Rabs constitute the largest branch of the Ras GTPase superfamily, with ten members in yeast and more than 60 in mammalian cells. They 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) that 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. We have proposed 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 Sec2, the GEF that activates the downstream Rab, Sec4, as well as Gyp1, the GAP that inactivates the upstream Rab, Ypt1. The net effect of these counter-current cascades is a programmed series of Rab transitions that lead to critical changes in the functional identity of the membrane as it flows along the exocytic pathway. Phosphoinositide 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 in a positive feedback loop. PI (4) P distribution together with Sec2 phosphorylation determine when each regulatory circuit is used. We propose three aims: 1. we will define the role of two protein kinases in the regulation of Sec2 and other components of the secretory machinery. 2. We will test the effects of rewiring the rab regulatory circuits to evaluate several models concerning the role of rabs in the control of membrane traffic. 3. We will determine the molecular mechanisms underlying several stages of secretory vesicle maturation.
Membrane traffic is required for a broad range of essential cellular functions and the regulation of membrane traffic by Rab GTPases is therefore relevant to major human diseases, including cancer, diabetes and neural degeneration. Additional diseases have been directly attributed to specific defects in Rab expression, Rab modification, Rab regulation and Rab effectors and a number of clinically important human pathogens have evolved to exploit and disrupt our Rab regulatory pathways to promote their own intracellular agenda and to evade host defenses. We will analyze how Rab proteins are functionally linked to one another through regulatory networks to control membrane traffic.
Showing the most recent 10 out of 18 publications
