The Membrane Proteins, Spt23p and Mga2p, Play Distinct Roles in the Activation of Saccharomyces cerevisiae OLE1 Gene Expression

Abstract

The Saccharomyces OLE1 gene encodes the ⌬-9 fatty acid desaturase, an enzyme that converts saturated fatty acyl-CoAs into cis-⌬-9 unsaturated fatty acids. OLE1 gene expression is regulated by unsaturated fatty acids, which repress transcription and destabilize the OLE1 mRNA. Expression of OLE1 is activated by N-terminal proteolytic fragments of two homologous endoplasmic reticulum membrane proteins, Spt23p and Mga2p. Disruption of either gene does not significantly affect cell growth or fatty acid metabolism; cells that contain null alleles of both genes, however, are unsaturated fatty acid auxotrophs. An analysis of spt23⌬ and mga2⌬ strains shows that Spt23p and Mga2p differentially activate and regulate OLE1 transcription. In glucose-grown cells, both genes activate transcription to similar levels of activity. Expressed alone, Mga2p induces high levels of OLE1 transcription in cells exposed to cobalt or grown in glycerolcontaining medium. Spt23p expressed alone activates OLE1 transcription to levels similar to those in wild type cells. OLE1 expression is strongly repressed by unsaturated fatty acids in spt23⌬ or mga2⌬ cells, under all growth conditions. To test if OLE1 expression is controlled by fatty acids at the level of membrane proteolysis, soluble N-terminal fragments of Spt23p and Mga2p that lack their membrane-spanning regions (⌬tm) were expressed under the control of their native promoters in spt23⌬;mga2⌬ cells. Under those conditions, Mga2p⌬tm acts as a powerful transcription activator that is strongly repressed by unsaturated fatty acids. By comparison, the Spt23p⌬tm polypeptide weakly activates transcription and shows little regulation by unsaturated fatty acids. Co-expression of the two soluble fragments results in activation to levels observed with the Mga2p⌬tm protein alone. The fatty acid repression of transcription under those conditions is attenuated by Spt23⌬tm, however, suggesting that the two proteins may interact to modulate OLE1 gene expression.

Regulation of Unsaturated Fatty Acid Biosynthesis in Saccharomyces

“…Although neither protein appears to have a DNA binding domain, the N-terminal fragments specifically activate OLE1 transcription presumably by influencing chromatin structure or by associating with DNA binding proteins that are bound to elements on the OLE1 promoter (5,6). Disruption of either SPT23 or MGA2 does not affect the growth rate or production of fatty acids under normal laboratory growth conditions (7). Disruption of both genes, however, blocks expression of Ole1p and results in a synthetic auxotrophy that can be overcome by adding unsaturated fatty acids to the medium or expressing OLE1 under a different promoter (4).…”

“…Spt23p and Mga2p are intrinsic ER membrane proteins that are integrated into the membrane through a C-terminal membrane spanning domain (4,7). Both proteins are cleaved by a novel ubiquitin-mediated mechanism that releases soluble N-terminal fragments from the membrane (4).…”

Overexpression of OLE1 Enhances Cytoplasmic Membrane Stability and Confers Resistance to Cadmium in Saccharomyces cerevisiae

“…In support of the aforementioned hypothesis, the correlation between OLE1 expression levels and cadmium resistance was investigated. Because the OLE1 null mutant is nonviable under standard conditions and a deficiency of MGA2 or SPT23 (important transcriptional regulators of OLE1) results in serious reduction of OLE1 expression levels (20), cadmium resistance tests were carried out with mga2Δ and spt23Δ mutants. As shown in Fig.…”

Maintenance and Regulation of mRNA Stability of the Saccharomyces cerevisiae OLE1 Gene Requires Multiple Elements within the Transcript That Act through Translation-independent Mechanisms

“…8. In a second reaction the OLE1-coding sequence beyond the start of TM regions (ϩ301 bp) and OLE1 3Ј-UTR (1 kb) was amplified using primers prMV132 and prMV048 from plasmid pMV071.…”

“…In the yeast Saccharomyces cerevisiae, unsaturated fatty acids are formed by Ole1p, a membrane-bound ⌬-9 desaturase that converts long chain saturated fatty acyl-CoA substrates into monounsaturated species (1,2). Our laboratory has shown that the OLE1 gene is regulated by a number of mechanisms, including transcriptional and mRNA stability controls (3)(4)(5)(6)(7)(8).…”