This dissertation focuses on the biochemical and genetic characterization of the protein-protein interaction in the budding yeast Saccharomyces cerevisiae between the cytosolic translation initiation guanine nucleotide exchange factor eIF2B and the endoplasmic reticulum (ER) membrane-embedded very-long-chain fatty acid (VLCFA) synthesis beta-keto-reductase enzyme YBR159W (IFA38). The dissertation is divided between the physical characterization of the interaction and examination of the functional consequences the ybr159wΔ deletion has on the yeast cells physiology. I first look at how the interaction is occurring in yeast. I utilize yeast 2-hybrid analysis to show that eIF2B subunits GCD6 and GCD7 interact with YBR159W. My experiments show that eIF2B does not interact with other VLCFA synthesis enzymes and that YBR159W does not interact directly with the other canonical components of the eIF2B complex. Compared to a wild type strain, a ybr159wΔ null yeast strain has a reduced growth rate and the hallmarks of a reduced translation activity including reduced 35S-methionine incorporation and low levels of polyribosomes. It is unknown if the reduced translation rate is a direct or indirect consequence of the ybr159wΔ mutation. The total cellular abundance of eIF2B complex is reduced in a ybr159wΔ null strain but the stoichiometry of the eIF2B complex and its enzymatic activity appears equivalent to wild-type. Deletion of YBR159W or other VLCFA synthesis enzymes significantly alters sphingolipid production in yeast. Deletion of the eIF2B subunit GCN3 does not cause a significant change in sphingolipid production in yeast. In the second section, I examine what effect YBR159W has on the localization of the cytoplasmic eIF2B complex. In yeast, eIF2B forms one or two large foci known as eIF2B bodies. I discover that YBR159W is important for either the formation or maintenance of the eIF2B body. In ybr159wΔ null yeast, eIF2B forms many smaller foci throughout the cell. Other VLCFA synthesis enzyme mutants display this same phenotype. I also find that a fraction of the eIF2B complex associates with lipid membranes. This lipid association is not dependent on the presence of YBR159W and is not mediated by rough ER bound ribosomes. Further experiments are required to determine the mechanistic and functional role of YBR159W interacting with eIF2B.
|Date||04 June 2013|
|Creators||Browne, Christopher Michael|
|Contributors||Nicholas J. Reiter, Claus Schneider, Kevin L. Schey, P. Anthony Weil, Andrew J. Link|
|Source Sets||Vanderbilt University Theses|
|Rights||unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.|
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