microbiol14:189–194
    [Google Scholar]
  6. Bleve G., Zacheo G., Cappello M. S., Dellaglio F., Grieco F.. 2005; Subcellular localization and functional expression of the glycerol uptake protein 1 ( GUP1 ) of Saccharomyces cerevisiae tagged with green fluorescent protein. Biochem J390:145–155
    [Google Scholar]
  7. Bonangelino C. J., Chavez E. M., Bonifacino J. S.. 2002; Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae . Mol Biol Cell13:2486–2501
    [Google Scholar]
  8. Bosson R., Jaquenoud M., Conzelmann A.. 2006; GUP1 of Saccharomyces cerevisiae encodes an O -acyltransferase involved in remodeling of the GPI anchor. Mol Biol Cell17:2636–2645
    [Google Scholar]
  9. Carbrey J. M., Cormack B. P., Agre P.. 2001; Aquaporin in Candida , characterization of a functional water channel protein. Yeast18:1391–1396
    [Google Scholar]
  10. Castro I. M., Loureiro-Dias M. C.. 1991; Glycerol utilization in Fusarium oxysporum var. lini : regulation of transport and metabolism. J Gen Microbiol137:1497–1502
    [Google Scholar]
  11. Davis D., Edwards J. E. Jr,, Mitchell A. P., Ibrahim A. S.. 2000; Candida albicans RIM101 pH response pathway is required for host–pathogen interactions. Infect Immun68:5953–5959
    [Google Scholar]
  12. Enjalbert B., Smith D. A., Cornell M. J., Alam I., Nicholls S., Brown A. J. P., Quinn J.. 2006; Role of Hog1 stress-activated protein kinase in the global transcription response to stress in the fungal pathogen Candida albicans . Mol Biol Cell17:1018–1032
    [Google Scholar]
  13. Fan J., Whiteway M., Shen S. H.. 2005; Disruption of a gene encoding glycerol 3-phosphatase from Candida albicans impairs intracellular glycerol accumulation salt-tolerance. FEMS Microbiol Lett245:107–116
    [Google Scholar]
  14. Ferea T. L., Botstein D., Brown P. O., Rosenzweig R. F.. 1999; Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc Natl Acad Sci U S A96:9721–9726
    [Google Scholar]
  15. Ferreira C., Lucas C.. 2007; Glucose repression over Saccharomyces cerevisiae glycerol/H+ symporter gene STL1 is overcome by high temperature. FEBS Lett581:1923–1927
    [Google Scholar]
  16. Ferreira C., Lucas C.. 2008; The yeast O -acyltransferase Gup1p interferes in lipid metabolism with direct consequences on the sphingolipid-sterol-ordered domains integrity/assembly. Biochim Biophys Acta 1778;2648–2653
    [Google Scholar]
  17. Ferreira C., van Voorst F., Martins A., Neves L., Oliveira R., Kielland-Brandt M. C., Lucas C., Brandt A.. 2005; A member of the sugar transporter family, Stl1p is the glycerol/H+ symporter in Saccharomyces cerevisiae . Mol Biol Cell16:2068–2076
    [Google Scholar]
  18. Ferreira C., Silva S., van Voorst F., Aguiar C., Kielland-Brandt M. C., Brandt A., Lucas C.. 2006; Absence of Gup1p in Saccharomyces cerevisiae results in defective cell wall composition, assembly, stability and morphology. FEMS Yeast Res6:1027–1038
    [Google Scholar]
  19. Fonzi W. A., Irwin M. Y.. 1993; Isogenic strain construction and gene mapping in Candida albicans . Genetics134:717–728
    [Google Scholar]
  20. Gasch A. P., Spellman P. T., Kao C. M., Carmel-Harel O., Eisen M. B., Storz G., Botstein D., Brown P. O.. 2000; Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell11:4241–4257
    [Google Scholar]
  21. Gillum A. M., Tsay E. Y., Kirsch D. R.. 1984; Isolation of the Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura 3 and E. coli pyrF mutations. Mol Gen Genet198:179–182
