RAB GERANYLGERANYL TRANSFERASE CATALYZES THE GERANYLGERANYLATION OF ADJACENT CYSTEINES IN THE SMALL GTPASES RAB1A, RAB3A, AND RAB5A

Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C termini, They differ from other geranylgeranylated small GTPases in several important respe cts. (i) Most Rab proteins contain two adjacent cysteine residues with in one of the following C-terminal sequence moths: -XXCC, -XCXC, or -C CXX; (ii) a Rab protein that ends in a -XCXC moth has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransfe rase. Whether this enzyme catalyzes the geranylgeranylation of both cy steines is unknown. We addressed this question by direct structural an alysis of in vitro prenylated proteins, We incubated recombinant Rab g eranylgeranyltransferase, Rab escort protein, and [1-H-3]geranylgerany l pyrophosphate with recombinant wild-type Rab1A (-XXCC), Rab3A (-XCXC ), or Rab5A (-CCXX) and treated each labeled protein with trypsin, We then analyzed the resulting peptides by HPLC and electrospray mass spe ctrometry and found that for each protein both C-terminal adjacent cys teines were geranylgeranylated, These results indicate that Rab gerany lgeranyltransferase/Rab escort protein catalyzes the geranylgeranylati on of both cysteines in Rab proteins with three distinct C-terminal mo ths and suggest that other Rab proteins with these motifs may be simil arly modified.