Six distinct ATPases
reside within the proteasomal regulatory particle, which has been
proposed to recognize ubiquitinated proteolytic substrates, unfold them,
and initiate their translocation into the proteolytic chamber of the
core particle. The ATPases have been proposed to mediate substrate
unfolding. To explore the roles of ATP in proteasome function, we
introduced the same site-directed mutation into the ATP-binding motif of
each ATPase. The mutations had dramatic effects: nonconservative
substitutions of the active-site Lys in the Walker A motif are lethal in
four of six cases, and conferred a strong growth defect in two cases.
Thus, the ATPase activities are not generally redundant, despite their
multiplicity and sequence similarity. Degradation of a specific
substrate can be inhibited by active site mutations in multiple ATPases,
indicating that the ATPases cooperate in the degradation of individual
substrates. The phenotypic defects were strikingly varied. The most
divergent was that of the rpt1 mutant, which was strongly growth
defective despite showing no general defect in protein turnover. The
rpt1 mutant also displayed a unique cell cycle defect. Proteasomes have
been purified from the mutant strains and characterized in vitro.
Unexpectedly, a dramatic inhibition of peptidase activity was seen with
proteasomes purified from an rpt2 mutant, consistent with a defect in
channel gating. In summary, ATP promotes substrate breakdown through
multipe mechanisms, as reflected by the markedly differentiated in vivo
and in vitro phenotypes of the ATPase mutants. In particular, specific
proteasomal ATPases have functions other than unfolding of the
proteolytic substrate.
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