Molecular cloning and characterization of the yeast gene for squalene synthetase.

Abstract

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

Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase.

“…The hydrophobicity plot of Arabidopsis enzyme (data not shown) constructed using the Kyte-Doolittle algorithm (28) was remarkably similar to those of the reported squalene synthases from yeast to man (14)(15)(16)(17)(18)(19). The C-terminal hydrophobic domain is especially characteristic, because it likely functions as an anchor in the endoplasmic reticulum membrane (15,29).…”

“…1 shows the alignment of the amino acid sequences of squalene synthases from Arabidopsis and other species. The amino acid sequence of the Arabidopsis enzyme was 42.5% (over 407 amino acids), 42.2% (over 408 amino acids), 42.2% (over 405 amino acids), 41.6% (over 380 amino acids), and 44.3% (over 325 amino acids) identical to those of the mouse (11), rat (14), human (15)(16)(17), budding yeast (18,19), and fission yeast (15) enzymes, respectively. The conserved segments, A, B, and C (14), or III, IV, and V (15), of known squalene synthases are overlined in Fig.…”

“…Amino acid sequence alignment of squalene synthase polypeptides from Arabidopsis, budding yeast (18,19), fission yeast (15), mouse (11), rat (14), and man (15)(16)(17). Amino acid residues common to more than four polypeptides are denoted as consensus ones.…”

“…The activity of squalene formation was assayed as follows. The reaction mixture contained, in a total volume of 100 ,ul, 11.4 ,uM [ (14)(15)(16)(17) and yeasts (15,18,19). Several approaches could have been taken for the cDNA cloning of plant squalene synthase.…”

Biochemistry, cell biology and molecular biology of lipids ofSaccharomyces cerevisiae

“…As the first enzyme in a linear pathway, this step would be expected to be an ideal place for regulation. 108 The enzyme competes with other enzymes for the FPP substrate and has been shown to respond to cellular sterol content in a manner similar to that shown by HMGCoA reductase. 214 Studies of this type present specific limitations, since yeast synthesize sterol only under aerobic conditions and are unable to import sterol under these conditions.…”