Molecular dissection of guanine nucleotide dissociation inhibitor functionin vivo - Rab-independent binding to membranes and role of Rab recycling factors

Guanine nucleotide dissociation inhibitor (GDI) is an essential protein req uired for the recycling of Rab GTPases mediating the targeting and fusion o f vesicles in the exocytic and endocytic pathways. Using site-directed muta genesis of yeast GDI1, we demonstrate that amino acid residues required for Rab recognition in vitro are critical for function in vivo in Saccharomyce s cerevisiae. Analysis of the effects of Rab-binding mutants on function in vivo reveals that only a small pool of recycling Rab protein is essential for growth, and that the rates of recycling of distinct Rabs are differenti ally sensitive to GDI. Furthermore, we find that membrane association of Gd i1p is Rab-independent. Mutant Gdi1 proteins unable to bind Rabs were able to associate with cellular membranes as efficiently as wild-type Gdi1p, yet caused a striking loss of the endogenous cytosolic Gdi1p-Rab pools leading to dominant inhibition of growth when expressed at levels of the normal, e ndogenous pool. These results demonstrate a potential role for a new recycl ing factor in the retrieval of Rab-GDP from membranes, and illustrate the i mportance of multiple effecters in regulating GDI function in Rab delivery and retrieval from membranes.