Proteasomes are the major protein-degrading complexes in the cytosol and regulate many cellular processes. To examine the functional importance of the MC14/MECL-1 proteasome active site subunits, cell lines expressing a catalytically inactive form of MECL-1 were established. Whereas mutant MECL-1 was readily incorporated into cytosolic proteasomes, replacing the constitutive MC14 subunit, removal of the prosequence was incomplete indicating that its processing required autocatalytic cleavage. Functional analyses showed that the absence of the MC14/MECL-1 active sites abrogated proteasomal trypsin-like activity, but did not affect other catalytic activities. Our data demonstrate a conservation of cleavage specificity between mammalian and yeast proteasomes.