Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.
Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.
Brondyk
W H
WH
Department of Pathology, Vermont Cancer Center, University of Vermont College of Medicine, Burlington 05405-0068.
McKiernan
C J
CJ
Fortner
K A
KA
Stabila
P
P
Holz
R W
RW
Macara
I G
IG
D000313:Adrenal Medulla / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D015723:Gene Library; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016366:Open Reading Frames; D017354:Point Mutation; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D011916:Rats, Inbred F344; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000096:biosynthesis; D020692:rab3 GTP-Binding Proteins
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C092115:Rab3ip protein, rat; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins