THE RAB3A GTPASE INTERACTS WITH MULTIPLE FACTORS THROUGH THE SAME EFFECTOR DOMAIN - MUTATIONAL ANALYSIS OF CROSS-LINKING OF RAB3A TO A PUTATIVE TARGET PROTEIN
Cj. Mckiernan et al., THE RAB3A GTPASE INTERACTS WITH MULTIPLE FACTORS THROUGH THE SAME EFFECTOR DOMAIN - MUTATIONAL ANALYSIS OF CROSS-LINKING OF RAB3A TO A PUTATIVE TARGET PROTEIN, The Journal of biological chemistry, 268(32), 1993, pp. 24449-24452
Rab3A/smg25A is a small Ras-like guanine nucleotide binding protein im
plicated in the control of regulated secretion from cells. Rab3A is ap
proximately 30% cytosolic and 70% associated with the membranes of sec
retory vesicles. It cross-links specifically to a rat brain membrane p
rotein of about 85 kilodaltons (p85). To identify epitopes on Rab3A th
at are important for its interaction with this putative target protein
, we have determined the effects of point mutations on the cross-linki
ng efficiency of Rab3A to p85. Rab3A, which was preincubated with a no
n-hydrolyzable analog of GTP, cross-linked more efficiently to p85 tha
n did Rab3A.GDP. Rab3A mutants that had decreased nucleotide binding a
lso exhibited poor cross-linking to p85. Mutations in the effector dom
ain, a site important for the interaction of Rab3A with its guanine nu
cleotide releasing factor, guanine nucleotide dissociation inhibitor,
and GTPase-activating protein, eliminated the ability of Rab3A to cros
s-link to p85. However, short peptides corresponding to the effector d
omain did not reduce cross-linking efficiency when present at a concen
tration of 50 muM.