Redundancy of mammalian proteasome β 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, beta (5)/X, beta (1)/Y, or beta (2)/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 beta active site proteasome inhibitors and a reconstituted degradation system, we now show that all three active subunits can independently contribute to ER-associated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR). Complete inactivation (> 99.5 %) of the beta (5)/X subunit decreased the rate of ATP-dependent conversion of CFTR to trichloroacetic acid soluble fragments by only 40%. Similarly, proteasomes containing only active beta (1)/Y or beta (2)/Z subunits degraded CFTR at similar to 50% of the rate observed for fully functional proteasomes. Simultaneous inhibition (> 93%) of all three beta subunits blocked CFTR degradation by similar to 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 greater than or equal to 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.