Molecular cloning, expression, and characterization of the cDNA for the rat hepatic squalene synthase.
Amino acid sequence information was obtained for the NH2 terminus, and for endogenous peptides generated by trypsin digestion, of a purified, truncated form of rat hepatic squalene synthase (RSS, EC 2.5.1.21) (Shechter, I., Klinger, E., Rucker, M. L., Engstrom, R. G., Spirito, J. A., Islam, M. A., Boettcher, B. R., and Weinstein, D. B. (1992) J. Biol. Chem. 267, 8628-8635). Degenerate primers, based on the amino acid sequences, were synthesized and used for the amplification and sequencing of a 1708-base pair (bp) cDNA for RSS from the rat hepatoma cell line H35. An open reading frame of 1248 bp encoding 416 amino acids (M(r) = 48,103) was detected for RSS. We have constructed a pRSS1327 expression vector by molecular cloning of a 1327-bp cDNA, which includes sequences of the entire coding region for RSS, into pBluescript. Expression in Escherichia coli of a functional, full-length RSS was confirmed by immunoblot analysis and enzymatic activity. We present and evaluate a model for the secondary structure of RSS and its possible membrane orientation. The model predicts a 315-residue domain at the center of the protein that contains the catalytic site and is released in a soluble form by partial proteolysis. The 33-residue NH2-terminal and 98-residue COOH-terminal sections are not involved in catalysis. Sequence analysis of the catalytic domain of RSS indicate three regions with high homology to sequences in a number of functionally distinct proteins that utilize polyprenyl diphosphate substrates.
McKenzie
T L
TL
Eleanor Roosevelt Institute, Denver, Colorado 80206.
Jiang
G
G
Straubhaar
J R
JR
Conrad
D G
DG
Shechter
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000737:chemistry / Q000235:genetics*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017433:Protein Structure, Secondary; D051381:Rats; D017386:Sequence Homology, Amino Acid
D004247:DNA; D013186:Farnesyl-Diphosphate Farnesyltransferase