Glucose-Induced Monoubiquitination of the Saccharomyces cerevisiae Galactose Transporter Is Sufficient To Signal Its Internalization

Abstract

In Saccharomyces cerevisiae, the addition of glucose to cells growing on galactose induces internalization of the galactose transporter Gal2p and its subsequent proteolysis in the vacuole. Here we report that the essential step in Gal2p down-regulation is its ubiquitination through the Ubc1p-Ubc4p-Ubc5p triad of ubiquitinconjugating enzymes and Npi1/Rsp5p ubiquitin-protein ligase. Moreover, Gal2p appears to be stabilized in mutant cells defective in the ubiquitin-hydrolase Npi2p/Doa4p, and the mutant phenotype can be reversed by overexpression of ubiquitin. An analysis of the fate of Gal2p in cells overexpressing wild-type ubiquitin as well as its variants incompetent to form polyubiquitin chains showed that monoubiquitination of Gal2p is sufficient to signal internalization of the protein into the endocytic pathway.The covalent linkage of ubiquitin to a variety of eukaryotic proteins targets them for degradation. This process involves sequential transfer of the ubiquitin moiety to specific lysine residues of substrate proteins via an E1-E2-E3 enzyme (ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin-protein ligase) thioester cascade and culminates in formation of an isopeptide bond between the C-terminal glycine of ubiquitin and the ⑀-amino group of lysine(s) on the substrate protein. Specificity in substrate recognition resides largely at the level of E3Јs, and an additional degree of combinatorial specificity probably results from specific E2-E3 interactions. Attachment of ubiquitin to substrate proteins has distinct mechanistic roles in two completely different intracellular proteolytic pathways. Ubiquitination of short-lived cytosolic and nuclear proteins, as well as proteins that undergo endoplasmic reticulum-associated degradation, marks them for hydrolysis by the multicatalytic, multisubunit protease, the 26S proteasome (15,18,20,37,38). For most of the 26S proteasome substrates, the attachment of a polyubiquitin chain in which at least four ubiquitin molecules must be linked by amide bonds between Lys48 of ubiquitin molecules and the C-terminal carboxyl group of the next following ubiquitin facilitates their binding to the proteasome (15,20,37,54). Ubiquitination of many plasma membrane receptors and nutrient transporters, however, signals their internalization into the endocytic pathway and subsequent proteolysis in the vacuole (5,16,43). For all short-lived plasma membrane proteins examined to date, a single ubiquitin moiety or a very short ubiquitin chain appears to suffice to trigger their endocytotic uptake. These plasma membrane proteins are often internalized constitutively. Modulation of the rate of their internalization, induced by a change in nutrient availability, usually from less desirable nitrogen or carbon sources or starvation conditions to preferred sources or rich medium, by binding of their own substrates, or by cell stress, plays the primary role in controlling nutrient uptake (21). Because the ubiquitin itself is a long-lived protein in the yeast Saccharomyces...

Yeast Chfr homologs retard cell cycle at G1and G2/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination

“…Surprisingly, deletion of ubc5 did not suppress Chf2 overexpression even though Ubc4 and Ubc5 are >90% identical in sequence and are reportedly redundant in multiple functions. 5,17,20,56 As Ubc13 and Mms2 function as a heterodimer, 15 the similar phenotypes of ubc13 and mms2 can be easily rationalized.…”

“…Considering the high sequence similarity of Ubc4 and Ubc5 and the frequently observed overlapping functions of Ubc4 and Ubc5, 5,17,20,56 assignment of Ubc4 to the G 1 function of Chf2 and genetic exclusion of Ubc5 was surprising. However, the expression of UBC4 mRNA is highest in exponentially growing cells, while UBC5 mRNA is primarily expressed in stationary phase.…”

“…Many membrane receptors are regulated by mono-or di-ubiquitination which establishes a signal for internalization and lysosomal degradation. [4][5][6][7] Polyubiquitination can occur at multiple primary sites on target proteins. There are seven Lys residues in Ub-all seven have been observed to be modified by polyubiquitination in yeast.…”

“…11,12 Adding complexity to the E2-dependence of the Ub system, in some cases, endocytosis of plasma membrane receptors is mediated by Ubc4 and Ubc5 monoubiquitination. 4,5,20 Biochemical analysis indicates that individual E2 enzymes can functionally pair with multiple E3 ligases 21 while several purified E3 ligases function in vitro with multiple E2 conjugating enzymes. 22 However, the apparent promiscuity of in vitro reactions casts doubt on reactions that have not been validated by genetic analysis.…”

The Rsp5 E3 Ligase Mediates Turnover of Low Affinity Phosphate Transporters in Saccharomyces cerevisiae

“…This ubiquitin-mediated protein degradation pathway is distinct from the ubiquitin proteasome pathway (11). As examples of the ubiquitin-endosome pathway, the yeast Rsp5 E3 ubiquitin ligase is involved in turnover of the Gap1 general amino acid permease (12,13), the Fur4 uracil permease (14 -16), the Tat2 tryptophan permease (17), the Gal2 galactose transporter (18,19), and the Mal61 maltose transporter (20,21). Accordingly, ubiquitin-mediated endocytosis appears to be a general mechanism to respond to changes in nutrient availability.…”