Rewiring a Rab regulatory network reveals a possible inhibitory role for the vesicle tether, Uso1

Ypt1 and Sec4 are essential Rab GTPases that control the early and late stages of the yeast secretory pathway, respectively. A chimera consisting of Ypt1 with the switch I domain of Sec4, Ypt1-SW1^Sec4, is efficiently activated in vitro by the Sec4 exchange factor, Sec2. This should lead to its ectopic activation in vivo and thereby disrupt membrane traffic. Nonetheless early studies found that yeast expressing Ypt1-SW1^Sec4 as the sole copy of YPT1 exhibit no growth defect. To resolve this conundrum, we have analyzed yeast expressing various levels of Ypt1-SW1^Sec4 We show that even normal expression of Ypt1-SW1^Sec4 leads to kinetic transport defects at a late stage of the pathway, with secretory vesicles accumulating near exocytic sites. Higher levels are toxic. Toxicity is suppressed by truncation of Uso1, a vesicle tether required for endoplasmic reticulum-Golgi traffic. The globular head of Uso1 binds to Ypt1 and its coiled-coil tail binds to the Golgi-associated SNARE, Sed5. We propose that when Uso1 is inappropriately recruited to secretory vesicles by Ypt1-SW1^Sec4, the extended coiled-coil tail blocks docking to the plasma membrane. This putative inhibitory function could serve to increase the fidelity of vesicle docking.