The degradation of membrane proteins from the mammalian endoplasmic reticulum

Abstract

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.

(cont.) In contrast, US 11 only degrades MHC class I molecules possessing a cytoplasmic tail of 30 amino acids. These data support a model that US2 and US 11 act through distinct degradative mechanisms. To define modular functional domains of US2 and US 11, lumenal or cytoplasmic domains were reciprocally exchanged between the viral molecules and between each viral molecule and their degradation substrate, the HLA-A2 heavy chain. Most chimeric molecules were not targeted for degradation when expressed alone or with their complementary construct. However, the US 11 molecule in which the cytoplasmic tail was exchanged for that of HLA-A2 was rapidly dislocated from the ER to the cytosol. In addition, lumenal GFP possessing the transmembrane domain of US 11 and the cytoplasmic tail of HLA-A2 was rapidly dislocated from the ER to the cytosol. Mutation of the critical glutamine residue in the transmembrane domain of US 11 essential for destruction of MHC class I molecules resulted in a marked stabilization of the GFP construct.