Dj. Witter et Cd. Poulter, YEAST GERANYLGERANYLTRANSFERASE TYPE-II - STEADY-STATE KINETIC-STUDIES OF THE RECOMBINANT ENZYME, Biochemistry, 35(32), 1996, pp. 10454-10463
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.