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Mutant farnesyltransferase beta subunit of Saccharomyces cerevisiae that can substitute for geranylgeranyltransferase type I beta subunit

Author:
Mitsuzawa, H., Esson, K., Tamanoi, F.
Source:
Proceedings of the National Academy of Sciences of the United States of America 1995 v.92 no.5 pp. 1704-1708
ISSN:
0027-8424
Subject:
proteins, structural genes, site-directed mutagenesis, Saccharomyces cerevisiae, genetic complementation, mutants, binding sites, alkyl (aryl) transferases, enzyme activity
Abstract:
The protein farnesyltransferase (PFT) beta-subunit gene of Saccharomyces cerevisiae, DPR1, was randomly mutagenized by PCR to construct a mutant DPR1 gene library on a high-copy plasmid. The library was screened for suppression of the temperature sensitivity conferred by a mutation in the protein geranylgeranyltransferase type I (PGGT-I) beta-subunit gene, CAL1. A mutant DPR1 gene was identified whose product contained a single amino acid change of Ser-159 to Asn. This mutant gene also suppressed a call disruption even on a low-copy plasmid, suggesting that the product (designated S159N) can substitute for PGGT-I beta subunit in vivo. Its ability to act as a PFT is not drastically reduced, since the mutant gene still complemented a dpr1 disruption. Results of in vitro assays demonstrate that the mutant enzyme has increased activity to farnesylate, a substrate for PGGT-I. On the other hand, the ability to farnesylate its own substrate is reduced. The increased ability to utilize the PGGT-I substrate is due to its increased affinity for the protein substrate. In addition, the mutant enzyme shows a severalfold increase in the sensitivity to a peptidomimetic inhibitor that acts as a competitor of the protein substrate. These results point to the importance of the beta subunit of PFT for the binding of a protein substrate and demonstrate that Ser-159 of DPR1 product is critical for its substrate specificity.
Agid:
2355520