Genetics, Vol. 157, 91-101, January 2001, Copyright © 2001

WW Domains of Rsp5p Define Different Functions: Determination of Roles in Fluid Phase and Uracil Permease Endocytosis in Saccharomyces cerevisiae

Beata Gajewskaa, Joanna Kaminskaa, Alicja Jesionowskaa, Nancy C. Martinb, Anita K. Hopperc, and Teresa Zoladeka
a Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland,
b Department of Biochemistry, University of Louisville, Louisville, Kentucky 40292
c Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Corresponding author: Teresa Zoladek, Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland., teresa{at}ibb.waw.pl (E-mail)

Communicating editor: F. WINSTON

Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30°. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p.





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