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-enco ded uracil permease. This permease undergoes endocytosis and subsequen t degradation in cells subjected to adverse conditions. The data prese nted here show that uracil permease also undergoes basal turnover unde r normal growth conditions. Both basal and induced turnover depend on the essential Npi1p/Rsp5p ubiquitin-protein ligase. Epitope-tagged ubi quitin variants have been used to show that uracil permease is ubiquit inated in vivo. The ubiquitin-permease conjugates that are readily dem onstrated in wild type cells were barely detectable in npi1 mutant cel ls, indicating that uracil permease may be a physiological substrate o f the Npi1p ubiquitin ligase. The lack of ubiquitination of the permea se in npi1 cells resulted in an increase in active, i.e. plasma membra ne-located, permease, suggesting that there is a direct relationship b etween ubiquitination and removal of the permease from tile plasma mem brane. The accumulation of ubiquitin-permease conjugates in thermosens itive act1 mutant cells, deficient in the internalization step of endo cytosis is consistent with this idea. On the other hand, the degradati on of uracil permease does not require a functional proteasome since t he permease was not stabilized in either pre1 pre2 or cim3 and cim5 mu tant cells that have impaired catalytic (pre) or regulatory (cim) prot easome subunits. In contrast, both basal and stress-stimulated turnove r rates were greatly reduced in pep4 mutant cells having defective vac uolar 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.