    [Google Scholar]
  22. Gomes K. N., Freitas S., Pais T., Fietto J., Totola A., Arantes R., Martins A., Lucas C., Schuller D.. other authors 2005; Deficiency of Pkc1 activity affects glycerol metabolism in Saccharomyces cerevisiae . FEMS Yeast Res5:767–776
    [Google Scholar]
  23. Haurie V., Perrot M., Mini T., Jenö P., Sagliocco F., Boucherie H.. 2001; The transcriptional activator Cat8p provides a major contribution to the reprogramming of carbon metabolism during the diauxic shift in Saccharomyces cerevisiae . J Biol Chem276:76–85
    [Google Scholar]
  24. Hofmann K.. 2000; A superfamily of membrane-bound O -acyltransferases with implications for Wnt signalling. Trends Biochem Sci25:111–112
    [Google Scholar]
  25. Holst B., Lunde C., Lages F., Oliveira R., Lucas C., Kielland-Brandt M.. 2000; GUP1 and its close homologue GUP2 , encoding multimembrane-spanning proteins involved in active glycerol uptake in Saccharomyces cerevisiae . Mol Microbiol37:108–124
    [Google Scholar]
  26. Kayingo G., Wong B.. 2005; The MAP kinase Hog1p differentially regulates stress-induced production and accumulation of glycerol and d-arabitol in Candida albicans . Microbiology151:2987–2999
    [Google Scholar]
  27. Kayingo G., Bill R., Calamita G., Hohmann S., Prior B. A.. 2001a; Microbial water and glycerol channels. Curr Topics Membr51:335–370
    [Google Scholar]
  28. Kayingo G., Kilian S. G., Prior B. A.. 2001b; Conservation and release of osmolytes by yeasts during hypo-osmotic stress. Arch Microbiol177:29–55
    [Google Scholar]
  29. Kayingo G., Sirotkin V., Hohmann S., Prior B. A.. 2004; Accumulation and release of the osmolyte glycerol is independent of the putative MIP channel Spac977.17p in Schizosaccharomyces pombe . Antonie Van Leeuwenhoek85:85–92
    [Google Scholar]
  30. Kiehn T. E., Bernard M., Gold J. W. M., Armstrong D.. 1979; Candidiasis detection by gas-liquid chromatography of d-arabinitol, a fungal metabolite in human serum. Science206:577–580
    [Google Scholar]
  31. Lages F., Lucas C.. 1995; Characterization of a glycerol/H+ symport in the halotolerant yeast Pichia sorbitophila . Yeast11:111–119
    [Google Scholar]
  32. Lages F., Lucas C.. 1997; Contribution to the physiological characterization of glycerol active uptake in Saccharomyces cerevisiae . Biochim Biophys Acta1322:8–18
    [Google Scholar]
  33. Lages F., Silva-Graça M., Lucas C.. 1999; Active glycerol uptake is a mechanism underlying halotolerance in yeasts, a study of 42 species. Microbiology145:2577–2585
    [Google Scholar]
  34. Lucas C., da Costa M., van Uden N.. 1990; Osmoregulatory active sodium-glycerol co-transport in the halotolerant yeast Debaryomyces hansenii . Yeast6:187–191
    [Google Scholar]
  35. Luyten K., Albertyn J., Skibbe W. F., Prior B. A., Ramos J., Thevelein J. M., Hohmann S.. 1995; Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress. EMBO J14:1360–1371
    [Google Scholar]
  36. Nelissen B., de Wachter R., Goffeau A.. 1997; Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae . FEMS Microbiol Rev21:113–134
    [Google Scholar]
  37. Neves L., Oliveira R., Lucas C.. 2004; Yeast orthologues associated with glycerol transport and metabolism. FEMS Yeast Res5:51–62
    [Google Scholar]
  38. Ni L., Snyder M.. 2001; A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae . Mol Biol Cell12:2147–2170
    [Google Scholar]
