Functional Domains of the Rsp5 Ubiquitin-Protein Ligase

RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 ubiquitin-protein ligase. Hect E3 proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity. We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the ubiquitin-thioester intermediate. Mutations within the hect domain that affect either the ability to form a ubiquitin-thioester or to catalyze substrate ubiquitination abrogate in vivo function, strongly suggesting that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linked to its essential function. The amino-terminal region of Rsp5 contains three WW domains and a C2 calcium-binding domain. Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and ubiquitination lie within the region required for in vivo function. Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role in the recognition of at least some of the substrates of Rsp5, including those related to its essential function. In addition, we show that haploid yeast strains bearing complete disruptions of either of two other hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.     

The HECT Domain of the Ubiquitin Ligase Rsp5 Contributes to Substrate Recognition

Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligase-substrate interactions. Rsp5 interacts directly with its substrate Cps1 via a novel interaction mode. Perturbation of this mode of interaction revealed a compensatory role for the Rsp5 adaptor Bsd2. These results highlight the ability of Rsp5 to interact with substrates via multiple modalities, suggesting additional mechanisms of regulating this interaction and relevant outcomes.     

Enhancement of Stress Tolerance in Saccharomyces cerevisiae by Overexpression of Ubiquitin Ligase Rsp5 and Ubiquitin-Conjugating Enzymes

Yeast Saccharomyces cerevisiae cells overexpressing essential ubiquitin ligase Rsp5 or ubiquitin-conjugating enzymes (Ubc1-Ubc13) showed tolerance to various stresses. Co-overexpression of Rsp5 and Ubc1, Ubc2, Ubc3, Ubc5, Ubc6, Ubc9, Ubc10, Ubc11, Ubc12, or Ubc13 further enhanced stress tolerance. These results suggest that overexpression of ubiquitin-related enzymes might be a useful method for breeding novel stress-resistant strains.     

Cerebral Cavernous Malformation 2 Protein Promotes Smad Ubiquitin Regulatory Factor 1-mediated RhoA Degradation in Endothelial Cells

Mutation of CCM2 predisposes individuals to cerebral cavernous malformations, vascular abnormalities that cause seizures and hemorrhagic stroke. CCM2 has been proposed to regulate the activity of RhoA for maintenance of vascular integrity. Herein, we define a novel mechanism where the CCM2 phosphotyrosine binding (PTB) domain binds the ubiquitin ligase (E3) Smurf1, controlling RhoA degradation. Brain endothelial cells with knockdown of CCM2 have increased RhoA protein and display impaired directed cell migration. CCM2 binding of Smurf1 increases Smurf1-mediated degradation of RhoA. CCM2 does not significantly alter the catalytic activity of Smurf1, nor is CCM2 a Smurf1 substrate. Rather the CCM2-Smurf1 interaction functions to localize Smurf1 for RhoA degradation. These findings provide a molecular mechanism for the pathogenesis of cerebral cavernous malformations (CCM) resulting from loss of CCM2-mediated localization of Smurf1, which controls RhoA degradation required for maintenance of normal endothelial cell physiology.We previously characterized a scaffold-like protein named osmosensing scaffold for MEKK3 (OSM) for its ability to bind actin and localize to Rac-containing membrane ruffles and its obligate requirement for p38 activation in response to hyperosmotic stress (1). Subsequently, the gene encoding OSM, CCM2, was found to be mutated in the human disease cerebral cavernous malformations (CCM)² (2). Cerebral cavernous malformations are vascular lesions of the central nervous system characterized as clusters of dilated, thin walled blood vessels. CCM lesions are fragile and prone to vascular leakiness and rupture, leading to hemorrhages that cause seizure and stroke (3, 4).Recently, CCM2 knockdown endothelial cells were shown to have increased activation of RhoA (5), although the mechanism was not defined. Herein, we demonstrate a molecular mechanism for activation of this pathway. Through a novel CCM2 PTB domain interaction with the Smurf1 homologous to the E6-AP C terminus (HECT) domain, we now show that CCM2 binds the E3 ligase Smurf1 for the control of RhoA degradation.     

A Cycle of Ubiquitination Regulates Adaptor Function of the Nedd4-Family Ubiquitin Ligase Rsp5

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The Catalytic Subunit of Drosophila Glutamate-Cysteine Ligase Is a Nucleocytoplasmic Shuttling Protein

GSH concentration is considerably lower in the nucleus than in the cytoplasm; however, it is significantly elevated during active cell proliferation. The main purpose of this study was to understand the mechanism underlying these variations in nuclear/cytoplasmic distribution of GSH. The rate-limiting step in the de novo GSH biosynthesis pathway is catalyzed by glutamate cysteine ligase (GCL), a heterodimer, composed of a catalytic subunit (GCLc) and a modulatory subunit (GCLm). In Drosophila, GCLc, but not GCLm, contains a nuclear localization signal (NLS). Drosophila S2 cells, constitutively expressing regular GCLc protein or expressing GCLc protein with a mutated NLS motif, were generated by transfection. In quiescent S2 cells, GCLc is aggregated in the perinuclear cytosol and the nucleus, whereas GLCm resides solely in the cytosol. In actively proliferating S2 cells, expressing the normal NLS motif, GCLc migrates from the perinuclear cytoplasm into the nucleus, and the nuclear GSH level becomes elevated; in contrast, in proliferating cells, expressing the mutated NLS motif, neither does the GCLc migrate into the nucleus nor does the nuclear GSH amount rise. In S2 cells expressing wild type GCLc, perturbation of cellular redox state by exposure to cadmium resulted in the migration of GCLc into the nucleus but not in cells expressing GCLc with the mutated NLS motif. Overall, results indicated that GSH biosynthesis in the nucleus is associated with migration of only the GCLc subunit from the cytoplasm into the nucleus, and this migration requires the presence of an intact NLS.The tripeptide, γ-glutamylcysteinylglycine or GSH, is the most abundant intracellular nonprotein thiol. It serves multiple physiological functions, including maintenance of redox homeostasis, providing reducing equivalents for the elimination of reactive oxygen species, protection against electrophilic xenobiotics, maintenance of protein structure, and storage/transport of l-cysteine. GSH is synthesized de novo by two ATP-dependent consecutive reactions: ligation of l-glutamate to l-cysteine by the activity of glutamate-cysteine ligase (GCL²; EC 6.3.2.2), a rate-limiting step in the pathway, followed by the coupling of glycine to γ-glutamylcysteine by glutathione synthase (EC 6.3.2.3). The GCL holoenzyme is heterodimeric, consisting of a catalytic (GCLc) and a modifier (GCLm) subunit, each encoded by a unique gene. The intracellular ratios of GLCc and GCLm are, however, not necessarily equimolar and may be altered under conditions of oxidative stress. Although GCLc by itself is fully competent to catalyze the biosynthesis of GSH (1), dimerization with GCLm lowers the K_m for glutamate and also decreases sensitivity to feedback inhibition by GSH (2).Overexpression of GCL subunits in cultured cells has been reported to increase GSH production and confer enhanced protection against oxidative stress (3), apoptosis (4), and oxidant-induced DNA lesions (5). We have shown that overexpressions of GCLc or GCLm boost GSH biosynthesis and extend life span in Drosophila melanogaster (6). In contrast, inhibition of GCL activity results in decreased GSH levels, enhanced susceptibility to oxidative or nitrosative stress, increased DNA damage, and cell cycle arrest (7, 8). The homozygous knock-out for the catalytic GCLc subunit is embryonic lethal (9).GSH is predominantly synthesized in the cytoplasm, but its levels greatly vary among the different intracellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cytosol (10–12). Currently available evidence suggests the existence of two alternate mechanisms for the differential distribution of GSH in subcellular compartments: (i) GSH may first be synthesized in the cytoplasm and then transported into the organelles either actively or via passive diffusion; (ii) GSH may be synthesized in those organelles that display activities of GCL and glutathione synthase (13, 14).It has been demonstrated that GSH levels in the nucleus of proliferating cells are much higher than those in the confluent cells (15); however, the mechanism underlying this variation is presently unclear. In the present study, we provide evidence that GSH is synthesized in the nucleus and that this synthesis is dependent upon the shuttling of the GCLc, but not the GCLm, subunit from the cytoplasm to the nucleus. The ability of GCLc to migrate into the nucleus is due to the presence of a nuclear localization signal (NLS).     

Mdp1, a Saccharomyces Cerevisiae Gene Involved in Mitochondrial/Cytoplasmic Protein Distribution, Is Identical to the Ubiquitin-Protein Ligase Gene Rsp5

