Submitted on April 19, 2005
Revised on June 23, 2005
Accepted on June 23, 2005
Erg28p is a key protein in the yeast sterol biosynthetic enzyme complex
Caiqing Mo and Martin Bard
Biology Dept., Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202
Corresponding Author: mbard{at}iupui.edu
Previously, a microarray expression study in the yeast Saccharomyces cerevisiae indicated that the ERG28 gene was strongly co-regulated with ergosterol biosynthesis. Subsequently Erg28p was shown to function as an ER transmembrane protein, acting as a scaffold to tether the C-4 demethylation enzymatic complex and also to interact with a downstream enzyme, Erg6p. In order to understand all possible protein interactions involving Erg28p in sterol biosynthesis, a yeast two-hybrid system designed to assess interactions between membrane proteins was employed. The Erg28p fusion protein was used as bait, to assess interactions with all fourteen sterol biosynthetic proteins in a pairwise study based on a two reporter systems as well as western blots demonstrating release of a transcription factor. Our results indicated that Erg28p not only interacted with the C-4 demethylation enzymes, and Erg6p, but also Erg11p and Erg1p. Interactions between Erg28p and seven ergosterol biosynthetic enzymes were confirmed by coimmunoprecipitation experiments. Further, 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.
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