MOLECULAR, FUNCTIONAL AND EVOLUTIONARY CHARACTERIZATION OF THE GENE ENCODING HMG-COA REDUCTASE IN THE FISSION YEAST, SCHIZOSACCHAROMYCES-POMBE

The synthesis of mevalonate, a molecule required for both sterol and i soprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase, hmg1+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Sp ecifically, hmg1+ was isolated on the basis of its ability to confer r esistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmg1+ was an essential gene. Thi s result provided evidence that, unlike Saccharomyces cerevisiae, S. p ombe contained only a single functional HMG-CoA reductase gene. The pr esence of a single HMG-CoA reductase gene was confirmed by genomic hyb ridization analysis. As observed for the S. cerevisiae HMG1p, the hmg1 + protein induced membrane proliferations known as karmellae. A previo usly undescribed 'feed-forward' regulation was observed in which eleva ted levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmg1+ pro tein in the cell and conferred partial resistance to lovastatin. The a mino acid sequences of yeast and human HMG-CoA reductase were highly d ivergent in the membrane domains, but were extensively conserved in th e catalytic domains. We tested whether the gene duplication that produ ced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specify ing post-divergence duplication had significantly higher likelihood. M oreover, phylogenetic analyses of available HMG-CoA reductase sequence s also suggested that the lineages of S. pombe and S. cerevisiae diver ged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurr ed only approximately 56 million years ago. To date, S. pombe is the o nly unicellular eukaryote that has been found to contain a single HMG- CoA reductase gene. Consequently, S. pombe may provide important oppor tunities to study aspects of the regulation of sterol biosynthesis tha t have been difficult to address in other organisms and serve as a tes t organism to identify novel therapies for modulating cholesterol synt hesis.