Ubiquitylation motifs on target plasma membrane proteins

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Target lysines for ubiquitylation have been identified in a number of cases. Investigations aiming to define the cis signals required for ubiquitylation have revealed that an acidic stretch in the linker region connecting the two halves of the ABC-transporter Ste6p [89], and an N-terminal acidic PEST-like sequence in Fur4p [90] and in the maltose permease Mal61p [81] are essential. Furthermore, many yeast transporters are phosphorylated. It is known at least for soluble proteins that phosphorylation, notably within PEST sequences, is frequently linked to ubiquitylation [9]. The PEST sequence of Fur4p displays serine phosphorylation in its PEST region, a modification required for permease ubiquitylation at nearby lysines [71, 90]. Phosphorylation in this sequence is partly dependent on the redundant Yck1p/Yck2p casein kinase I homologs [71]. Similarly, the unique target Lys in the Zrt1p zinc transporter is 14 amino acids away from a short acidic sequence containing several serines required for Zrt1p ubiquitylation [91]. Two phospho-acceptor residues in the linker region of Ste6p are also important for phosphorylation, efficient ubiquitylation, and internalization [92, 93]. Finally, the acidic, Ser-rich sequence, SINNDAKSS of Ste2p is the target of both phosphorylation partly due to Yck1p/Yck2p, and ubiquitylation [94]. So, when documented, critical Lys residues are generally located within or adjacent to these acidic sequences potentially important for recognition by the ubiquitylation machinery.

The Lys residue of the cis signal SINNDAKSS, is one of the major ubiquitylation sites in the full-length Ste2p receptor [80]. A motif similar to the SINNDAKSS sequence, DAKTI, has been identified in the acidic region of Ste6p required for ubiquitylation of the protein [89], although a Lys-to-Arg mutation within this motif had only a minor effect on Ste6p turnover (suggesting the involvement of additional Lys residues). Two Lys residues included in similar sequences, ERKS and EYKS, have been shown to be essential, together with three other nearby lysines, in the ubiquitylation and turnover of the tryptophan permease, Tat2p [95]. One of the two adjacent lysine residues required for Fur4p ubiquitylation is also included in a similar sequence, EYKSS. We suggest that Lys residues included in D/EXKS/T motifs are probably primary targets for the ubiquitylation of plasma membrane proteins, at least in yeast. In a proteomics approach, attempts have recently been made to identify ubiquitylated proteins in cells with 6His-tagged ubiquitin as their sole ubiquitin [88]. Potentially ubiquitylated proteins (retained on Ni colunms), included 12 plasma membrane transporters. Precise ubiquitylation sites were identified in six transporters including three proteins that have already been shown to be ubiquitylated. Strikingly, the target Lys identified in these transporters lie in very acidic motifs, often rich in Ser residues. Phosphorylation sites were also identified within nine transporters. For two of these transporters the ubiquitylated target Lys lie very close to the identified phosphorylated amino acids and lie in a D/EXKS/T motif. It remains to define whether the identified target Lys are ubiquitylated at the plasma membrane, as ubiquitylation is also involved in other trafficking steps. The very same physiological condition can trigger both Rsp5p-dependent ubiquitylation and internalization of a given plasma membrane transporter, and ubiquitylation at an intracellular location leading to diversion of neosynthesized transporter for lysosome/vacuolar degradation pathway without passing through the plasma membrane (reviewed in [50]). The target Lys in Gap1p identified in the above study differs from the two target Lys shown to be ubiquitylated at the cell surface following the addition of ammonium to cells cultured in nitrogen-poor medium [72], and the function of this modification remains to be determined. Uracil permease, Fur4p undergoes both substrate-induced plasma membrane internalization, and direct vacuolar routing if synthesized de novo in the presence of its substrate [5]. The ubiquitylation required for endosome sorting and vacuolar degradation, also Rsp5p-dependent,  did not require prior phosphorylation of the PEST sequence of Fur4p [5], whereas plasma membrane ubiquitylation did [90]. The two target Lys for plasma membrane ubiquitylation [71] were also sites of ubiquitylation for endosome sorting, together with other Lys [5]. Thus, even for the same protein, the sequences required for ubiquitylation by the same enzyme are not the same at different intracellular locations. Obviously, the proteomic approach, performed for cells cultured in standard conditions, should be complemented with data obtained in defined physiological conditions corresponding to known trafficking of a given transporter. If this study requires complementary analyses, it constitutes an important step in determining whether the ubiquitylation sites in plasma membrane transporters display some signature. The data currently available, conforting prior studies, highlight the link between the phosphorylation, D/EXKS/T motifs, and ubiquitylation, possibly at plasma membrane, of some transporters.