The mechanisms mediating polarized delivery of vesicles to cell surface domains are poorly understood in animal cells. We have previously shown that expression of Rab8 promotes the formation of new cell surface domains through reorganization of actin and microtubules. To unravel the function of Rab8, we used the yeast two-hybrid system to search for potential Rab8-specific activators. We identified a coil-coiled protein (Rabin8), homologous to the rat Rabin3 that stimulated nucleotide exchange on Rab8 but not on Rab3A and Rab5. Furthermore, we show that rat Rabin3 has exchange activity on Rab8 but not on Rab3A, supporting the view that rat Rabin3 is the rat equivalent of human Rabin8. Rabin8 localized to the cortical actin and expression of Rabin8 resulted in remodeling of actin and the formation of polarized cell surface domains. Activation of PKC by phorbol esters enhanced translocation of both Rabin8 and Rab8-specific vesicles to the outer edge of lamellipodial structures. Moreover, coexpression of Rabin8 with dominant negative Rab8 (T22N) redistributes Rabin8 from cortical actin to Rab8-specific vesicles and promotes their polarized transport to cell protrusions. The C-terminal region of Rabin8 plays an essential role in this transport. We propose that Rabin8 is a Rab8-specific activator that is connected to processes that mediate polarized membrane traffic to dynamic cell surface structures.

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Saccharomyces cerevisiae cells grown at 24 degrees C acquire thermotolerance and survive exposure to 50 degrees C, but only if they are first incubated at 30 degrees C, the temperature where heat shock genes are activated. We show here that the enzymatic activity of a secretory beta‐lactamase fusion protein, pre‐accumulated at 37 degrees C in the endoplasmic reticulum, was abolished by exposure of the cells to 50 degrees C. When the cells were returned to 24 degrees C, beta‐lactamase activity was resumed. Reactivation occurred in the endoplasmic reticulum, but not in the Golgi apparatus. It was dependent on metabolic energy, but did not require de novo protein synthesis. According to co‐immunoprecipitation experiments, immuno‐globulin‐binding protein (BiP/Kar2p) was associated with the fusion protein. We suggest that recovery from thermal insult involves, in addition to cytoplasmic and nuclear events, refolding of heat‐damaged proteins in the endoplasmic reticulum by a heat‐resistant machinery, which forms part of a fundamental survival mechanism.