WW domains of Rsp5p define different functions: Determination of roles in fluid phase and uracil permease endocytosis in Saccharomyces cerevisiae

Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, c ontains three WW domains that mediate protein-protein interactions. To dete rmine if these domains adapt Rsp5p to a subset of substrates involved in nu merous cellular processes, we generated mutations in individual or combinat ions of the WW domains. The rsp5-w1, rsp5-w2 and rsp5-w3 mutant alleles com plement RSP5 deletions at 30 degrees. Thus, individual WW domains are not e ssential. Each rsp5-w mutation caused temperature-sensitive growth. Among v ariants with mutations in multiple WW domains, only rsk5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functi ons. To determine whether rsp5-w mutations affect endocytosis, fluid ph;lsc and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is im portant for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, th e WW1 domain affects fluid phase endocytosis, but it does not appear to fun ction in Fur4p endocytosis. Thus, various WW domains play different roles i n the endocytosis of these two substrates. Rsp5p is located in the cytoplas m in a punctate pattern that does not change during the cell cycle. Alterin g WW domains does not change the location of Rsp5p.