Rabs represent 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 effectors proteins such as cytoskeletal motors, vesicle tethering proteins and regulators of SNARE complex assembly. Rabs, in turn, are regulated by specific guanine nucleotide exchange proteins (GEFs) that catalyze the displacement of GDP and binding of GTP and GTPase activating proteins (GAPs) that stimulate the slow intrinsic rate of GTP hydrolysis. Recent work from our lab has demonstrated that different Rabs 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 is a programmed series of Rab conversions that lead to changes in the functional identity of the membrane as it flows along the exocytic pathway. We have also shown that the Golgi pool of phosphatidylinositol 4-phosphate (PI4P) works in concert with Ypt32 to recruit Sec2 and to control a regulatory switch in Sec2 function. We propose five specific aims to address the molecular mechanisms by which Rabs are networked to one another and by which the distribution of PI4P is spatially defined. 1. We will explore the role of phosphorylation in the regulation of Sec2 function. Phosphomimetic and non- phosphorylatable alleles will be tested for their interactions with Sec4, Ypt32, Sec15 and PI4P. 2. The Sec4 GEF Sec2 binds to the Sec4 effectors Sec15 leading to a positive feedback loop. To test the role of this mechanism in membrane traffic we will generate Sec2 alleles specifically defective in Sec15 binding. 3. We have generated an allele of Ypt1 that can be activated by the Sec4 GEF, Sec2. We will determine if this mutation redirects the membrane association of Ypt1 and the effects of this "short circuit" on membrane traffic. 4. We have evidence for a second example of a Rab-GAP cascade and will screen all Rab GAPs against representative Rabs to identify more. We will test the effects of GAP disruption on overlap of Rab domains. 5. PI4P is normally restricted to the Golgi and is not enriched on Golgi-derived secretory vesicles. We will determine the mechanism by which PI4P is limited to the Golgi.

Public Health Relevance

Membrane traffic is required for a broad range of essential cellular functions, such as controlling the accessibility of cell surface receptors, the translocaton of glucose transporters in response to insulin, antigen presentation, neuronal transmission and the establishment and maintenance of epithelial cell polarity. The regulation of membrane traffic by Rab GTPases is therefore directly relevant to a broad range of human diseases including cancer, diabetes and neural degeneration. We will analyze how Rab proteins are functionally linked to one another through regulatory networks to control membrane traffic.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM082861-07
Application #
8692849
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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Shen, David; Yuan, Hua; Hutagalung, Alex et al. (2013) The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p. J Cell Biol 202:509-26
Mizuno-Yamasaki, Emi; Rivera-Molina, Felix; Novick, Peter (2012) GTPase networks in membrane traffic. Annu Rev Biochem 81:637-59
Hutagalung, Alex H; Novick, Peter J (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91:119-49
Sclafani, Anthony; Chen, Shuliang; Rivera-Molina, Felix et al. (2010) Establishing a role for the GTPase Ypt1p at the late Golgi. Traffic 11:520-32
Mizuno-Yamasaki, Emi; Medkova, Martina; Coleman, Jeff et al. (2010) Phosphatidylinositol 4-phosphate controls both membrane recruitment and a regulatory switch of the Rab GEF Sec2p. Dev Cell 18:828-40
Williams, Daniel C; Novick, Peter J (2009) Analysis of SEC9 suppression reveals a relationship of SNARE function to cell physiology. PLoS One 4:e5449