The degradation of membrane proteins from the mammalian endoplasmic reticulum

Membrane glycoproteins of the secretory pathway that cannot adopt their native conformation are targeted for dislocation from the endoplasmic reticulum (ER) membrane for subsequent degradation by the cytosolic proteasome. This thesis investigates factors influencing the catalyzed destruction of MHC class I molecules by the HCMV glycoproteins US2 and US 11 and the degradation of the model substrate TCRuc. The ER chaperone calnexin, implicated in glycoprotein folding, and the ER chaperones protein disulfide isomerase and Erolu, implicated in substrate disulfide bond formation, were examined for their roles in protein dislocation. By targeting these ER chaperones with siRNA constructs, the cellular levels of these ER chaperones were significantly reduced. Nevertheless, the rates of degradation of TCRct and the US2- and US 11-catalyzed destruction of MHC class I molecules were similar to wild-type cells. The Unfolded Protein Response (UPR) transcriptionally regulates ER chaperones and is essential in S. cerevisiae for efficient degradation of model ER substrates. In mammalian cells, neither the ATF6-dependent response nor the IREltc-XBP-1-dependent response of the UPR was found to be essential for efficient degradation of TCRc, US2-, or US 11-catalyzed destruction of MHC class I molecules. Interestingly, the ATF6 response, but not the IRElct-XBP-1 response, is essential for cellular viability. To better define the substrate requirements of US2 and US 11, the 30 residue cytoplasmic tail of MHC class I molecules was mutated. US2 can degrade MHC class I molecules with a cytoplasmic tail shortened to 10 residues or lengthened, by the fusion of GFP to the C-terminus, to several hundred residues.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Biology

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2003

Author dc:creator

• Stern, Patrick J. (Patrick Joseph), 1972-

Advisor dc:contributor.advisor

• Hidde L. Ploegh.

• Biology.

Statement dc:rights

• 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.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/29361

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/29361