Two New Ypt GTPases Are Required for Exit From the Yeast trans-Golgi Compartment

Abstract

Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the transGolgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/ 32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.

Rab11 Is Required for Trans-Golgi Network–to–Plasma Membrane Transport and a Preferential Target for GDP Dissociation Inhibitor

“…Deletion of both Ypt31 and Ypt32 caused newly synthesized proteins to accumulate in the transGolgi, lending support to the suggested role for these proteins in Golgi vesicle budding (Jedd et al, 1997). This is analogous to what we observed for VSV G protein when mutant rab11S25N protein was expressed.…”

“…Taken together these data suggest a possible role for rab11 in the budding of a subset of TGN vesicles. Although rab proteins have been primarily associated with vesicletargeting functions, there are a number of examples in which the rab proteins have been suggested to influence vesicle budding (Plutner et al, 1991;Riederer et al, 1994;Jedd et al, 1995Jedd et al, , 1997. Perhaps the most compelling evidence derives from in vitro assays showing a direct role for rab5 in coated vesicle formation at the plasma membrane (McLauchlan et al, 1998).…”

“…Two yeast homologues of rab11 were originally isolated in a suppresser screen to identify further regulators of ER-to-Golgi transport (Benli et al, 1996;Jedd et al, 1997). Deletion of both Ypt31 and Ypt32 caused newly synthesized proteins to accumulate in the transGolgi, lending support to the suggested role for these proteins in Golgi vesicle budding (Jedd et al, 1997).…”

“…The recent finding that the closest homologues of rab11 in yeast, Ypt31/32, are required for exit from the trans-Golgi suggested that this issue needed to be examined more directly (Jedd et al, 1997). Here we analyzed the intracellular distribution of wild-type and mutant forms of rab11 and tested its functional involvement in exocytic transport in baby hamster kidney (BHK) cells.…”

Membrane Organization and Dynamics in Cell Polarity

“…In yeast, the Rab11 homologs, Ypt31 and Ypt32, are thought to regulate vesicle budding from the TGN (Jedd et al 1997). It was shown that the GTP-bound form of Ypt31/32 directly interacts with Myo2, a myosin V motor that mediates the polarized transport of secretory vesicles along actin cables; disruption of the interaction between Ypt31/32 and Myo2 blocks vesicle transport to daughter cells (Lipatova et al 2008).…”