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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded

Spring 2014

First Advisor

Dr. Daniel N. Hebert

Subject Categories

Cell Biology

Abstract

The endoplasmic reticulum (ER) is an organelle responsible for a variety of functions in all eukaryotic cells. Some of these functions are localized to specific regions in the ER, such as the ribosome-studded rough ER sheets or the ribosome-free smooth ER sheets. The smooth ER sheets have the ability to form ER tubules, which extend throughout the cell and make contact with other organelles. In order to accommodate these localized functional regions, a certain degree of heterogeneity and compartmentalization into sub-domains exists within the ER. Since the ER membrane and lumen are contiguous, the compartmentalization of the ER into sub-domain cannot exclusively be created by membrane barriers. Adapter proteins nucleate the formation of protein complexes to create sub-domains in the ER. Tetratricopeptide repeats (TPR) containing proteins are known to organize protein complexes involved in a wide range of cellular processes. While the human genome is predicted to encode ~ 180 TPR proteins, only two have previously been shown to localize to the ER. We hypothesized that there are additional TPR-containing adapter proteins that contribute to the organization or compartmentalization of ER processes. To this end, we screened an in silico library of putative TPR containing proteins from the Regan laboratory (Yale University) to identify proteins that potentially possess an N-terminal ER targeting signal sequence. This screen, combined with additional in silico approaches, identified nine putative ER proteins that contained between three to ten TPR motifs. While some of the identified proteins are soluble ER proteins (i.e. TTC13 and TTC17), others were found to reside in the ER membrane with their TPR domains facing the ER lumen (TMTC1-4). TMTC1 and TMTC2 were found to interact with the ER calcium pump SERCA2B, and TMTC2 with calnexin. Additionally, live cell calcium measurements showed a role for TMTC1 and TMTC2 in calcium homeostasis. Overall, this strategy was successful in identifying novel ER proteins with TPR motifs, and this approach can be applied to identify proteins with specific motifs in the ER or other organelles.

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