Redundancy of mammalian proteasome beta subunit function during endoplasmic reticulum associated degradation.
Misfolded proteins in the endoplasmic reticulum (ER) are degraded by N-terminal threonine proteases within the 26S proteasome. Each protease is formed by an activated beta subunit, beta5/X, beta1/Y, or beta2/Z, that exhibits chymotrypsin-like, peptidylglutamyl-peptide hydrolyzing, or trypsin-like activity, respectively. Little is known about the relative contribution of specific beta subunits in the degradation of endogenous protein substrates. Using active site proteasome inhibitors and a reconstituted degradation system, we now show that all three active beta subunits can independently contribute to ER-associated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR). Complete inactivation (>99.5%) of the beta5/X subunit decreased the rate of ATP-dependent conversion of CFTR to trichloroacetic acid soluble fragments by only 40%. Similarly, proteasomes containing only active beta1/Y or beta2/Z subunits degraded CFTR at approximately 50% of the rate observed for fully functional proteasomes. Simultaneous inhibition (>93%) of all three beta subunits blocked CFTR degradation by approximately 90%, and inhibition of both protease and ATPase activities was required to completely prevent generation of small peptide fragments. Our results demonstrate both a conserved hierarchy (ChT-L > PGPH > or = T-L) as well as a redundancy of beta subunit function and provide insight into the mechanism by which active site proteasome inhibitors influence degradation of endogenous protein substrates at the ER membrane.
Oberdorf
J
J
Molecular Medicine Division, Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Carlson
E J
EJ
Skach
W R
WR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002474:Cell-Free System; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology; D006031:Glycosylation; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008051:Lipid Bilayers / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology; D014452:Ubiquitins / Q000378:metabolism
C505032:CFTR protein, human; D007783:Lactones; D007976:Leupeptins; D008051:Lipid Bilayers; D009097:Multienzyme Complexes; D021122:Protein Subunits; D014452:Ubiquitins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006427:Hemin; D000255:Adenosine Triphosphate; D014357:Trypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D005990:Glycerol; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde