Ubiquitin‐Independent Entry into the Yeast Recycling Pathway

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Abstract
The yeast a‐factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand‐independent pathway links to vacuolar degradation of the receptor, while a ligand‐dependent uptake pathway links primarily to recycling and thus, receptor reutilization. Ste3p ubiquitination triggers its uptake into the constitutive pathway. The present work considers the role of the receptor ubiquitination associated with the Ste3p ligand‐dependent endocytosis mechanism. The doa4Δ mutation which reduces the cellular availability of ubiquitin blocks the Ste3p constitutive uptake. Uptake into the Ste3p ligand‐dependent recycling pathway, however, continues unimpaired. The ubiquitin independence of Ste3p ligand‐dependent uptake was further indicated by analysis of receptor mutants having Lys‐to‐Arg substitutions at all possible ubiquitin acceptor sites. Again, the ligand‐induced internalization was unimpaired. Furthermore, no discernible effect was seen on either recycling or on the slow PEP4‐dependent turnover of the receptor (for receptor internalized via the ligand‐dependent mechanism, trafficking to the vacuole/lysosome is the minor, alternate fate to recycling). However, one striking effect of the Lys‐to‐Arg mutations was noted. Following a prolonged exposure of the cells to the a‐factor ligand, rather than being delivered to the vacuolar lumen, the Lys‐to‐Arg receptor was found to localize instead to the limiting membrane of the vacuole. Thus, while receptor ubiquitination clearly is not required for either the a‐factor‐dependent uptake into recycling pathway or for the recycling itself, it does affect the routing of receptor to the vacuole, likely by affecting the routing through the late endosomal, multivesicular body: ubiquitinated receptor may be selected into the internal, lumenal vesicles, while unmodified receptor may be left to reside at the limiting external membrane.