Year of Award

2010

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Other Degree Name/Area of Focus

Integrative Microbiology and Biochemistry, Cellular and Molecular Biology

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

Jesse Hay

Commitee Members

Scott Wetzel, Mark Grimes, Scott Samuels, Darrell Jackson

Keywords

calcium, COPII, endoplasmic reticulum, ERGIC, Golgi, transport

Abstract

Calcium is an important regulatory ion which acts as a trigger or is required at a basal level for many membrane fusion events. The role of calcium, and in particular luminal calcium, in regulating fusion in the early secretory pathway has not been thoroughly investigated. Here, I show that calcium has an inhibitory effect on homotypic coat protein complex (COPII) vesicle fusion. In particular, chelation of luminal calcium as well as fast chelation of extra luminal calcium, result in a significant increase of COPII vesicle fusion. Specific inhibition of Sarco/endoplasmic reticulum calcium ATPases using cyclopiazonic acid in the presence of calcium free medium results in low luminal calcium while maintaining normal calcium concentration in the cytosol. This luminal calcium depletion results in a dramatic expansion of the endoplasmic reticulum to Golgi intermediate compartment (ERGIC) in the periphery of the cell visualized using the markers rbet1 and p24. Depletion of luminal calcium also results in major kinetic defects of VSV-G transport to the Golgi, as well as reconstitution of the Golgi after it has been dispersed using brefeldin A. These results suggest that luminal calcium regulates fusogenicity, structure, and function of COPII derived membranes in transport. However, bulk soluble cargo is not affected by luminal calcium depletion. This suggests that luminal calcium may act at the level of membrane protein sorting in the ERGIC. Possible mechanisms by which luminal calcium exerts these novel roles are discussed herein and include prolonged retention of the outer COPII shell Sec13/Sec31 with Apoptosis Linked Gene-2 (ALG-2) being a likely candidate as the calcium sensor on the outside of the vesicle.

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© Copyright 2010 Marvin Bentley