The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. The 26S proteasome consists of the proteolytic core particle (CP) and one or two 19S regulatory particles (RPs). Although the mechanisms of CP assembly are well described, the mechanism of RP assembly is largely unknown. Here, we show that four proteasome-interacting proteins (PIPs), Nas2/p27, Nas6/gankyrin, Rpn14/PAAF1, and Hsm3/S5b, bind specific Rpt subunits of the RP and interact each other genetically. Lack of these PIPs resulted in defective assembly of the 26S proteasome at an early stage, suggesting that these proteins are bona fide RP chaperones. Each of the RP chaperones formed distinct specific subassemblies of the base components and escorted them to mature RPs. Our results indicate that the RP assembly is a highly organized and elaborate process orchestrated by multiple proteasome-dedicated chaperones.

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The IRAI gene is a negative regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. To identify other genes involved in this pathway, we screened yeast genomic DNA libraries for genes that can suppress the heat shock sensitivity of the iral mutation on a multicopy vector. We identified IRA2, encoding a protein of 3,079 amino acids, that is 45% identical to the IRA] protein. The region homologous between the IRA] protein and ras GTPase-activating protein is also conserved in IRA2. IRA2 maps 11 centimorgans distal to the argl locus on the left arm of chromosome XV and was found to be allelic to gk4. Disruption of the IRA2 gene resulted in (i) increased sensitivity to heat shock and nitrogen starvation, (ii) sporulation defects, and (lii) suppression of the lethality of the cdc25 mutant. Analysis of disruption mutants of IRAI and IRA2 indicated that IRAI and IRA2 proteins additively regulate the RAS-cyclic AMP pathway in a negative fashion. Expression of the IRA2 domain homologous with GAP is sufficient for complementation of the heat shock sensitivity of ira2, suggesting that IRA down regulates RAS activity by stimulating the GTPase activity of RAS proteins.