Abstract
Cytochrome b5 is an amphipathic mobile membrane protein that is predominantly located at the endoplasmic reticulum surface. It is an essential component of a number of membrane-bound redox systems. In animal and fungal cells cytochrome b5 is thought to be an electron donor for sterol modifying enzymes and fatty acid desaturases. Disruption of the Saccharomyces cytochrome b5 gene, however, yielded cells that had no nutritional requirement for either sterols or unsaturated fatty acids. Expression of sterol and fatty acid-modifying genes was increased in the cytochrome b5-disrupted cells, however, suggesting that cytochrome b5 may play some nonessential role in these functions. Unsaturated fatty acids in yeast are formed by Ole1p, an oxygen-dependent delta-9 fatty acid desaturase that is an intrinsic endoplasmic reticulum membrane protein. Although the yeast delta-9 fatty acid desaturase does not appear to require cytochrome b5, introduction of the rat liver stearoyl-CoA desaturase gene into an ole1-disrupted, cytochrome b5-disrupted yeast strain revealed that this enzyme specifically requires cytochrome b5 to function. Comparison of the coding sequences of the yeast and rat desaturase genes showed that the yeast protein contains a 113-amino acid carboxyl-terminal extension not found in the rat enzyme. That extension has regions of strong homology to cytochrome b5, particularly in the heme binding and electron transfer motifs. Truncation or disruption of the desaturase cytochrome b5-like domain in cells that contain the wild type diffusible b5 produced unsaturated fatty acid auxotrophy, suggesting that the cytochrome b5-like domain of Ole1p plays an essential role in the desaturase reaction.
Highlights
Cytochrome b5 is a ubiquitous eukaryotic protein that appears to be an essential component of a number of endoplasmic reticulum-linked redox enzyme systems
Cytochrome b5 appears to play some role in the regulation of these endoplasmic reticulum-based electron transport systems, as disruption of the cytochrome b5 gene causes significant changes in the level of gene expression in the cytochrome P450-like ERG11 gene and in the fatty acid desaturase gene
There were no significant differences in growth rates or differences in unsaturated fatty acid levels and fatty acid compositions between wild type and cytochrome b5-disrupted cells
Summary
The Saccharomyces cerevisiae strains used in this study were derived from W303 1a and 1b (Table I). W303–1a W303–1b DTY-10a DTY-11a AMY-1a AMY-1␣ AMY-2a AMY-2␣ AMY-3␣ AMY-4␣ AMY-5␣. Used for mating, sporulation, complementation, and construction of strains bearing the appropriate mutations [10]. Cell growth conditions and growth medium for E. coli and yeast have been previously described [11]. Yeast were transformed by electroporation (Life Technologies, Inc. cell porator) according to the manufacturer’s instructions. Standard molecular biological techniques were used as described in Refs. Standard molecular biological techniques were used as described in Refs. 12 and 13
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