Molecular, Functional and Evolutionary Characterization of the Gene Encoding HMG-CoA Reductase in the Fission Yeast, Schizosaccharomyces pombe

Abstract

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase, <i>hmg1+</i>, from the fission yeast <i>Schizosaccharomyces pombe</i> (Accession Number L76979). Specifically, <i>hmg1+</i> was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that <i>hmg1+</i> was an essential gene. This result provided evidence that, unlike <i>Saccharomyces cerevisiae</i>, <i>S. pombe</i> contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the <i>S. cerevisiae</i> HMG1p, the <i>hmg1+</i> protein induced membrane proliferations known as karmellae. A previously undescribed 'feed-forward' regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of <i>hmg1+</i> protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in <i>S. cerevisiae</i> occurred before or after <i>S. pombe</i> and <i>S. cerevisiae</i> diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of <i>S. pombe</i> and <i>S. cerevisiae</i> diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, <i>S. pombe</i> is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, <i>S. pombe</i> may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.