WW domains 2 and 3 of Rsp5p play overlapping roles in binding to the LPKY motif of Spt23p and Mga2p.
Rsp5p of Saccharomyces cerevisiae is a member of the C2-WW-HECT family of ubiquitin ligases and it interacts with targets via its WW domains. Spt23p and Mga2p are Rsp5p substrates and Rsp5p activates the OLE1 inducing functions of these membrane-localized transcription factors by ubiquitination. Although it is known that Rsp5p binds Mga2p and Spt23p via an imperfect WW domain-binding site (LPKY) that is located within the carboxy-terminal domain of the proteins, it remains unclear which WW domains mediate binding. We show that Rsp5p mutants harboring mutations in single WW domains are Spt23p/Mga2p binding and ubiquitination proficient. This is also the case for WW domains 1/2 and WW domains 1/3 mutants. However, disrupting WW domains 2 and 3 abrogates a physical and functional interaction with substrates in vitro and in cells. We also show that abrogation of WW domains 2 and 3 eliminates the activity of an Rsp5p dominant-negative mutant and an rsp5 WW domain 2/3 mutant is unable to rescue the proliferative defects of rsp5 Delta cells. Interestingly, while rsp5 Delta cells are able to grow on oleic acid containing YPD media, they as well as those transformed with the WW domain 2/3 mutant are unable to proliferate on oleic acid containing synthetic drop-out media. We conclude from these studies that WW domains 2 and 3 of Rsp5p play overlapping roles in binding to the LPKY site on Spt23p and Mga2p. Also, we propose that WW domains 2 and 3 perform yet to be defined essential function(s) outside of the OLE1 pathway when cells are grown in nutrient restrictive media.
Bhattacharya
Sabyasachi
S
Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, United States.
Zoladek
Teresa
T
Haines
Dale S
DS
D004271:DNA, Fungal; D056827:Endosomal Sorting Complexes Required for Transport; D004790:Enzyme Induction; D044943:Fatty Acid Desaturases / Q000378:metabolism*; D018014:Gene Transfer Techniques; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013230:Stearoyl-CoA Desaturase; D013379:Substrate Specificity; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D004271:DNA, Fungal; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D011994:Recombinant Proteins; C087570:SPT23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase; C029627:delta-9 fatty acid desaturase; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; C469287:RSP5 protein, S cerevisiae