Substrates of the ubiquitin-dependent
protein degradation system are digested by the 26S proteasome. The 20S
proteasome, the catalytic core of this essential protease complex, is
composed of four stacked heptameric rings. Its outer rings are made up
of alpha-type subunits, and the inner two rings of beta-type subunits.
In yeast, three of the seven beta subunits -- Doa3, Pup1 and Pre3
--house protease active sites. These three subunits are each
synthesized with an N-terminal propeptide sequence, which is processed
autocatalytically during proteasome assembly. We have demonstrated
that one role of these propeptides is to protect the subunit from
inactivation, by shielding the mature N-terminus from
acetylation. While the deletion of the Pup1 and Pre3 propeptides has
little if any effect on cell growth and protein degradation in vivo,
cells deleted for the Doa3 propeptide fail to grow. Previous work has
shown that the lethality of this mutation is likely due to the
inability of the propeptide-deleted Doa3 subunit to incorporate
efficiently into assembling proteasomes. In order to probe the
function of the Doa3 propeptide, we have constructed a series of
charged-to-alanine mutants in the propeptide coding region. These
mutants display a variety of growth phenotypes stemming from defects
in proteasome assembly. Several high copy suppressors of two of these
mutants have been identified.
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