Interactions between Rsp5p and its plasma membrane substrates

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Although much is already known about the putative ubiquitylation signals in yeast transporters and, to a lesser extent, the role played by Rsp5p domains in this process (see below), we still have no clear overall picture of the way in which Rsp5p interacts with transporters.

Rsp5 is the only member of the Nedd4 family of HECT-E3 proteins in yeast and is currently the only ubiquitin ligase shown to be involved in the posttranslational modification of yeast cell-surface proteins [21]. The three WW domains of Rsp5p have been shown to be important for Ste2p ubiquitylation [96], whereas only the second and moreover the third WW are required for the efficient ubiquitylation of Fur4p ([97] and Marchal and Urban-Grimal, unpublished data). These data suggest that the Rsp5p WW domains, or a subset of these domains, are involved, directly or indirectly, in substrate recognition. However, Rsp5p-WW domains are Group I WW that bind PPXY motifs [98] and yeast cell-surface proteins include generally no obvious PPXY motifs that could accommodate the direct binding of Rsp5p-WWs. Thus, there may be other, currently unknown motifs involved in the interaction of Rsp5p via its WW domains, or Rsp5p may interact with its plasma membrane substrates via a novel type of interaction, or with the assistance of adaptors. The Bul proteins are possible candidate adaptor proteins. Bul1p interacts physically with Rsp5p via its PPXY motif, and Bul2p is a homolog [99, 100] . Both Bul proteins contain a PPXY motif and a mutant Bul1p protein with an altered PY-motif has been shown to be defective in Rsp5p binding [100]. Bul1p has been shown to interact with Rsp5p functionally, biochemically, and genetically [99, 100]. Bul1p has been shown to assist Rsp5p in several of its many functions  in the cell [72, 101-104]. Both proteins are conjugated with ubiquitin, the role of this modification being unknown [88]. Deletion of BUL1 and BUL2 impairs cell-surface ubiquitylation and downregulation of the cell-surface transporters Gap1p [72] and Fui1p (Volland, personal communication) but has no effect on Fur4p (Castillon and Urban-Grimal, personal communication). Thus, the Bul proteins may be involved in the recognition of only a subset of of Rsp5p plasma membrane substrates.

A last possibility would be that productive Rsp5p-membrane proteins interaction at the cell surface could be only transient and weak as already postulated for Nedd4-Eps15 interaction [105]. No physical interaction has been found between Rsp5p and the cytoplasmic part of Fur4p containing the PEST signal for ubiquitylation (even if a PEST variant efficiently constitutively ubiquitylated in vivo after changing all Ser residues to Glu, which mimics phosphorylation, was used) (Castillon and Urban-Grimal, unpublished data). Rsp5p is peripherally associated with membranes and has been shown to function as part of a multimeric protein complex at the plasma membrane [96, 106]. One possiblity would be that at this location Rsp5p could be able to recognize any of its target lysines provided that they are uncovered following a change in the conformation of the substrate protein due to phosphorylation at a nearby site.

Whatever the mode of interaction between Rsp5p and its plasma membrane substrates, little is known about the regulation of this interaction. Ste2p displays ligand-induced hyperphosphorylation, which is required for subsequent ubiquitylation, and the downregulation of many transporters is also highly controlled. The cell-surface ubiquitylation of Gap1p, Zrt1p, the Mg++ transporter, sugar transporters and Fur4p is induced and/or accelerated in the presence of ammonium, zinc, magnesium, glucose, and uracil, respectively. Other plasma membrane transporters have been reported to undergo controlled downregulation, but it is not yet defined whether this downregulation depends on ubiquitylation events (reviewed in [50]). It is unclear whether such regulation depends on phosphorylation, or whether these transporters display specific conformational changes, exposing critical lysine residues. The possibility that Rsp5p activity is specifically regulated also remains to be addressed, by analogy with data reported in mammals (see below).