Date of Award

9-27-2012

Embargo Period

12-12-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Advisor(s)

Tina H. Lee

Abstract

The mammalian endoplasmic reticulum (ER) is the largest organelle in the cell, extending from the nuclear envelope throughout the cell periphery. The ER houses a wide variety of vital cell processes within a single membrane bound organelle. In order to accommodate these functions and respond to the demands of the cell, the ER is partitioned into dynamically regulated subdomains, each with its own distinct structure. Despite the likely importance of ER structure for its functions, few proteins have been identified as having a direct role in maintaining the structure of the ER and the consequences of alteration of normal ER structure are not well understood.

Here we identify Yip1A, a conserved membrane protein that cycles between the ER and early Golgi, as a likely regulator of ER organization. Yip1A depletion led to restructuring of ER membranes into micrometer-sized, concentrically stacked whorls. These structures are reminiscent of the ER whorls found in certain specialized secretory cell types, where the regulation and functional consequence of ER whorl formation is not understood. We found that membrane stacking and whorl formation after Yip1A depletion coincided with a marked slowing of coat protein (COP) II-mediated protein export from the ER. Furthermore, whorl formation driven by exogenous expression of an ER protein with no role in COPII function also delayed cargo export. Thus, it appears that Yip1A is required to prevent ER whorl formation and that whorl formation can in turn delay protein export from the organelle. Whether this is the function of ER whorls in tissues remains to be seen, however these results make Yip1A a good candidate for playing a role in their regulation.

To obtain insight into how Yip1A regulates ER whorl formation and to determine whether the mechanism might be shared with the yeast homologue Yip1p, we carried out a systematic mutational analysis of all residues in the protein. Two discrete sites (E95 and K146) were crucial for the control of ER whorl formation by Yip1A. Notably, the same residues were previously shown to be important for Yip1p-mediated viability in yeast, indicating a shared mechanism. On the other hand, a third site (E89) also essential for yeast viability was dispensable for Yip1A function in regulating whorl formation. Thus Yip1p/Yip1A may possess at least two distinct essential functions only one of which is required for regulation of ER structure. Of note, the sites required for control of ER whorl formation by Yip1A were dispensable for the binding of Yip1p to its established binding partners Yif1p and Ypt1/31p, whereas the site required for Yip1p to bind the same partners was dispensable for ER structuring by Yip1A. Based on these observations, we speculate that the function of Yip1A in regulating whorl formation is mediated by one or more distinct and yet-to-be identified binding partners.

Collectively, these findings indicate that a dispersed ER network is important for proper COPII-mediated protein export and that Yip1A has a conserved function between yeast and humans in maintaining proper ER network dispersal through prevention of ER whorl formation. These studies set up an important framework for determining the molecular mechanism of Yip1A as an ER structuring protein

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