Yeast Protein Geranylgeranyltransferase Type-I: Overproduction, Purification, and Characterization

Abstract

Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes alkylation of the cysteine residue in proteins containing a consensus C-terminal CaaX sequence ending in leucine by the C 20 hydrocarbon moiety in geranylgeranyl diphosphate (GGPP). The Saccharomyces cerevisiae genes encoding the α ( RAM2) and β ( CDC43) subunits of PGGTase-I were translationally coupled by overlapping the RAM2- CDC43 stop-start codons and by locating a ribosome-binding site near the 3′ end of RAM2. Recombinant PGGTase-I was overproduced in Escherichia coli to give ∼8% of total cellular protein and purified 12-fold to >95% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The purified heterodimer contained α- and β-subunits with molecular masses of 34 and 42 kDa, respectively. A continuous fluorescence assay was developed to measure PGGTase-I activity. The recombinant enzyme showed maximal activity at pH 7.5 and required both Mg 2+ and Zn 2+. Michaelis constants for GGPP (1.0 μM) and dansyl-Gly-Cys-Ile-Ile-Leu (2.4 μM) were similar to those reported for yeast protein farnesyltransferase (PFTase) with farnesyl diphosphate and dansyl-Gly-Cys-Val-Ile-Ala; V max = 0.20 μmol min −1 mg −1 for recombinant yeast PGGTase-I was similar to that reported for yeast PFTase.