Regulation of membrane traffic by Rab GEF and GAP cascades

Abstract

ASBTRACT Rab GTPases serve as master regulators of membrane traffic, each typically controlling several different aspects of a specific stage of membrane traffic by recruiting diverse effector proteins such as cytoskeletal motors, vesicle tethering proteins and regulators of SNARE complex assembly. Rabs, in turn, are regulated by specific guanine nucleotide exchange factors (GEFs), which catalyze the displacement of GDP and binding of GTP, as well as GTPase activating proteins (GAPs) that stimulate the slow intrinsic rate of GTP hydrolysis. Here I review our studies on the final stages of the yeast secretory pathway that have led us to propose that adjacent Rabs on a pathway are networked to one another through their regulators; specifically we have shown that the Rab, Ypt32, in its GTP-bound form recruits both Sec2, the GEF that activates the downstream Rab, Sec4, as well as Gyp1, the GAP that inactivates the upstream Rab, Ypt1. The postulated effect of these counter-current cascades is a programmed series of abrupt Rab transitions that lead to critical changes in the functional identity of the membrane as it flows along the exocytic pathway. Phosphoinositides also play key roles in the temporal and spatial regulation of membrane traffic. The Golgi pool of phosphatidylinositol 4-phosphate (PI(4)P) works in concert with Ypt32 to initially recruit Sec2, yet a subsequent drop in PI(4)P levels directs a regulatory switch in Sec2 function in which it binds to the Sec4 effector Sec15 generating a positive feedback loop. PI(4)P distribution together with Sec2 phosphorylation by a casein kinase determines when and where each regulatory circuit is used.

Keywords: GAP cascade; GEF cascade; GTPase activating protein; Rab; exchange factor; membrane traffic.

Figure 1.

(from left to right) Sec2 is recruited to membranes by the combination of Ypt32-GTP and PI(4)P. Sec2 phosphorylation by Yck1/2 is initially inhibited by the high level of PI(4)P. The secretory vesicle buds off and Sec2 activates Sec4, which then recruits its effector, Sec15. During vesicle maturation, PI(4)P is removed by Osh4 and Ypt32 is released from the vesicle. This permits Sec15 to interact with Sec2 and also allows Sec2 phosphorylation by Yck1/2, further enhancing the Sec2–Sec15 interaction. This process pushes the reaction forward and creates a positive-feedback loop that generates a micro-domain of high Sec4-GTP and high Sec15, facilitating the delivery, tethering, and fusion of the vesicle with the plasma membrane. Sec2 is dephosphorylated by an as yet unknown phosphatase that allows Sec2 to dissociate from Sec15 and thus dissociate from the vesicle. The dephosphorylated Sec2 is then available to associate with a new round of vesicles.