  39. Oelkers P., Tinkelenberg A., Erdeniz N., Cromley D., Billheimer J. T., Sturley S. L.. 2000; A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast. J Biol Chem275:15609–15612
    [Google Scholar]
  40. Palma M., Goffeau A., Spencer Martins I., Baret P. V.. 2007; A phylogenetic analysis of the sugar porters in hemiascomycetous yeasts. J Mol Microbiol Biotechnol12:241–248
    [Google Scholar]
  41. Rep M., Krantz M., Thevelein J. M., Hohmann S.. 2000; The transcriptional response of Saccharomyces cerevisiae to osmotic shock. Hot1p and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathway-dependent genes. J Biol Chem275:8290–8300
    [Google Scholar]
  42. Rønnow B., Kielland-Brandt M. C.. 1993; GUT2 , a gene for mitochondrial glycerol 3-phosphate dehydrogenase of Saccharomyces cerevisiae . Yeast9:1121–1130
    [Google Scholar]
  43. Sambrook J., Russell D. W.. 2001; Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
  44. San José C., Alonso R., Pérez-Díaz R. M., Pla J., Nombela C.. 1996; The mitogen-activated protein kinase homolog HOG1 gene controls glycerol accumulation in the pathogenic fungus Candida albicans . J Bacteriol178:5850–5852
    [Google Scholar]
  45. Silva-Graça M., Lucas C.. 2003; Physiological studies on long-term adaptation to salt stress in the extremely halotolerant yeast Candida versatilis CBS 4019(syn. C. halophila ). FEMS Yeast Res3:247–260
    [Google Scholar]
  46. Tamás M. J., Luyten K., Sutherland F. C., Hernandez A., Albertyn J., Valadi H., Li H., Prior B. A., Kilian S. G.. other authors 1999; Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation. Mol Microbiol31:1087–1104
    [Google Scholar]
  47. Tamás M. J., Rep M., Thevelein J. M., Hohmann S.. 2000; Stimulation of the yeast high osmolarity glycerol ( HOG ) pathway, evidence for a signal generated by a change in turgor rather than by water stress. FEBS Lett472:159–165
    [Google Scholar]
  48. Tang X.-M., Kayingo G., Prior B. A.. 2005; Functional analysis of the Zygosaccharomyces rouxii Fps1p homologue. Yeast22:571–581
    [Google Scholar]
  49. Thomas B. J., Rothstein R. J.. 1989; Elevated recombination rates in transcriptionally active DNA. Cell56:619–630
    [Google Scholar]
  50. van Zyl P. J., Kilian S. G., Prior B. A.. 1990; The role of an active transport mechanism in glycerol accumulation during osmoregulation by Zygosaccharomyces rouxii . Appl Microbiol Biotechnol34:231–235
    [Google Scholar]
  51. Wilson R. B., Davis D., Mitchell A. P.. 1999; Rapid hypothesis testing in Candida albicans through gene disruption with short homology regions. J Bacteriol181:1868–1874
    [Google Scholar]
  52. Wilson R. B., Davis D., Enloe B. M., Mitchell A. P.. 2000; A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions. Yeast16:65–70
    [Google Scholar]
  53. Wojda I., Alonso-Monge R., Bebelman J. P., Mager W. H., Siderius M.. 2003; Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways. Microbiology149:1193–1204
    [Google Scholar]
  54. Wong B., Bernard E. M., Gold J. W. M., Fong D., Armstrong D.. 1982; The arabinitol appearance rate in laboratory animals and humans, estimation from the arabinitol/creatinine ratio and relevance to the diagnosis of candidiasis. J Infect Dis146:353–359
    [Google Scholar]
  55. Yoshimoto H., Saltsman K., Gasch A. P., Li H. X., Ogawa N., Botstein D., Brown P. O., Cyert M. S.. 2002; Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae . J Biol Chem277:31079–31088
    [Google Scholar]
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