Interaction of the Doa4 deubiquitinating enzyme with the yeast 26S proteasome

The Saccharomyces cerevisiae Doa4 deubiquitinating enzyme is required for t he rapid degradation of protein substrates of the ubiquitin-proteasome path way. Previous work suggested that Doa4 functions late in the pathway, possi bly by deubiquitinating (poly)ubiquitin-substrate intermediates associated with the 26S proteasome. We now provide evidence for physical and functiona l interaction between Doa4 and the proteasome. Genetic interaction is indic ated by the mutual enhancement of defects associated with a deletion of DOA 4 or a proteasome mutation when the two mutations are combined. Physical as sociation of Doa4 and the proteasome was investigated with a new yeast 26S proteasome purification procedure, by which we find that a sizeable fractio n of Doa4 copurifies with the protease. Another yeast deubiquitinating enzy me, Ubp5, which is related in sequence to Dean, but cannot substitute for i t even when overproduced, does not associate with the proteasome. DOA4-UBP5 chimeras were made by a novel PCR/yeast recombination method and used to i dentify an N-terminal 310-residue domain of Doa4 that, when appended to the catalytic domain of Ubp5, conferred Doa4 function, consistent with Ubp enz ymes having a modular architecture. Unlike Ubp5, a functional Doa4-Ubp5 chi mera associates with the proteasome, suggesting that proteasome binding is important for Doa4 function. Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic int ermediates on the proteasome before or after initiation of substrate breakd own.