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Hidde L. Ploegh. Casagrande, Rocco (Rocco John), 1973- Massachusetts Institute of Technology. Dept. of Biology. Massachusetts Institute of Technology. Department of Biology 2005-08-23T21:26:54Z 2005-08-23T21:26:54Z 2001 2001 http://hdl.handle.net/1721.1/8580 49264403 Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001. Includes bibliographical references. The majority proteins that misfold in the endoplasmic reticulum (ER) are dislocated to the cytoplasm for degradation by the proteasome. This process of dislocation and degradation occurs in several steps. First, unfolded proteins need to be recognized in the ER. Following recognition, unfolded proteins are transported from the ER to the cytoplasm. Once in the cytoplasm, the misfolded proteins are processed and destroyed by the proteasome. This thesis examines each step of the process from the recognition of unfolded proteins in the ER, to the processing of proteins prior to degradation. We find that the murine MHC class I molecule H2-Kb expressed heterologously in yeast, is degraded in a proteasome-dependent fashion. Mere expression of H2-Kb induces the unfolded protein response. If the unfolded protein response is disrupted, degradation of H2-Kb and the misfolded, lumenal protein, T-CPY*, is greatly impaired, indicating that the unfolded protein response is essential for the recognition of unfolded proteins in the ER prior to degradation. The translocon has been implicated as the point of exit for unfolded proteins from the ER. We used existing strains that carry mutations in SEC61 that have demonstrated impaired degradation of the soluble ER protein, CPY*. We find that these strains demonstrate no defect in the degradation of the transmembrane protein HLA-A2, indicating that transmembrane and soluble proteins may interact with distinct aspects of the translocon prior to dislocation. It is hypothesized that N-linked glycans and ubiquitin must be removed from a protein prior to degradation by the proteaseome. However, we find that the disruption of PNGJ, the gene encoding yeast N-glycanase, has no effect on the degradation of CPY* or HLA-A2. To study how the removal of ubiquitin from proteins is involved in the degradation of unfolded proteins, we used a novel covalent inhibitor of de-ubiquitinating enzymes, ubiquitin-vinyl-sulfone. This inhibitor modifies 6 polypeptides in a yeast lysate, all 6 of which are known or putative de-ubiquitinating enzymes. The expression of these 6 de-ubiquitinating enzymes is not altered by heat shock, induction of the unfolded protein response or cell cycle arrest. by Rocca Casagrande. Ph.D. 178 leaves 14276066 bytes 14275823 bytes application/pdf application/pdf eng Massachusetts Institute of Technology M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 Biology. Protein degradation from the endoplasmic reticulum in yeast Thesis application/pdf false Publication unknown unknown <Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="24e33b43-b579-4256-ae4c-ebf28b732bb8"> <Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type> <Language>eng</Language> <Title>Protein degradation from the endoplasmic reticulum in yeast</Title> <PublishedIn> <Publication> </Publication> </PublishedIn> <PublicationDate>2001</PublicationDate> <Authors> <Author> <DisplayName>Casagrande, Rocco (Rocco John), 1973-</DisplayName> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> </Authors> <Editors> </Editors> <Publishers> <Publisher> <DisplayName>Massachusetts Institute of Technology</DisplayName> <OrgUnit /> </Publisher> </Publishers> <License>http://dspace.mit.edu/handle/1721.1/7582</License> <Keyword>Biology.</Keyword> <Abstract>The majority proteins that misfold in the endoplasmic reticulum (ER) are dislocated to the cytoplasm for degradation by the proteasome. This process of dislocation and degradation occurs in several steps. First, unfolded proteins need to be recognized in the ER. Following recognition, unfolded proteins are transported from the ER to the cytoplasm. Once in the cytoplasm, the misfolded proteins are processed and destroyed by the proteasome. This thesis examines each step of the process from the recognition of unfolded proteins in the ER, to the processing of proteins prior to degradation. We find that the murine MHC class I molecule H2-Kb expressed heterologously in yeast, is degraded in a proteasome-dependent fashion. Mere expression of H2-Kb induces the unfolded protein response. If the unfolded protein response is disrupted, degradation of H2-Kb and the misfolded, lumenal protein, T-CPY*, is greatly impaired, indicating that the unfolded protein response is essential for the recognition of unfolded proteins in the ER prior to degradation. The translocon has been implicated as the point of exit for unfolded proteins from the ER. We used existing strains that carry mutations in SEC61 that have demonstrated impaired degradation of the soluble ER protein, CPY*. We find that these strains demonstrate no defect in the degradation of the transmembrane protein HLA-A2, indicating that transmembrane and soluble proteins may interact with distinct aspects of the translocon prior to dislocation. It is hypothesized that N-linked glycans and ubiquitin must be removed from a protein prior to degradation by the proteaseome. However, we find that the disruption of PNGJ, the gene encoding yeast N-glycanase, has no effect on the degradation of CPY* or HLA-A2. To study how the removal of ubiquitin from proteins is involved in the degradation of unfolded proteins, we used a novel covalent inhibitor of de-ubiquitinating enzymes, ubiquitin-vinyl-sulfone. This inhibitor modifies 6 polypeptides in a yeast lysate, all 6 of which are known or putative de-ubiquitinating enzymes. The expression of these 6 de-ubiquitinating enzymes is not altered by heat shock, induction of the unfolded protein response or cell cycle arrest.</Abstract> <Access xmlns="http://purl.org/coar/access_right" > </Access> </Publication>