The Rab3A GTPase interacts with multiple factors through the same effector domain. Mutational analysis of cross-linking of Rab3A to a putative target protein.
Rab3A/smg25A is a small Ras-like guanine nucleotide binding protein implicated in the control of regulated secretion from cells. Rab3A is approximately 30% cytosolic and 70% associated with the membranes of secretory vesicles. It cross-links specifically to a rat brain membrane protein of about 85 kilodaltons (p85). To identify epitopes on Rab3A that are important for its interaction with this putative target protein, we have determined the effects of point mutations on the cross-linking efficiency of Rab3A to p85. Rab3A, which was preincubated with a non-hydrolyzable analog of GTP, cross-linked more efficiently to p85 than did Rab3A-GDP. Rab3A mutants that had decreased nucleotide binding also exhibited poor cross-linking to p85. Mutations in the effector domain, a site important for the interaction of Rab3A with its guanine nucleotide releasing factor, guanine nucleotide dissociation inhibitor, and GTPase-activating protein, eliminated the ability of Rab3A to cross-link to p85. However, short peptides corresponding to the effector domain did not reduce cross-linking efficiency when present at a concentration of 50 microM.
McKiernan
C J
CJ
Department of Pathology, University of Vermont Medical College, Burlington 05405-0068.
Brondyk
W H
WH
Macara
I G
IG
D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004252:DNA Mutational Analysis; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D051381:Rats; D020692:rab3 GTP-Binding Proteins
D003432:Cross-Linking Reagents; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins