The Rsp5 ubiquitin ligase is coupled to and antagonized by the Ubp2 deubiquitinating enzyme

Saccharomyces cerevisiae Rsp5 is an essential HECT ubiquitin ligase involved in several biological processes. To gain further insight into regulation of this enzyme, we identified proteins that copurified with epitope-tagged Rsp5. Ubp2, a deubiquitinating enzyme, was a prominent copurifying protein. Rup1, a previously uncharacterized UBA domain protein, was required for binding of Rsp5 to Ubp2 both in vitro and in vivo. Overexpression of Ubp2 or Rup1 in the rsp5-1 mutant elicited a strong growth defect, while overexpression of a catalytically inactive Ubp2 mutant or Rup1 deleted of the UBA domain did not, suggesting an antagonistic relationship between Rsp5 and the Ubp2/Rup1 complex. Consistent with this model, rsp5-1 temperature sensitivity was suppressed by either ubp2Delta or rup1Delta mutations. Ubp2 reversed Rsp5-catalyzed substrate ubiquitination in vitro, and Rsp5 and Ubp2 preferentially assembled and disassembled, respectively, K63-linked polyubiquitin chains. Together, these results indicate that Rsp5 activity is modulated by being physically coupled to the Rup1/Ubp2 deubiquitinating enzyme complex, representing a novel mode of regulation for an HECT ubiquitin ligase.     

Deubiquitination Step in the Endocytic Pathway of Yeast Plasma Membrane Proteins: Crucial Role of Doa4p Ubiquitin Isopeptidase

The Fur4p uracil permease, like most yeast plasma membrane proteins, undergoes ubiquitin-dependent endocytosis and is then targeted to the vacuole (equivalent to the mammalian lysosome) for degradation. The cell surface ubiquitination of Fur4p is mediated by the essential Rsp5p ubiquitin ligase. Ubiquitination of Fur4p occurs on two target lysines, which receive two ubiquitin moieties linked through ubiquitin Lys63, a type of linkage (termed UbK63) different from that involved in proteasome recognition. We report that pep4 cells deficient for vacuolar protease activities accumulate vacuolar unubiquitinated Fur4p. In contrast, pep4 cells lacking the Doa4p ubiquitin isopeptidase accumulate ubiquitin-conjugated Fur4p. These data suggest that Fur4p undergoes Doa4p-dependent deubiquitination prior to vacuolar degradation. Compared to pep4 cells, pep4 doa4 cells have huge amounts of membrane-bound ubiquitin conjugates. This indicates that Doa4p plays a general role in the deubiquitination of membrane-bound proteins, as suggested by reports describing the suppression of some doa4 phenotypes in endocytosis and vacuolar protein sorting mutants. Some of the small ubiquitin-linked peptides that are a hallmark of Doa4 deficiency are not present in rsp5 mutant cells or after overproduction of a variant ubiquitin modified at Lys 63 (UbK63R). These data suggest that the corresponding peptides are degradation products of Rsp5p substrates and probably of ubiquitin conjugates carrying UbK63 linkages. Doa4p thus appears to be involved in the deubiquitination of endocytosed plasma membrane proteins, some of them carrying UbK63 linkages.     

Protein-linked ubiquitin chain structure restricts activity of deubiquitinating enzymes

The attachment of lysine 48 (Lys(48))-linked polyubiquitin chains to proteins is a universal signal for degradation by the proteasome. Here, we report that long Lys(48)-linked chains are resistant to many deubiquitinating enzymes (DUBs). Representative enzymes from this group, Ubp15 from yeast and its human ortholog USP7, rapidly remove mono- and diubiquitin from substrates but are slow to remove longer Lys(48)-linked chains. This resistance is lost if the structure of Lys(48)-linked chains is disrupted by mutation of ubiquitin or if chains are linked through Lys(63). In contrast to Ubp15 and USP7, Ubp12 readily cleaves the ends of long chains, regardless of chain structure. We propose that the resistance to many DUBs of long, substrate-attached Lys(48)-linked chains helps ensure that proteins are maintained free from ubiquitin until a threshold of ubiquitin ligase activity enables degradation.     

The yeast ubiquitin protease,Ubp3p,promotes protein stability

Stu1p is a microtubule-associated protein required for spindle assembly. In this article we show that the temperature-sensitive stu1-5 allele is synthetically lethal in combination with ubp3, gim1-gim5, and kem1 mutations. The primary focus of this article is on the stu1-5 ubp3 interaction. Ubp3 is a deubiquitination enzyme and a member of a large family of cysteine proteases that cleave ubiquitin moieties from protein substrates. UBP3 is the only one of 16 UBP genes in yeast whose loss is synthetically lethal with stu1-5. Stu1p levels in stu1-5 cells are several-fold lower than the levels in wild-type cells and the stu1-5 temperature sensitivity can be rescued by additional copies of stu1-5. These results indicate that the primary effect of the stu1-5 mutation is to make the protein less stable. The levels of Stu1p are even lower in ubp3Delta stu1-5 cells, suggesting that Ubp3p plays a role in promoting protein stability. We also found that ubp3Delta produces growth defects in combination with mutations in other genes that decrease protein stability. Overall, these data support the idea that Ubp3p has a general role in the reversal of protein ubiquitination.     

Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein.

We have recently reported that the yeast plasma membrane uracil permease undergoes cell-surface ubiquitination, which is dependent on the Npi1/Rsp5 ubiquitin-protein ligase. Ubiquitination of this permease, like that of some other transporters and receptors, signals endocytosis of the protein, leading to its subsequent vacuolar degradation. This process does not involve the proteasome, which binds and degrades ubiquitin-protein conjugates carrying Lys48-linked ubiquitin chains. The data presented here show that ubiquitination and endocytosis of uracil permease are impaired in yeast cells lacking the Doa4p ubiquitin-isopeptidase. Both processes were rescued by overexpression of wild-type ubiquitin. Mutant ubiquitins carrying Lys-->Arg mutations at Lys29 and Lys48 restored normal permease ubiquitination. In contrast, a ubiquitin mutated at Lys63 did not restore permease polyubiquitination. Ubiquitin-permease conjugates are therefore extended through the Lys63 of ubiquitin. When polyubiquitination through Lys63 is blocked, the permease still undergoes endocytosis, but at a reduced rate. We have thus identified a natural target of Lys63-linked ubiquitin chains. We have also shown that monoubiquitination is sufficient to induce permease endocytosis, but that Lys63-linked ubiquitin chains appear to stimulate this process.     

Interaction of the Deubiquitinating Enzyme Ubp2 and the E3 Ligase Rsp5 Is Required for Transporter/Receptor Sorting in the Multivesicular Body Pathway

Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.     

Engineering of the yeast ubiquitin ligase Rsp5: isolation of a new variant that induces constitutive inactivation of the general amino acid permease Gap1

Rsp5 is an essential ubiquitin-protein ligase in Saccharomyces cerevisiae . We found previously that the Ala401Glu rsp 5 mutant is hypersensitive to various stresses that induce protein misfolding, suggesting that Rsp5 is a key enzyme for yeast cell growth under stress conditions. To isolate new Rsp5 variants as suppressors of the A401E mutant, PCR random mutagenesis was used in the rsp5 ^A401E gene, and the mutagenized plasmid library was introduced into rsp5 ^A401E cells. As a phenotypic suppressor of rsp5 ^A401E cells, we isolated a quadruple variant (Thr357Ala/Glu401Gly/Lys764Glu/Glu767Gly) on a minimal medium containing the toxic proline analogue azetidine-2-carboxylate (AZC). Site-directed mutagenesis experiments showed that the rsp5 ^T357A/K764E cells were much more tolerant to AZC than the wild-type cells, due to the smaller amounts of intracellular AZC. However, the T357A/K764E variant Rsp5 did not reverse the hypersensitivity of rsp5 ^A401E cells to other stresses such as high growth temperature, ethanol, and freezing treatment. Interestingly, immunoblot and localization analyses indicated that the general amino acid permease Gap1, which is involved in AZC uptake, was absent on the plasma membrane and degraded in the vacuole of rsp5 ^T357A/K764E cells before the addition of ammonium ions. These results suggest that the T357A/K764E variant Rsp5 induces constitutive inactivation of Gap1.     

