Characterization of the interaction of the monomeric GTP-binding protein Rab3a with geranylgeranyl transferase II.
The monomeric GTP-binding protein Rab3a controls exocytosis in neuroendocrine and neuronal cells. Like other members of the Rab family, Rab3a is posttranslationally modified by the addition of hydrophobic geranylgeranyl groups to its C-terminus. The geranylgeranylation reaction is catalysed by the heterotrimeric geranylgeranyl transferase II. We describe the cDNA cloning of the beta-subunit of human geranylgeranyl transferase II by means of the yeast two-hybrid system. The human enzyme, which is 49% and 96% similar to yeast and rat isoforms, respectively, can complement the beta-subunit deficiency in the yeast strain ANY119. Furthermore, by means of the two-hybrid system and in vitro geranylgeranylation reactions with purified recombinant rat geranylgeranyl transferase II, we have characterized Rab3a domains implicated in the interaction with geranylgeranyl transferase II. We find that the N-terminus, the effector loop, the hypervariable region of the C-terminus, and the geranylgeranyl-acceptor cysteines have roles in this interaction. The GDP-bound form of Rab3a is the preferred substrate of geranylgeranyl transferase II.
Johannes
L
L
Service de Neurobiologie Physico-Chimique, Centre National de la Recherche Scientifique UPR 9071, Paris, France.
Perez
F
F
Laran-Chich
M P
MP
Henry
J P
JP
Darchen
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D017368:Protein Prenylation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D020692:rab3 GTP-Binding Proteins
D017931:DNA Primers; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; C095279:Rab geranylgeranyl transferase beta-subunit; D011994:Recombinant Proteins; D014166:Transferases; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins