Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease.

Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. This permease undergoes endocytosis and subsequent degradation in cells subjected to adverse conditions. The data presented here show that uracil permease also undergoes basal turnover under normal growth conditions. Both basal and induced turnover depend on the essential Npi1p/Rsp5p ubiquitin-protein ligase. Epitope-tagged ubiquitin variants have been used to show that uracil permease is ubiquitinated in vivo. The ubiquitin-permease conjugates that are readily demonstrated in wild type cells were barely detectable in npi1 mutant cells, indicating that uracil permease may be a physiological substrate of the Npi1p ubiquitin ligase. The lack of ubiquitination of the permease in npi1 cells resulted in an increase in active, i.e. plasma membrane-located, permease, suggesting that there is a direct relationship between ubiquitination and removal of the permease from the plasma membrane. The accumulation of ubiquitin-permease conjugates in thermosensitive act1 mutant cells, deficient in the internalization step of endocytosis is consistent with this idea. On the other hand, the degradation of uracil permease does not require a functional proteasome since the permease was not stabilized in either pre1 pre2 or cim3 and cim5 mutant cells that have impaired catalytic (pre) or regulatory (cim) proteasome subunits. In contrast, both basal and stress-stimulated turnover rates were greatly reduced in pep4 mutant cells having defective vacuolar protease activities. We therefore propose that ubiquitination of uracil permease acts as a signal for endocytosis of the protein that is subsequently degraded in the vacuole.