Substrate- and ubiquitin-dependent trafficking of the yeast siderophore transporter Sit1

Eukaryotic plasma membrane transporters are subjected to a tightly regulated intracellular trafficking. The yeast siderophore iron transporter1 (Sit1) displays substrate-regulated trafficking. It is targeted to the plasma membrane or to a vacuolar degradative pathway when synthesized in the presence or absence of external substrate, respectively. Sorting of Sit1 to the vacuolar pathway is dependent on the clathrin adaptor Gga2, and more specifically on its C-GAT subdomain. Plasma membrane undergoes substrate-induced ubiquitylation dependent on the Rsp5 ubiquitin protein ligase. Sit1 is also ubiquitylated in an Rsp5-dependent manner in internal compartments when expressed in the absence of substrate. In several rsp5 mutants including cells deleted for RSP5 , Sit1 expressed in the absence of substrate is correctly targeted to the endosomal pathway but its sorting to multivesicular bodies (MVBs) is impaired. Consequently, it displays endosome to plasma membrane targeting, with kinetics similar to those observed in vps mutants defective for MVB sorting. Plasma membrane Sit1 is modified by Lys63-linked ubiquitin chains. We also show for the first time in yeast that modification by this latter type of ubiquitin chains is required directly or indirectly for efficient MVB sorting, as it is for efficient internalization at the plasma membrane.     

Functional dissection of a HECT ubiquitin E3 ligase

Ubiquitination is one of the most prevalent protein post-translational modifications in eukaryotes, and its malfunction is associated with a variety of human diseases. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitination remain largely unknown. Here we used a combination of yeast proteome chip assays, genetic screening, and in vitro/in vivo biochemical analyses to identify and characterize eight novel in vivo substrates of the ubiquitinating enzyme Rsp5, a homolog of the human ubiquitin-ligating enzyme Nedd4, in yeast. Our analysis of the effects of a deubiquitinating enzyme, Ubp2, demonstrated that an accumulation of Lys-63-linked polyubiquitin chains results in processed forms of two substrates, Sla1 and Ygr068c. Finally we showed that the localization of another newly identified substrate, Rnr2, is Rsp5-dependent. We believe that our approach constitutes a paradigm for the functional dissection of an enzyme with pleiotropic effects.     

Pleiotropic effects of Ubp6 loss on drug sensitivities and yeast prion are due to depletion of the free ubiquitin pool

Mutation of the mouse Usp14 gene, encoding the homolog of yeast deubiquitinating enzyme Ubp6, causes ataxia. Here we show that deletion of the UBP6 gene in Saccharomyces cerevisiae causes sensitivity to a broad range of toxic compounds and antagonizes phenotypic expression and de novo induction of the yeast prion [PSI+], a functionally defective self-perpetuating isoform of the translation termination factor Sup35. Conversely, overexpression of ubiquitin (Ub) increases phenotypic expression and induction of [PSI+] in the wild type cells and suppresses all tested ubp6Delta defects, indicating that they are primarily due to depletion of cellular Ub levels. Several lines of evidence suggest that Ubp6 functions on the proteasome. First, Ub levels in the ubp6Delta cells can be partly restored by proteasome inhibitors, suggesting that deletion of Ubp6 decreases Ub levels by increasing proteasome-dependent degradation of Ub. Second, fluorescence microscopy analysis shows that Ubp6-GFP fusion protein is localized to the nucleus of yeast cell, as are most proteasomes. Third, the N-terminal Ub-like domain, although it is not required for nuclear localization of Ubp6, targets Ubp6 to the proteasome and cannot be functionally replaced by Ub. The human ortholog of Ubp6, USP14, probably plays a similar role in higher eukaryotes, since it fully compensates for ubp6Delta defects and binds to the yeast proteasome. These data link the Ub system to prion expression and propagation and have broad implications for other neuronal inclusion body diseases.     

