CDC43 and RAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase.
The question regarding the identity of the alpha and beta subunits of the yeast type I protein geranylgeranyltransferase was explored using prokaryotic expression of candidate genes. The Saccharomyces cerevisiae CDC43 and RAM2 genes were expressed in Escherichia coli and cell extracts examined for the ability to transfer [3H]geranylgeranyl diphosphate to an appropriate CaaX protein substrate. Individual expression of each gene yielded no activity; however, co-expression of the two genes resulted in high levels of [3H] geranylgeranyl incorporation into the substrate protein Ras-Cys-Val-Val-Leu. The activity was partially purified yielding approximately 12,600 units/liter. The partially purified enzyme geranylgeranylated the Ras-Cys-Val-Val-Leu, Ras-Cys-Ala-Ile-Leu, Ras-Cys-Ile-Ile-Leu, and Ras-Cys-Thr-Ile-Leu substrates but not the Ras-Cys-Val-Leu-Ser or Ras-Ser-Val-Leu-Ser substrates. The protein geranylgeranyltransferase was highly specific for geranylgeranyl diphosphate and poorly transferred farnesyl. The recombinant enzyme was indistinguishable from the native type I geranylgeranyltransferase in yeast extracts. As has been reported for the protein farnesyltransferase, the yeast type I protein geranylgeranyltransferase is also a magnesium-requiring, zinc metalloenzyme. Interestingly, the recombinant enzyme functioned with calcium as the only divalent cation, although addition of zinc increased calcium-dependent activity 2-fold.
Mayer
M L
ML
Department of Medicine, Indiana University, Indianapolis 46202-5121.
Caplin
B E
BE
Marshall
M S
MS
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013045:Species Specificity; D013379:Substrate Specificity; D014166:Transferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; C077378:RAM2 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; C065203:CDC43 protein, S cerevisiae