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Originally published In Press as doi:10.1194/jlr.M500153-JLR200 on July 1, 2005

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Journal of Lipid Research, Vol. 46, 1991-1998, September 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Erg28p is a key protein in the yeast sterol biosynthetic enzyme complex

Caiqing Mo and Martin Bard1

Biology Department, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202

Published, JLR Papers in Press, July 1, 2005. DOI 10.1194/jlr.M500153-JLR200

1 To whom correspondence should be addressed. e-mail: mbard{at}iupui.edu

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.

Abbreviations: 3-AT, 3-aminotriazole; CSM, complete synthetic medium; Cub, C-terminal ubiquitin; ER, endoplasmic reticulum; HA, peptide from human hemagglutinin; Nub, N-terminal ubiquitin; TF, transcription factor containing protein A-LexA-VP16; X-Gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside; YTH, yeast two-hybrid

Supplementary key words ergosterol biosynthesis • membrane proteins • split-ubiquitin membrane yeast two-hybrid system


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