Abstract
Previously, a microarray expression study in the yeast Saccharomyces cerevisiae indicated that the ERG28 gene was strongly coregulated with ergosterol biosynthesis. Subsequently, Erg28p was shown to function as an endoplasmic reticulum transmembrane protein, acting as a scaffold to tether the C-4 demethylation enzymatic complex and also to interact with a downstream enzyme, Erg6p. To understand all possible protein interactions involving Erg28p in sterol biosynthesis, a yeast two-hybrid system designed to assess interactions between membrane proteins was used. The Erg28p fusion protein was used as bait to assess interactions with all 14 sterol biosynthetic proteins in a pairwise study based on two reporter systems as well as Western blots demonstrating the release of a transcription factor. Our results indicated that Erg28p not only interacted with the C-4 demethylation enzymes and Erg6p but also with Erg11p and Erg1p. Interactions between Erg28p and seven ergosterol biosynthetic enzymes were confirmed by coimmunoprecipitation experiments. Furthermore, by comparing the reporter gene expression levels, we demonstrate that Erg28p is most closely associated with Erg27p, Erg25p, Erg11p, and Erg6p and less with Erg26p and Erg1p. Based on these results, we suggest that many if not all sterol biosynthetic proteins may be tethered as a large complex.
Highlights
A microarray expression study in the yeast Saccharomyces cerevisiae indicated that the ERG28 gene was strongly coregulated with ergosterol biosynthesis
The split-ubiquitin membrane yeast two-hybrid (YTH) system is an invaluable tool in assessing membrane protein-protein interactions because it requires a direct interaction between two proteins
In the case of ergosterol biosynthesis, virtually nothing is known regarding the topology of enzymes leading to ergosterol
Summary
A microarray expression study in the yeast Saccharomyces cerevisiae indicated that the ERG28 gene was strongly coregulated with ergosterol biosynthesis. Erg28p was shown to function as an endoplasmic reticulum transmembrane protein, acting as a scaffold to tether the C-4 demethylation enzymatic complex and to interact with a downstream enzyme, Erg6p. By comparing the reporter gene expression levels, we demonstrate that Erg28p is most closely associated with Erg27p, Erg25p, Erg11p, and Erg6p and less with Erg26p and Erg1p Based on these results, we suggest that many if not all sterol biosynthetic proteins may be tethered as a large complex.—Mo, C., and M. In a yeast microarray expression profile study, the gene YER044C encoding a 148 amino acid protein with two transmembrane domains, designated ERG28, was found to be highly coregulated with other ergosterol biosynthetic genes [8]. Because of the prevalence and conservation of ERG28 among fungi, animal, and plant sterol biosynthetic path-
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