Substrate characterization of the Saccharomyces cerevisiae protein farnesyltransferase and type-I protein geranylgeranyltransferase.
The in vitro substrate preferences of recombinant S. cerevisiae protein farnesyltransferase and type-I protein geranylgeranyl-transferase were determined. Proteins ending in 16 different CaaX sequences (C = cysteine, a = aliphatic amino acid, X = variable amino acids) were used to determine the protein substrate preferences of these S. cerevisiae prenyltransferases. The identities of the attached prenyl groups were confirmed by iodomethane treatment of prenylated substrates and reverse-phase HPLC. The CaaX preference of each recombinant yeast enzyme was found to be nearly identical to the reported preferences of purified mammalian protein farnesyltransferase and type-I protein geranylgeranyltransferase. S. cerevisiae farnesyltransferase preferentially farnesylated CaaX sequences ending in methionine, cysteine or serine. The farnesyltransferase also attached geranylgeranyl to some CaaX sequences ending in methionine, leucine and cysteine. The S. cerevisiae type-I geranylgeranyltransferase preferentially geranylgeranylated CaaX sequences ending in leucine and to a lesser degree methionine. Yeast extracts were found to contain prenylating activities identical to those observed with the recombinant enzymes. Farnesyltransferase activity in yeast extracts exceeded type-I geranylgeranyltransferase activity by at least 3-fold, resulting in prenylation of leucine-ending CaaX substrates with a mixture of 75% geranylgeranyl and 25% farnesyl. These results suggest that some substrate overlap may occur between the S. cerevisiae protein farnesyltransferase and the type-I protein geranylgeranyltransferase in vivo.
Caplin
B E
BE
Department of Medicine, Indiana University, Indianapolis 46202-5121.
Hettich
L A
LA
Marshall
M S
MS
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D051231:Farnesyltranstransferase; D008969:Molecular Sequence Data; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*
D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D051231:Farnesyltranstransferase