Ubiquitination Mediated by the Npi1p/Rsp5p Ubiquitin-protein Ligase Is Required for Endocytosis of the Yeast Uracil Permease

Abstract

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.

Uptake of the ATP-Binding Cassette (ABC) Transporter Ste6 into the Yeast Vacuole Is Blocked in the doa4 Mutant

“…The most suggestive evidence for a role of ubiquitination as a trigger of endocytosis has been gained from experiments on the ␣-pheromone receptor Ste2 (Hicke and Riezman, 1996). Additional evidence for a role of ubiquitination in endocytosis has been obtained for several other yeast plasma membrane proteins, including the a-factor receptor Ste3 (Roth and Davis, 1996), uracil permease (Fur4) (Galan et al, 1996), the general amino acid permease (Gap1; Hein et al, 1995), and the multidrug transporter Pdr5 (Egner and Kuchler, 1996). Ubiquitination has also been directly implicated in endocytosis and lysosomal degradation of the mammalian growth hormone receptor (Strous et al, 1996).…”

A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.

“…Equally important to their conjugation, ubiquitin subunits are also removed by ubiquitin carboxyl-terminal hydrolases (isopeptidases), making ubiquitination a reversible and regulatable posttranslational modification (Hershko and Ciechanover, 1992;Wilkinson, 1995). While ubiquitinmediated proteolysis is the best-studied function of ubiquitin conjugation, it has also been implicated in regulating a host of other cellular processes, including endocytosis and vacuolar targeting, protein kinase activation, protein import into mitochondria, and peroxisome biogenesis (Ciechanover, 1994;Wilkinson, 1995;Chen et al, 1996;Egner and Kuchler, 1996;Galan et al, 1996;Hicke and Riezman, 1996;Roth and Davis, 1996;Strous et al, 1996). The utility of the ubiquitin system raises the question of whether parallel pathways exist, using novel ubiquitin-like proteins.…”

“…Whereas the primary fate of ubiquitinated proteins is degradation by the 26S proteosome (Ciechanover, 1994;Jentsch and Schlenker, 1995), it has long been recognized that ubiquitination is likely to have roles beyond proteolysis. Ubiquitin conjugation has been recently shown to act as a signal for endocytosis and vacuolar targeting (Egner and Kuchler, 1996;Galan et al, 1996;Hicke and Riezman, 1996;Roth and Davis, 1996;Strous et al, 1996) and as regulator of IKB kinase activity (Chen et al, 1996). In addition to functions other than proteolysis, a recurring question related to the ubiquitin system has been whether substrates other than ubiquitin may be used in parallel pathways to regulate as yet unrecognized cellular activities.…”

Human ubiquitin‐protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin‐conjugating enzymes

“…(i) Saccharomyces cerevisiae Rsp5 was originally identified as a revertant of mutations in the Spt3 gene encoding a TFIID-binding protein (Eisenmann et al 1992). Rsp5 is also required for the degradation of Gap1 and Fur4 permeases (Hein et al 1995), the endocytosis of uracil permease (Galan et al 1996), and the minichromosome maintenance (Yashiroda et al 1996). The large subunit of RNA polymerase II is a substrate of Rsp5 , and Schizosaccharomyces pombe Pub1 seems to be involved in the degradation of cdc25 phosphatase (Nefsky & Beach 1996).…”