As shown in Fig 1B, disruption of individual WW domains did not disrupt an Rsp5p-Spt23p or Rsp5p-Mga2p binding. 1/2 and WW domains 1/3 mutants. However, disrupting WW domains 2 and 3 abrogates a physical and functional interaction with substrates 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 WW domain 2/3 mutant is unable to rescue the proliferative defects of cells. Interestingly, while cells are able to grow on oleic acid containing YPD media, they as Saracatinib (AZD0530) 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 pathway when cells are grown in nutrient restrictive media. is an essential protein and one of the most intensely studied of the Nedd4 family (www.yeastgenome.org). The gene has been isolated from numerous genetic screens and has been postulated to play an important role in a plethora of biochemical and cellular events. Surprisingly, we still have very little knowledge of direct Rsp5p targets and how their modification affects specific biochemical processes. Spt23p and Mga2p have emerged as direct and physiologically relevant targets of Rsp5p and the ligase is required for activation of their function (Hoppe et al., 2000; Shcherbik, Zoladek, Nickels, & Haines, 2003; Shcherbik, Kee, Lyon, Huibregtse, & Haines, 2004,). These homologous proteins are endoplasmic reticulum (ER) localized transcription factors that play overlapping roles in up-regulating the expression of cells can be rescued, at least in part, on nutrient rich media by the addition of oleic acid (Hoppe et al, 2000). Rsp5p interacts with substrates via its WW domains (Harvey, & Kumar, 1999; Rotin, Staub, Haguenauer-Tsapis, 2000; Ingham, Gish, & Pawson, 2004). WW domains are protein interaction modules that are approximately forty amino acids in length (Kay, Williamson, & Sudol, 2000; Macias, Wiesner, & Sudol, 2002; Dupre, Urban-Grimal, & Haguenauer-Tsapis, 2004) and its name refers to two strictly conserved tryptophan residues that are spaced 20C22 amino acids apart. In Saracatinib (AZD0530) addition to these amino acids, WW domains contain two highly conserved tyrosine/phenylalanine residues that are located in the middle of the domain as well as a number of semi-conserved amino acids, some of which differ between the various groups (Kay, Williamson, & Sudol, 2000; Macias, Wiesner, & Sudol, 2002; Dupre, Urban-Grimal, & Haguenauer-Tsapis, 2004). Nevertheless, the amino acids that comprise this domain adopt a triple stranded anti-parallel -sheet that forms a hydrophobic pocket for substrate binding. WW domains have been classified into at least four groups based on their binding specificity (Kato, Ito, Kawai, Nagata, & Tanokura, 2002; Kato et al, 2004). Group I binds to poly-proline motifs that contain a tyrosine residue separated by any amino acid (e.g. PPXY); groups II and III associates with longer poly-proline motifs; CYFIP1 and group IV interacts with phosphorylated serine and threonine that are flanked by a proline residue. Rsp5p harbors three group I WW domains and they play unique roles in Rsp5p function. Although disruption of any of the three impairs plasma membrane receptor ubiquitination-mediated endocytosis, mutation of WW domain 1 or 3, severely impairs transport of fluid phase markers to the vacuole Saracatinib (AZD0530) (Dunn, & Hicke, 2001; Gajewska et al, 2001). In addition, mutations in WW domains 2 or 3 3 alter sorting of cargo to multivesicular bodies (Gajewska et al, 2001; Morvan, Froissard, Haguenauer-Tsapis, & Urban-Grimal, 2004) and mRNA nuclear export (Rodriguez, Gwizdek, Haguenauer-Tsapis & Dargemont, 2003). In terms Saracatinib (AZD0530) of mediating interaction with substrates, WW domain 2 performs a dominant role in binding to the carboxy-terminal domain of Rbp1, the large Saracatinib (AZD0530) subunit of RNA polymerase II (Wang, Yang, & Huibregtse, 1999). Considering that each individual WW domain does not perform an essential function (Wang, Yang, & Huibregtse, 1999;.
As shown in Fig 1B, disruption of individual WW domains did not disrupt an Rsp5p-Spt23p or Rsp5p-Mga2p binding