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 β subunit, β5/X, β1/Y, or β2/Z, that exhibits chymotrypsin-like, peptidylglutamyl-peptide hydrolyzing, or trypsin-like activity, respectively. Little is known about the relative contribution of specific β 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 β subunits can independently contribute to ER-associated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR). Complete inactivation (> 99.5%) of the β5/X subunit decreased the rate of ATP-dependent conversion of CFTR to trichloroacetic acid soluble fragments by only 40%. Similarly, proteasomes containing only active β1/Y or β2/Z subunits degraded CFTR at ∼50% of the rate observed for fully functional proteasomes. Simultaneous inhibition (> 93%) of all three β subunits blocked CFTR degradation by ∼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 ≥ T-L) as well as a redundancy of β subunit function and provide insight into the mechanism by which active site proteasome inhibitors influence degradation of endogenous protein substrates at the ER membrane.
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