Proteasome
deubiquitinating activity - Intrinsic or associated?
Adi Guterman,
Michael Glickman
Dept. of Biology, The Technion, I.T.T., Haifa, 32000, Israel
(adigute@tx.technion.ac.il)
Substrates
destined for degradation by the 26S proteasome are first labeled with
polyubiquitin chains. These chains can be dismantled by deubiquitinating
enzymes (DUBs). We find that proteasomes purified from yeast are able to carry
out DUB activity that is ATP-dependent and sensitive to Ubiquitin-aldehyde,
NEM, and phenanthroline. A number of reports have identified various
proteasome-associated DUBs responsible for processing of polyubiquitin chains.
These DUBs include members of the UCH (Uch37/p37), UBP (Ubp6/Usp15) or a
metalloprotease-resembling (Rpn11) families. So far, only Rpn11 has been
characterized as a stochiometric component of highly purified proteasomes. We
now show that the majority of naturally occurring Ubp6 in whole cell extracts
is also associated with proteasomes, and that purified 26S holoenzymes indeed
contain Ubp6. In an attempt to study the relative contributions of Rpn11 and
Ubp6 to proteasome function, we purified proteasomes from a mutant in the
alleged MPN+ catalytic domain of Rpn11, as well as from the ubp6 null. Detailed
characterization of DUB activity found in proteasomes from these strains
supports previous identification of Rpn11 as a putative metalloprotease and
Ubp6 as a cysteine protease. Interestingly, both mutations significantly lower
the inherent DUB activity of purified proteasomes but do not abolish it
completely, suggesting that Rpn11 and Ubp6 serve complementary role.