Transcriptional Profiling Identifies Two Members of the ATP-binding Cassette Transporter Superfamily Required for Sterol Uptake in Yeast

Abstract

In contrast to lipoprotein-mediated sterol uptake, free sterol influx by eukaryotic cells is poorly understood. To identify components of non-lipoprotein-mediated sterol uptake, we utilized strains of Saccharomyces cerevisiae that accumulate exogenous sterol due to a neomorphic mutation in the transcription factor, UPC2. Two congenic upc2-1strains, differing quantitatively in aerobic sterol uptake due to a modifying mutation in the HAP1 transcription factor, were compared using DNA microarrays. We identified 9 genes as responsive toUPC2 that were also induced under anaerobiosis, when sterol uptake is essential. Deletion mutants in these genes were assessed for sterol influx in the upc2-1 background. UPC2itself was up-regulated under these conditions and was required for aerobic sterol influx. Deletion of the ATP-binding cassette transporters YOR011w (AUS1) orPDR11, or a putative cell wall protein encoded byDAN1, significantly reduced sterol influx. Sodium azide and vanadate inhibited sterol uptake, consistent with the participation of ATP-binding cassette transporters. We hypothesized that the physiological role of Aus1p and Pdr11p is to mediate sterol uptake when sterol biosynthesis is compromised. Accordingly, expression ofAUS1 or PDR11 was required for anaerobic growth and sterol uptake. We proposed similar molecules may be important components of sterol uptake in all eukaryotes. In contrast to lipoprotein-mediated sterol uptake, free sterol influx by eukaryotic cells is poorly understood. To identify components of non-lipoprotein-mediated sterol uptake, we utilized strains of Saccharomyces cerevisiae that accumulate exogenous sterol due to a neomorphic mutation in the transcription factor, UPC2. Two congenic upc2-1strains, differing quantitatively in aerobic sterol uptake due to a modifying mutation in the HAP1 transcription factor, were compared using DNA microarrays. We identified 9 genes as responsive toUPC2 that were also induced under anaerobiosis, when sterol uptake is essential. Deletion mutants in these genes were assessed for sterol influx in the upc2-1 background. UPC2itself was up-regulated under these conditions and was required for aerobic sterol influx. Deletion of the ATP-binding cassette transporters YOR011w (AUS1) orPDR11, or a putative cell wall protein encoded byDAN1, significantly reduced sterol influx. Sodium azide and vanadate inhibited sterol uptake, consistent with the participation of ATP-binding cassette transporters. We hypothesized that the physiological role of Aus1p and Pdr11p is to mediate sterol uptake when sterol biosynthesis is compromised. Accordingly, expression ofAUS1 or PDR11 was required for anaerobic growth and sterol uptake. We proposed similar molecules may be important components of sterol uptake in all eukaryotes. ATP-binding cassette open reading frame Unesterified sterol is an essential component of all eukaryotic membranes where it influences membrane fluidity and the activity and localization of many proteins. In mammalian cells, the processes that control the supply of this commodity are precisely regulated, primarily at the transcriptional level. Although the endogenous synthesis and storage (as ester) of cholesterol are critical aspects of this homeostasis, it is the uptake of exogenous cholesterol that presents the greatest challenge to this equilibrium. The endocytosis of cholesterol-containing low density lipoprotein particles by the low density lipoprotein receptor or the selective uptake of cholesteryl ester from high density lipoproteins by the scavenger receptor type BI are transcriptionally regulated events (1Krieger M. Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (458) Google Scholar, 2Repa J.J. Turley S.D. Lobaccaro J.A. Medina J., Li, L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1144) Google Scholar, 3Chinetti G. Gbaguidi F.G. Griglio S. Mallat Z. Antonucci M. Poulain P. Chapman J. Fruchart J.C. Tedgui A. Najib-Fruchart J. Staels B. Circulation. 2000; 101: 2411-2417Crossref PubMed Scopus (385) Google Scholar). The reverse process of sterol efflux to high density lipoproteins requires ABCA1, a member of the ATP-binding cassette (ABC)1 family of membrane transporters which is also regulated transcriptionally (4Schmitz G. Kaminski W.E. Orso E. Curr. Opin. Lipidol. 2000; 11: 493-501Crossref PubMed Scopus (115) Google Scholar). In contrast, the mechanisms and regulation of non-lipoprotein-mediated free sterol influx are poorly understood. This saturable process is of major relevance to the uptake of dietary cholesterol from bile acid micelles into the brush border membranes of enterocytes (5Hernandez M. Montenegro J. Steiner M. Kim D. Sparrow C. Detmers P.A. Wright S.D. Chao Y.S. Biochim. Biophys. Acta. 2000; 1486: 232-242Crossref PubMed Scopus (55) Google Scholar). However, the molecular components of this phenomenon have yet to be identified despite the observation that it can be inhibited by small molecules such as sterol glycosides (6Detmers P.A. Patel S. Hernandez M. Montenegro J. Lisnock J.M. Pikounis B. Steiner M. Kim D. Sparrow C. Chao Y.S. Wright S.D. Biochim. Biophys. Acta. 2000; 1486: 243-252Crossref PubMed Scopus (48) Google Scholar).The budding yeast, Saccharomyces cerevisiae, exhibits many aspects of sterol homeostasis in common with higher eukaryotes and has been a useful model for the study of sterol trafficking (7Sturley S.L. Curr. Opin. Lipidol. 1998; 9: 85-91Crossref PubMed Scopus (12) Google Scholar, 8Sturley S.L. Biochim. Biophys. Acta. 2000; 1529: 155-163Crossref PubMed Scopus (71) Google Scholar). Normal or “wild-type” strains of S. cerevisiae do not significantly accumulate exogenous sterol from the media when grown aerobically but, instead, satisfy their sterol requirements by way of ergosterol biosynthesis. This phenomenon is referred to as aerobic sterol exclusion (9Lorenz R.T. Parks L.W. Lipids. 1991; 26: 598-603Crossref PubMed Scopus (55) Google Scholar). However, when sterol biosynthesis is compromised, for example by anaerobic growth, sterol uptake is required for viability (10Gollub E.G. Trocha P. Liu P.K. Sprinson D.B. Biochem. Biophys. Res. Commun. 1974; 56: 471-477Crossref PubMed Scopus (30) Google Scholar). Completely anaerobic systems are difficult to maintain and allow for little experimental manipulation. For this reason, studies of sterol influx in yeast have primarily employed sterol auxotrophs or mutants in heme synthesis which accumulate sterol from the media when grown aerobically (11Lorenz R.T. Rodriguez R.J. Lewis T.A. Parks L.W. J. Bacteriol. 1986; 167: 981-985Crossref PubMed Google Scholar). In the background of heme and sterol competence, aerobic sterol influx has been achieved using theupc2-1 mutant strain (12Lewis T.L. Keesler G.A. Fenner G.P. Parks L.W. Yeast. 1988; 4: 93-106Crossref PubMed Scopus (61) Google Scholar), the molecular basis of which is a single nucleotide change in the UPC2 (UPtakeControl) open reading frame (13Crowley J.H. Leak F.W., Jr. Shianna K.V. Tove S. Parks L.W. J. Bacteriol. 1998; 180: 4177-4183Crossref PubMed Google Scholar). UPC2 encodes a member of a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain. Upc2p activates transcription via binding to sterol regulatory elements of some enzymes of ergosterol biosynthesis (14Vik A. Rine J. Mol. Cell. Biol. 2001; 21: 6395-6405Crossref PubMed Scopus (196) Google Scholar). Deletion of the UPC2 gene does not result in sterol uptake, suggesting that the upc2-1 allele (a glycine to aspartic acid change at residue 888) is a gain-of-function mutation (13Crowley J.H. Leak F.W., Jr. Shianna K.V. Tove S. Parks L.W. J. Bacteriol. 1998; 180: 4177-4183Crossref PubMed Google Scholar).We hypothesized that sterol uptake in yeast results from altered transcriptional regulation of one or more targets of UPC2. We therefore conducted a genome-wide transcriptional analysis of normal and upc2-1 mutant strains grown under aerobic conditions. By virtue of this expression profiling, we have identified a subset of 9 genes regulated by UPC2 and induced by anaerobiosis that are putative components of free sterol influx. Based on the null phenotypes of each candidate gene, the major determinants of sterol uptake are two members of the ABC superfamily of membrane transporters, AUS1 andPDR11. Unesterified sterol is an essential component of all eukaryotic membranes where it influences membrane fluidity and the activity and localization of many proteins. In mammalian cells, the processes that control the supply of this commodity are precisely regulated, primarily at the transcriptional level. Although the endogenous synthesis and storage (as ester) of cholesterol are critical aspects of this homeostasis, it is the uptake of exogenous cholesterol that presents the greatest challenge to this equilibrium. The endocytosis of cholesterol-containing low density lipoprotein particles by the low density lipoprotein receptor or the selective uptake of cholesteryl ester from high density lipoproteins by the scavenger receptor type BI are transcriptionally regulated events (1Krieger M. Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (458) Google Scholar, 2Repa J.J. Turley S.D. Lobaccaro J.A. Medina J., Li, L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1144) Google Scholar, 3Chinetti G. Gbaguidi F.G. Griglio S. Mallat Z. Antonucci M. Poulain P. Chapman J. Fruchart J.C. Tedgui A. Najib-Fruchart J. Staels B. Circulation. 2000; 101: 2411-2417Crossref PubMed Scopus (385) Google Scholar). The reverse process of sterol efflux to high density lipoproteins requires ABCA1, a member of the ATP-binding cassette (ABC)1 family of membrane transporters which is also regulated transcriptionally (4Schmitz G. Kaminski W.E. Orso E. Curr. Opin. Lipidol. 2000; 11: 493-501Crossref PubMed Scopus (115) Google Scholar). In contrast, the mechanisms and regulation of non-lipoprotein-mediated free sterol influx are poorly understood. This saturable process is of major relevance to the uptake of dietary cholesterol from bile acid micelles into the brush border membranes of enterocytes (5Hernandez M. Montenegro J. Steiner M. Kim D. Sparrow C. Detmers P.A. Wright S.D. Chao Y.S. Biochim. Biophys. Acta. 2000; 1486: 232-242Crossref PubMed Scopus (55) Google Scholar). However, the molecular components of this phenomenon have yet to be identified despite the observation that it can be inhibited by small molecules such as sterol glycosides (6Detmers P.A. Patel S. Hernandez M. Montenegro J. Lisnock J.M. Pikounis B. Steiner M. Kim D. Sparrow C. Chao Y.S. Wright S.D. Biochim. Biophys. Acta. 2000; 1486: 243-252Crossref PubMed Scopus (48) Google Scholar). The budding yeast, Saccharomyces cerevisiae, exhibits many aspects of sterol homeostasis in common with higher eukaryotes and has been a useful model for the study of sterol trafficking (7Sturley S.L. Curr. Opin. Lipidol. 1998; 9: 85-91Crossref PubMed Scopus (12) Google Scholar, 8Sturley S.L. Biochim. Biophys. Acta. 2000; 1529: 155-163Crossref PubMed Scopus (71) Google Scholar). Normal or “wild-type” strains of S. cerevisiae do not significantly accumulate exogenous sterol from the media when grown aerobically but, instead, satisfy their sterol requirements by way of ergosterol biosynthesis. This phenomenon is referred to as aerobic sterol exclusion (9Lorenz R.T. Parks L.W. Lipids. 1991; 26: 598-603Crossref PubMed Scopus (55) Google Scholar). However, when sterol biosynthesis is compromised, for example by anaerobic growth, sterol uptake is required for viability (10Gollub E.G. Trocha P. Liu P.K. Sprinson D.B. Biochem. Biophys. Res. Commun. 1974; 56: 471-477Crossref PubMed Scopus (30) Google Scholar). Completely anaerobic systems are difficult to maintain and allow for little experimental manipulation. For this reason, studies of sterol influx in yeast have primarily employed sterol auxotrophs or mutants in heme synthesis which accumulate sterol from the media when grown aerobically (11Lorenz R.T. Rodriguez R.J. Lewis T.A. Parks L.W. J. Bacteriol. 1986; 167: 981-985Crossref PubMed Google Scholar). In the background of heme and sterol competence, aerobic sterol influx has been achieved using theupc2-1 mutant strain (12Lewis T.L. Keesler G.A. Fenner G.P. Parks L.W. Yeast. 1988; 4: 93-106Crossref PubMed Scopus (61) Google Scholar), the molecular basis of which is a single nucleotide change in the UPC2 (UPtakeControl) open reading frame (13Crowley J.H. Leak F.W., Jr. Shianna K.V. Tove S. Parks L.W. J. Bacteriol. 1998; 180: 4177-4183Crossref PubMed Google Scholar). UPC2 encodes a member of a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain. Upc2p activates transcription via binding to sterol regulatory elements of some enzymes of ergosterol biosynthesis (14Vik A. Rine J. Mol. Cell. Biol. 2001; 21: 6395-6405Crossref PubMed Scopus (196) Google Scholar). Deletion of the UPC2 gene does not result in sterol uptake, suggesting that the upc2-1 allele (a glycine to aspartic acid change at residue 888) is a gain-of-function mutation (13Crowley J.H. Leak F.W., Jr. Shianna K.V. Tove S. Parks L.W. J. Bacteriol. 1998; 180: 4177-4183Crossref PubMed Google Scholar). We hypothesized that sterol uptake in yeast results from altered transcriptional regulation of one or more targets of UPC2. We therefore conducted a genome-wide transcriptional analysis of normal and upc2-1 mutant strains grown under aerobic conditions. By virtue of this expression profiling, we have identified a subset of 9 genes regulated by UPC2 and induced by anaerobiosis that are putative components of free sterol influx. Based on the null phenotypes of each candidate gene, the major determinants of sterol uptake are two members of the ABC superfamily of membrane transporters, AUS1 andPDR11. We thank Vladin Miljkovic of the GeneChip array facility of the Institute of Cancer Genetics for expert assistance.