YEAST GERANYLGERANYLTRANSFERASE TYPE-II - STEADY-STATE KINETIC-STUDIES OF THE RECOMBINANT ENZYME

Rab proteins in mammalian cells, or Ypt1p and Sec4p in yeast, regulate vesicular traffic. Prenylation of these small GTP-binding proteins is required for membrane attachment and subsequent biological activity. Yeast protein geranylgeranyltransferase type-II (PGCTase-II) catalyzes the prenylation of Ypt1p in the presence of an escort protein, Msi4p. The genes encoding the alpha- (BET4) and beta- (BET2) subunits of PGC Tase-II were translationally coupled by overlapping the BET4-BET2 stop /start codons and by adding a ribosome-binding site near the 3'-end of BET4 that fused an -EEF C-terminal alpha-tubulin epitope to Bet4p. Ac tive recombinant heterodimer was purified by chromatography on DE52 an d anti-alpha-tubulin columns. Recombinant Msi4p with an N-terminal pol yhistidine leader was purified on a Ni2+-Sepharose column, followed by gel filtration and ion exchange chromatography. An escort protein, Ms i4p, was necessary for geranylgeranylation of Ypt1p by yeast PGGTase-I I. Michaelis constants for GCPP and Ypt1p were 1.6 and 1.1 mu M, respe ctively; V-max = 1.7 nmol min(-1) mg(-1) for yeast PGGTase-II. Typical Michaelis-Menten behavior was also seen for the enzyme for varied con centrations of Msi4p, with a maximal catalytic activity seen for a 10- fold excess of escort protein over enzyme: In contrast to previous rep orts, PGGTase-II requires both Zn2+ and Mg2+ for maximal activity, alt hough Zn2+ becomes inhibitory at concentrations above similar to 10 mu M. Prenylated Ypt1p obtained after incubation of Ypt1p with PGCTase-I I, Msi4p, and geranylgeranyl diphosphate was digested with trypsin. Th e C-terminal peptide fragment from modified Ypt1p was purified by HPLC and analyzed by electrospray mass spectrometry. The mass of the fragm ent is consistent with the 12-mer C-terminal amino acid fragment predi cted from proteolysis by trypsin with both cysteine residues modified by geranylgeranyl moieties.