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, 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 degrees. 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.
Gajewska
B
B
Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.
Kamińska
J
J
Jesionowska
A
A
Martin
N C
NC
Hopper
A K
AK
Zoładek
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002453:Cell Cycle; D004271:DNA, Fungal / Q000235:genetics; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D033704:Nucleotide Transport Proteins; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
D004271:DNA, Fungal; D056827:Endosomal Sorting Complexes Required for Transport; C035528:FUR4 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033704:Nucleotide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; C469287:RSP5 protein, S cerevisiae