WW domains of Rsp5p define different functions: determination of roles in fluid phase and uracil permease endocytosis in Saccharomyces cerevisiae

Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30 degrees. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p.     

A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting

In yeast, the sorting of transmembrane proteins into the multivesicular body (MVB) internal vesicles requires their ubiquitylation by the ubiquitin ligase Rsp5. This allows their recognition by the ubiquitin-binding domains (UBDs) of several endosomal sorting complex required for transport (ESCRT) subunits. K63-linked ubiquitin (K63Ub) chains decorate several MVB cargoes, and accordingly we show that they localize prominently to the class E compartment, which accumulates ubiquitylated cargoes in cells lacking ESCRT components. Conversely, yeast cells unable to generate K63Ub chains displayed MVB sorting defects. These properties are conserved among eukaryotes, as the mammalian melanosomal MVB cargo MART-1 is modified by K63Ub chains and partly missorted when the genesis of these chains is inhibited. We show that all yeast UBD-containing ESCRT proteins undergo ubiquitylation and deubiquitylation, some being modified through the opposing activities of Rsp5 and the ubiquitin isopeptidase Ubp2, which are known to assemble and disassemble preferentially K63Ub chains, respectively. A failure to generate K63Ub chains in yeast leads to an MVB ultrastructure alteration. Our work thus unravels a double function of K63Ub chains in cargo sorting and MVB biogenesis.     

Loss of Ubp3 increases silencing,decreases unequal recombination in rDNA,and shortens the replicative life span in Saccharomyces cerevisiae

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.     

Is the Rsp5 ubiquitin ligase involved in the regulation of ribophagy?

Under nutrient limiting conditions, cytoplasmic components are randomly sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for degradation and recycling. In the last few years, however, it has been observed that several cytoplasmic components such as organelles, pathogens or specific protein complexes can also be selectively targeted for degradation by autophagy-related pathways (reviewed in ref. 1). We have recently shown that in S. cerevisiae, mature ribosomes are subject to such selective degradation by autophagy under starvation conditions, in a process that we termed 'ribophagy.'(2) By genetic screening, we found that selective degradation of 60S large ribosomal subunits depends on the ubiquitin protease Ubp3 and its cofactor Bre5, implying that ribophagy is regulated by ubiquitin-dependent steps. Interestingly, several ubiquitinated proteins accumulate in ribosome fractions isolated from ubp3Delta cells, suggesting that the regulation of ribophagy by ubiquitin may be direct. Here we present data on a potential role of the ubiquitin ligase Rsp5 as a positive regulator of ribophagy, and discuss the possible involvement of ubiquitin as a signaling molecule in this process.     

Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities

The ubiquitin ligase Hul5 was recently identified as a component of the proteasome, a multisubunit protease that degrades ubiquitin-protein conjugates. We report here a proteasome-dependent conjugating activity of Hul5 that endows proteasomes with the capacity to extend ubiquitin chains. hul5 mutants show reduced degradation of multiple proteasome substrates in vivo, suggesting that the polyubiquitin signal that targets substrates to the proteasome can be productively amplified at the proteasome. However, the products of Hul5 conjugation are subject to disassembly by a proteasome-bound deubiquitinating enzyme, Ubp6. A hul5 null mutation suppresses a ubp6 null mutation, suggesting that a balance of chain-extending and chain-trimming activities is required for proper proteasome function. As the association of Hul5 with proteasomes was found to be strongly stabilized by Ubp6, these enzymes may be situated in proximity to one another. We propose that through dynamic remodeling of ubiquitin chains, proteasomes actively regulate substrate commitment to degradation.