Abstract
Rab proteins comprise a family of small GTPases that serve a regulatory role in membrane traffic. These proteins are in part cytosolic and in part associated with the membranes of specific exocytic and endocytic organelles. Smg p25A/rab3A GDI, a cytosolic protein which inhibits the dissociation of GDP from smg p25A/rab3A, Sec4p, and rab11, has also been found to prevent association of rab3A with the membrane. In this study, we have used Madin-Darby canine kidney cells permeabilized with the bacterial toxin streptolysin O to test the general activity of rab3A GDI in modulating the membrane association of various small GTP-binding proteins. Rab3A GDP dissociation inhibitor (GDI) removed from the membrane all rab proteins we have tested and inhibited the membrane binding of in vitro translated rab proteins. However, rab3A GDI had a limited effect on the membrane association of a mutant rab5 protein which contained a farnesylated cysteine motif. Finally, we found that, although rab3A GDI resides primarily in the cytosol, it is also associated with compartments of the exocytic and endocytic pathways. Since rab3A GDI can modulate the membrane association of various rab proteins, we propose to rename it rab GDI.
Highlights
This hypothesis has originally been supported by the finding that serve a regulatory role in membrtarnaeffic
We studied the effect of rab3A GDP dissociation inhibitor (GDI) on endogenous rab2, rab5, rab7, rab8, rab9, and rabll proteins by quantitative immunoblot analysis using specific affinity-purified antibodies
Conversion of the geranylgeranylated cysteine motif of rab5 to -CVLS, whichis a substrate for farnesylation, dramaticallyreduced binding of the mutantprotein to themembrane of permeabilized MDCK cells. These results suggest that thehigher hydrophobicity of the addition of one or two 20-carbon isoprenoid groups increases the hydrophobicity of rab proteins allowing them to become tightlymembrane associated, this process can be reversed by rab GDI
Summary
This hypothesis has originally been supported by the finding that serve a regulatory role in membrtarnaeffic. In the case of rab3A, the machinery on the vesicle and on the acceptor compartment COOH-terminal region of this protein has been found to be (Bourne, 1988;Bourne et al, 1990) According to this model, required for the interaction with rab3A GDI Owing to the structural divergence in the COOH terminus of rab proteins, thesefindings raise the question of the This complex wouldthen be recognized bya docking machin- specificity of rab3A GDI for the various members of the rab ery on the target membrane. We show that rab3A the GTPase would be returned to the donor membrane via GDI removed all endogenous rab proteinswe have tested from the cytosol to perform multiple rounds of vesicular transport. Rab and rab5-CVLS-RNA were in vitro translated for 1 h a t 30 "C in 100-p1reactions containing [3H]farnesylpyrophosphate
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