Academic journal article Genetics

Cumulative Mutations Affecting Sterol Biosynthesis in the Yeast Saccharomyces Cerevisiae Result in Synthetic Lethality That Is Suppressed by Alterations in Sphingolipid Profiles

Academic journal article Genetics

Cumulative Mutations Affecting Sterol Biosynthesis in the Yeast Saccharomyces Cerevisiae Result in Synthetic Lethality That Is Suppressed by Alterations in Sphingolipid Profiles

Article excerpt

ABSTRACT

UPC2 and ECM22 belong to a Zn(2)-Cys(6) family of fungal transcription factors and have been implicated in the regulation of sterol synthesis in Saccharomyces cerevisiae and Candida albicans. Previous reports suggest that double deletion of these genes in S. cerevisiae is lethal depending on the genetic background of the strain. In this investigation we demonstrate that lethality of upc2Δ ecm22Δ in the S288c genetic background is attributable to a mutation in the HAP1 transcription factor. In addition we demonstrate that strains containing upc2Δ ecm22Δ are also inviable when carrying deletions of ERG6 and ERG28 but not when carrying deletions of ERG3, ERG4, or ERG5. It has previously been demonstrated that UPC2 and ECM22 regulate S. cerevisiae ERG2 and ERG3 and that the erg2Δ upc2Δ ecm22Δ triple mutant is also synthetically lethal. We used transposon mutagenesis to isolate viable suppressors of hap1Δ, erg2Δ, erg6Δ, and erg28Δ in the upc2Δ ecm22Δ genetic background. Mutations in two genes (YND1 and GDA1) encoding apyrases were found to suppress the synthetic lethality of three of these triple mutants but not erg2Δ upc2Δ ecm22Δ. We show that deletion of YND1, like deletion of GDA1, alters the sphingolipid profiles, suggesting that changes in sphingolipids compensate for lethality produced by changes in sterol composition and abundance.

THE yeast Saccharomyces cerevisiae has provided a powerful model system to study the biochemistry of lipid biosynthesis. Many genes encoding the enzymes for sterol, fatty acid, phospholipid, and sphingolipid synthesis have been isolated, their products characterized, and the orthologous genes in human cells identified (DAUM et al. 1998; KELLEY and HERMAN 2001). However, less is known about the regulation of lipid synthesis and transport between organelles. Recently, two transcription factors, Upc2p and Ecm22p, were implicated in the coordination of these processes in this model organism (VIK and RINE 2001; WILCOX et al. 2002).

Upc2p and Ecm22p are members of the Zn[2]-CyS[6] binuclear cluster family of transcription factors (TODD and ANDRIANOPOULOS 1997) and share significant amino acid sequence identity (45%). A semidominant allele of UPC2 (upc2-1) was demonstrated to confer aerobic sterol influx, a process normally restricted to anaerobiosis (LEWIS et al. 1988; CROWLEY et al. 1998), while a lesser role of Ecm22p in sterol transport has also been reported (SHIANNA et al. 2001). Microarray analyses of the upc2-1 mutant identified novel genes involved in sterol influx, including AUS1 and PDR11, which encode ABC transporters required for anaerobic sterol influx (WILCOX et al. 2002).

In addition to a role in sterol transport, an involvement of Upc2p and Ecm22p in regulating sterol biosynthesis has been suggested (VIK and RINE 2000). Both transcription factors have been referred to as sterol regulatory element (SRE)binding proteins (SREBPs), responsible for regulating the transcription of the ergosterol biosynthetic genes ERG2 and ERG3 via binding to an 11-bp SRE (VIK and RINE 2001). However, these proteins lack sequence conservation with the analogous mammalian or recently identified Schizosaccharomyces pombe SREBPs (BROWN and GOLDSTEIN 1997; HUGHES et al. 2005); whether they exhibit functional similarity remains to be established. Despite this, a large number of ergosterol biosynthetic genes, such as ERG1, ERG6, ERG8, ERG12, ERG13, and ERG25 contain this SRE and are likely under UPC2/ECM22 transcriptional control. In Candida albicans, loss of UPC2 resulted in an inability to upregulate ERG2 and ERG11 in response to azole treatment. Similarly, a 7-bp SRE core element also appears in many of the same ergosterol biosynthetic genes in C. albicans (SILVEr et al. 2004).

Upc2p has further been proposed to regulate the transcription of a number of anaerobically expressed genes (KWAST et al. 1998). For example, Upc2p induces the expression of the anaerobic cell wall-related genes in the PAU family (ABRAMOVA et al. …

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