Squalene synthetase (farnesyl-diphosphate: farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) is a critical branch point enzyme of isoprenoid biosynthesis that is thought to regulate the flux of isoprene intermediates through the sterol pathway. The structural gene for this squalene by a two-step reductive condensation of two molecules of FDP. It has been isolated from many sources and its reaction mechanism has been investigated in some detail (3, 4). Squalene synthetase solubilized from membranes of bakers' yeast, Saccharomyces cerevisiae, has been described (4-8) and the purified enzyme was reported to have a molecular mass of "50 kDa (5-8). Yeast with defects in squalene synthetase were identified by screening a collection of mutants blocked in ergosterol biosynthesis (9). Mutants designated erg9 were unable to convert radiolabeled mevalonate into squalene but did accumulate farnesol, a breakdown product of FDP. Because these squalene synthetase mutants were also obligate ergosterol auxotrophs, they were used in a phenotype complementation approach to clone the ERG9 gene. In this report, we describe the structure of squalene synthetase deduced from its primary sequence and explore the consequences of genomic deletion of this gene in S. cerevisiae. * MATERIALS AND METHODSStrains. The following strains of S. cerevisiae were used in this study: DC67 (MA Ta ERG9 leu24 adel lys2 cir,) was from J. Hicks; W303-1A (MA Ta) and W303-1B (MATa) (both ERG9 leu2-3,112 ade2-1 ura3-1 trpl-J his3-11,15) were from R. Rothstein; SGY336 (MATa erg9-1) was from F. Karst; JRY527 (MATa ERG9 ura3-52 ade2-101 his3-d200 lys2-801 Met-) was from J. Rine; SC14089 (MATa ERG9) was from J.Tkacz; SGY1161 an ERG9/ERG9 diploid was from a mating of W303-1A and W303-1B; SGY969 an ERG9/erg9 diploid was constructed from SGY336 by mating to W303-1B; SGY1011 (MATa erg9 leu2-3,112 ade2-1 ura3-1 his3-11,15) was a segregant from SGY969. Standard Plasmid-borne disruptions of the ERG9 gene were constructed by replacement of a 1.0-kb EcoRI/Sca I fragment of

Squalene synthetase (farnesyl diphosphate:farnesyl diphosphate farnesyltransferase; EC 2.5.1.21) is thought to represent a major control point of isoprene and sterol biosynthesis in eukaryotes. We demonstrate structural and functional conservation between the enzymes from humans, a budding yeast (Saccharomyces cerevisiae), and a fission yeast (Schizosaccharomyces pombe). The amino acid sequences of the human and S. pombe proteins deduced from cloned cDNAs were compared to those of the known S. cerevisiae protein. All are predicted to encode C-terminal membrane-spanning proteins of approximately 50 kDa with similar hydropathy profiles. Extensive sequence conservation exists in regions of the enzyme proposed to interact with its prenyl substrates (i.e., two farnesyl diphosphate molecules). Many of the highly conserved regions are also present in phytoene and prephytoene diphosphate synthetases, enzymes which catalyze prenyl substrate condensation reactions analogous to that of squalene synthetase. Expression of cDNA clones encoding S. pombe or hybrid human-S. cerevisiae squalene synthetases reversed the ergosterol requirement of S. cerevisiae cells bearing ERG9 gene disruptions, showing that these enzymes can functionally replace the S. cerevisiae enzyme.

Saccharomyces cerevisiae strains that contain the erg8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis. A (10,24,33,50). This enzyme functions in the elaboration of isoprene subunits, which are used for the synthesis of a variety of essential compounds including sterols, dolichols, and ubiquinones. Isoprene-derived molecules are also used in some species for the covalent modification of tRNAs (17) and specific proteins, including ras and a-factor in S. cerevisiae (1,27,46 (12,47,53). Some evidence suggests that in both yeast and mammalian cells, significant pathway regulation may be mediated through additional enzymes of sterol biosynthesis, including mevalonate kinase and squalene synthetase (18,20,52

Squalene synthetase (farnesyl diphosphate:farnesyl diphosphate farnesyltransferase; EC 2.5.1.21) is thought to represent a major control point of isoprene and sterol biosynthesis in eukaryotes. We demonstrate structural and functional conservation between the enzymes from humans, a budding yeast (Saccharomyces cerevisiae), and a fission yeast (Schizosaccharomyces pombe). The amino acid sequences of the human and S. pombe proteins deduced from cloned cDNAs were compared to those of the known S. cerevisiae protein. All are predicted to encode C-terminal membrane-spanning proteins of approximately 50 kDa with similar hydropathy profiles. Extensive sequence conservation exists in regions of the enzyme proposed to interact with its prenyl substrates (i.e., two farnesyl diphosphate molecules). Many of the highly conserved regions are also present in phytoene and prephytoene diphosphate synthetases, enzymes which catalyze prenyl substrate condensation reactions analogous to that of squalene synthetase. Expression of cDNA clones encoding S. pombe or hybrid human-S. cerevisiae squalene synthetases reversed the ergosterol requirement of S. cerevisiae cells bearing ERG9 gene disruptions, showing that these enzymes can functionally replace the S. cerevisiae enzyme. Inhibition of sterol synthesis in S. cerevisiae and S. pombe cells or in cultured human fibroblasts by treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor lovastatin resulted in elevated levels of squalene synthetase mRNA in all three cell types.

Saccharomyces cerevisiae strains that contain the ery8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis. A plasmid bearing the yeast ERG8 gene was isolated from a YCp50 genomic library by functional complementation of the erg8-1 mutant strain. Genetic analysis demonstrated that integrated copies of an ERG8 plasmid mapped to the erg8 locus, confirming the identity of this clone. Southern analysis showed that ERG8 was a single-copy gene. Subcloning and DNA sequencing defined the functional ERG8 regulon as an 850-bp upstream region and an adjacent 1,272-bp open reading frame. The deduced 424-amino-acid ERG8 protein showed no homology to known proteins except within a putative ATP-binding domain present in many kinases. Disruption of the chromosomal ERG8 coding region by integration of URA3 or HIS3 marker fragments was lethal in haploid cells, indicating that this gene is essential. Expression of the ERG8 gene in S. cerevisiae from the galactose-inducible galactokinase (GAL1) promoter resulted in 1,000-fold-elevated levels of phosphomevalonate kinase enzyme activity. Overproduction of a soluble protein with the predicted 48-kDa size for phosphomevalonate kinase was also observed in the yeast cells.