Candida albicans Spt23p Controls the Expression of the Ole1p Δ9 Fatty Acid Desaturase and Regulates Unsaturated Fatty Acid Biosynthesis

Chan-Seok Oh,Charles E Martin

doi:10.1074/jbc.m510746200

Abstract

In Saccharomyces cerevisiae the endoplasmic reticulum membrane proteins scSpt23p and scMga2p control the formation of unsaturated fatty acids by a mechanism that involves their release from the membrane by ubiquitin-mediated proteolysis. The resulting soluble polypeptides act as transcription activators that specifically control the expression of scOLE1, a gene that encodes scOle1p, a Delta9 fatty acid desaturase that forms cis-monounsaturated fatty acids (9Z-16:1 and 9Z-18:1) from saturated fatty acyl-CoA precursors. ScOle1p is the only long chain fatty acid desaturase in Saccharomyces and its membrane and storage lipids contain only saturated fatty acids and the monounsaturated products of that enzyme. Most other fungi, however, express multiple endoplasmic reticulum desaturases, including enzymes that form both mono- and polyunsaturated fatty acids. These typically include Delta12 and Delta15 enzymes that form the polyunsaturated species, 9Z,12Z-18:2, and 9Z,12Z,15Z-18:3, which are the most abundant fatty acids in membrane and storage lipids. An analysis of genomic DNA sequences shows that Candida albicans has a single homologue of the Saccharomyces scSPT23 and scMGA2 genes that we designate here as caSPT23. This study describes the characterization of the caSPT23 gene product and shows that it can repair the unsaturated fatty acid auxotrophy when it is expressed in a Saccharomyces scspt23Delta;scmga2Delta strain. In addition we show caSPT23 is essential for the expression of one of the two Delta9 desaturase homologues in Candida and potentially other functions associated with fatty acid metabolism.

Highlights

Release from the endoplasmic reticulum by a mechanism that involves the ubiquitination of the membrane-bound precursor followed by a selective proteolytic cleavage by the 26 S proteosome [7]
Given the highly specific and essential regulatory role of the scSpt23p/scMga2p proteins in regulating unsaturated fatty acid formation, we examined the hypothesis that caSpt23p exerts a similar control over the expression of the ⌬9 enzyme and possibly other desaturases
We further show that repression of caSPT23 in Candida albicans blocks the expression of the caOLE1 desaturase, producing growth defects and loss of viability that can be repaired by a mixture of unsaturated fatty acids

Summary

EXPERIMENTAL PROCEDURES

Strains and Growth Media—C. albicans and S. cerevisiae strains and plasmids used in this study are shown in Tables 1 and 2. Saccharomyces strains were grown at 30 °C on synthetic dextrose) or SG (synthetic galactose) drop out medium as previously described [12]. Candida yeast phase cells were grown at 30 °C in synthetic dextrose or synthetic maltose drop-out medium with the addition of 80 ␮M uridine [13]. In experiments employing fatty acid supplements, the growth medium used in liquid cultures contained 0.1% Tergitol/Nonidet P-40 (Sigma) to disperse the fatty acids. Tergitol/Nonidet P-40 is not derived from fatty acids or fatty alcohols and is apparently not metabolized by Candida. Escherichia coli NovaBlue Singles௡ competent cells were obtained from Novagen. Ura3/ura, his1::hisG/his1::hisG, arg4::hisG/arg4::hisG ura3/ura, his1::hisG/his1::hisG, arg4::hisG/arg4::hisG, SPT23/spt23⌬::HIS1 ura3/ura, his1::hisG/his1::hisG, arg4::hisG/arg4::hisG, spt23⌬::HIS1;ARG4/MAL2p/SPT23 ura3/ura, his1::hisG/his1::hisG, arg4::hisG/arg4::hisG, spt23⌬::HIS1;ARG4/MAL2p/3xHA/SPT23

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RESULTS

DISCUSSION