Analysis of a gene encoding Rpn10 of the fission yeast proteasome reveals that the polyubiquitin-binding site of this subunit is essential when Rpn12/Mts3 activity is compromised

Substrates are targeted for proteolysis by the ubiquitin pathway by the add ition of a polyubiquitin chain before being degraded by the 26 S proteasome , Previously, a subunit of the proteasome, S5a, was identified that was abl e to bind to polyubiquitin in vitro and thus proposed to act as a substrate recognition component. Deletion of the corresponding Saccharomyces cerevis iae gene, MCB1/RPN10, rendered cells viable indicating that other proteasom al polyubiquitin receptors must exist. In this study, we describe pus1(+), the fission yeast homologue of RPN10, This gene is also not required for ce ll viability; however, the Delta pus1 mutant is synthetically lethal with m utations in other proteasomal component-encoding genes, namely mts3, pad1, and mts4 (RPN12, RPN11, and RPN1), Overexpression of pus1(+) is able to res cue mts3-1 at 32 degrees C but overexpression of a cDNA encoding a version of Pus1 that does not bind to polyubiquitin cannot and leads to greatly red uced viability when used to rescue the mts3-1 Delta pus1 double mutant. The Mts3 protein was unable to bind to polyubiquitin in vitro, but the Pus1 an d Mts3 proteins were found to bind to one another in vitro, which taken tog ether with the genetic data suggests that they are also closely associated in vivo.