All ETDs from UAB

Advisory Committee Chair

Elizabeth Sztul

Advisory Committee Members

Anne Burton

Jim Collawn

Kevin L Kirk

Julian C Rayner

Document Type

Dissertation

Date of Award

2008

Degree Name by School

Doctor of Philosophy (PhD) School of Medicine

Abstract

Metazoan cells are characterized with elaborate network of intracellular membranous compartments. These membranes allow the cell to spatially separate antagonistic processes and environments, and maintain sequential order of reactions necessary for maturation and secretion of biosynthetic cargo. The core transport machinery consists of coat proteins, tethering factors, SNAREs and small Ras-like GTPases. We have explored the function of the tethering factor p115 in organellogenesis and in secretory traffic. The p115-depleted system was utilized to explore structurefunction relationships within p115. Here, we analyzed the architecture of the Golgi after RNAi induced depletion of p115. We show that in p115 depleted cells Golgi disrupts in to dynamic perinuclear structures that retain the cis-trans polarity of the normal Golgi complex, and can reassemble after treatment with Brefeldin A and subsequent washout. We also characterized the impact of p115 depletion on the traffic and secretion of biosynthetic cargo. Our studies revealed that p115 depletion has a selective effect on cargo traffic - we observed ER retention of transmembrane VSV-G protein with no effect on trafficking of soluble proteins. The p115 tethering factor does not appear to have an enzymatic activity and acts through the interaction with its binding partners. The C-terminal coiled-coil region of p115 is critical for binding to Rab1 GTPase and SNAREs, and has been shown to be essential for p115 function in Golgi biogenesis. We used p115 deletion mutant to analyze iii the importance of CC3 and CC4 for p115 function. Expression of the p115/1-766 mutant, lacking CC3-4 region, in p115 depleted cells or in control cells disrupted Golgi in to scattered punctate fragments, which suggest a dominant-negative nature of the mutant. Interestingly, the p115/1-766 mutant supported ER exit of transmembrane VSV-G but arrested it in dispersed punctuate structures, without any detectable effect on transport of soluble cargo. Our studies provide the first evidence that the CC3-4 region is necessary for the function of p115. Combining our findings with the current knowledge on p115 functional domains, we propose a model where p115 not only increases the fidelity of SNARE-pin formation, but also functions in proteins sorting during ER exit of cargo proteins.

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