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 respects. (i) Most Rab proteins contain two adjacent cysteine residues within one of the following C-terminal sequence motifs: -XXCC, -XCXC, or -CCXX; (ii) a Rab protein that ends in a -XCXC motif has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransferase. Whether this enzyme catalyzes the geranylgeranylation of both cysteines is unknown. We addressed this question by direct structural analysis of in vitro prenylated proteins. We incubated recombinant Rab geranylgeranyltransferase, Rab escort protein, and [1-3H]geranylgeranyl 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 spectrometry and found that for each protein both C-terminal adjacent cysteines were geranylgeranylated. These results indicate that Rab geranylgeranyltransferase/Rab escort protein catalyzes the geranylgeranylation of both cysteines in Rab proteins with three distinct C-terminal motifs and suggest that other Rab proteins with these motifs may be similarly modified.
Farnsworth
C C
CC
Howard Hughes Medical Institute, University of Washington, Seattle 98195.
Seabra
M C
MC
Ericsson
L H
LH
Gelb
M H
MH
Glomset
J A
JA
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014166:Transferases / Q000378:metabolism*; D014357:Trypsin; D020761:rab1 GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C075607:Rab geranylgeranyltransferase; D014357:Trypsin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D003545:Cysteine