title: Regulation of selective protein degradation in the endoplasmic reticulum by redox potential.
abstract: Recent studies show that the endoplasmic reticulum (ER) contains proteases, but it is not understood how these enzymes are regulated. In this report we study the selective ER degradation of the subunits (alpha beta gamma delta epsilon zeta) of the T-cell antigen receptor (TCR). When analyzed in vivo, unassembled subunits of the TCR fail to reach the Golgi apparatus and show a differential sensitivity to degradation after synthesis. The alpha, beta, and delta subunits are degraded rapidly, while gamma, epsilon, and zeta are stable. To study the regulation of proteolysis in more detail, beta, gamma, delta, and epsilon subunits were expressed alone in fibroblasts and their selective degradation analyzed in vitro. The beta and delta chains were degraded in the complete absence of vesicular transport, indicating their degradation in the ER membrane compartment. Proteolysis was unaffected by GTP gamma S (guanosine 5'-O-(thiotriphosphate)), EDTA, or depletion of ATP. The gamma and epsilon subunits were stable under the same in vitro conditions, indicating that the assay reconstituted selective protein degradation within the ER. Furthermore, the results showed that the gamma and epsilon subunits did not escape degradation by being transported from the ER to pre-Golgi, or cis-Golgi, membrane compartments. Structural determinants of ER degradation contained within the membrane anchor of the TCR beta subunit were only active in permeabilized cells when reducing agents were added to the assay. Surprisingly, reducing conditions disrupted the regulation of ER proteolysis and induced rapid ER degradation of the stable CD3 gamma subunit and of a control interleukin 2 receptor chimera. Taken together, the results indicated that the ER membrane compartment regulates the selective degradation of newly synthesized proteins. Importantly, the stability of proteins retained in the ER was highly sensitive to redox conditions. It is possible that the redox buffer within the ER lumen may regulate ER protein degradation in vivo.
authors.affiliation: Beth Israel Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215.
authors.forename: J
authors.initials: J
authors.lastname: Young
authors.forename: L P
authors.initials: LP
authors.lastname: Kane
authors.forename: M
authors.initials: M
authors.lastname: Exley
authors.forename: T
authors.initials: T
authors.lastname: Wileman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015703:Antigens, CD / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology; D010084:Oxidation-Reduction; D011817:Rabbits / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000378:metabolism; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D014162:Transfection
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D009842:Oligopeptides; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; D016692:Receptors, Antigen, T-Cell, gamma-delta; D011994:Recombinant Proteins; D010450:Endopeptidases; D005978:Glutathione; D004229:Dithiothreitol