Alteration of the subcellular distribution of Mod5p-I, a tRNA modification enzyme, member of the sorting isozyme family, affects tRNA-mediated nonsense suppression. Altered suppression efficiency was used to identify MDP genes, which, when mutant, change the mitochondrial/cytosolic distribution of Mod5p-I,KR6. MDP2 is the previously identified VRP1, which encodes verprolin, required for proper organization of the actin cytoskeleton. MDP3 is identical to PAN1, which encodes a protein involved in initiation of translation and actin cytoskeleton organization. We report here the cloning and characterization of wild-type and mutant MDP1 alleles and the isolation and characterization of a multicopy suppressor of mdp1 mutations. MDP1 is identical to RSP5, which encodes ubiquitin-protein ligase, and mdp1 mutations are suppressed by high copy expression of ubiquitin. All four characterized mdp1 mutations cause missense changes located in the hect domain of Rsp5p that is highly conserved among ubiquitin-protein ligases. In addition to its well-known function in protein turnover, ubiquitination has been proposed to play roles in subcellular sorting of proteins via endocytosis and in delivery of proteins to peroxisomes, the endoplasmic reticulum and mitochondria. mdp1, as well as mdp2/vrp1 and mdp3/pan1 mutations, affect endocytosis. Further, mdp1 mutations show synthetic interactions with mdp2/vrp1 and mdp3/pan1. Identification of MDP1 as RSP5, along with our previous identification of MDP2/VRP1 and MDP3/PAN1, implicate interactions of the ubiquitin system, the actin cytoskeleton and protein synthesis in the subcellular distribution of proteins.     

The Nedd4-Type Rsp5p Ubiquitin Ligase Inhibits Tombusvirus Replication by Regulating Degradation of the p92 Replication Protein and Decreasing the Activity of the Tombusvirus Replicase

Recent in vitro proteomics screens revealed that many host proteins could interact with the replication proteins of Tomato bushy stunt virus (TBSV), which is a small, plus-stranded RNA virus (Z. Li, D. Barajas, T. Panavas, D. A. Herbst, and P. D. Nagy, J. Virol. 82:6911-6926, 2008). To further our understanding of the roles of host factors in TBSV replication, we have tested the effect of Rsp5p, which is a member of the Nedd4 family of E3 ubiquitin ligases. The full-length Rsp5p, via its WW domain, is shown to interact with p33 and the central portion of p92^pol replication proteins. We find that overexpression of Rsp5p inhibits TBSV replication in Saccharomyces cerevisiae yeast, while downregulation of Rsp5p leads to increased TBSV accumulation. The inhibition is caused by Rsp5p-guided degradation of p92^pol, while the negative effect on the p33 level is less pronounced. Interestingly, recombinant Rsp5p also inhibits TBSV RNA replication in a cell-free replication assay, likely due to its ability to bind to p33 and p92^pol. We show that the WW domain of Rsp5p, which is involved in protein interactions, is responsible for inhibition of TBSV replication, whereas the HECT domain, involved in protein ubiquitination, is not necessary for Rsp5p-mediated inhibition of viral replication. Overall, our data suggest that direct binding between Rsp5p and p92^pol reduces the stability of p92^pol, with consequent inhibition of TBSV replicase activity.Various interactions with their host cells are critical for plus-stranded (+)RNA viruses as they attempt to utilize the host translation machinery to produce viral proteins, gain access to the resources of the host cells, co-opt host proteins, and subvert host membranes (1, 17). Additional levels of interaction between virus and host reflect antiviral responses which may involve innate immunity, as well as other antiviral processes and factors. On-going research with several model viruses is striving to map all the interactions between viruses and hosts and characterize the functions of the co-opted host factors. In this regard, recent research has led to the identification of a large number of host proteins which affect the replication of various (+)RNA viruses and minus-stranded RNA viruses (4, 5, 9, 11, 22, 35, 39). The roles and functions of most of the host proteins identified as being involved in RNA virus replication, however, are currently unknown.Tombusviruses, such as Tomato bushy stunt virus (TBSV), are among the most advanced model systems in relation to the identification of host factors affecting (+)RNA virus replication. The TBSV genome codes for only five proteins, two of which are the replication proteins translated directly from the genomic RNA (45). One of these replication proteins is the abundant p33 replication cofactor; the other is the RNA-dependent RNA polymerase (RdRp) p92^pol. Due to the overlapping expression strategy, p33 is identical with the N-terminal portion of the larger p92^pol protein (Fig. ​(Fig.1A).1A). Both replication proteins contain an RNA-binding motif (arginine-proline-rich motif), phosphorylation sites that affect RNA binding by the p33 protein, a p33-p33/p92 interaction domain, and two transmembrane domains (Fig. ​(Fig.1A)1A) (18, 19, 32, 36, 37). Three short stretches of amino acids in p33 and p92^pol are involved in binding to the Pex19p host protein that facilitates the transportation of p33 and p92^pol from the cytosol to the cytosolic surface of the peroxisomes, the site of replicase complex formation and viral RNA replication (25). The essential nature of the above-named domains for obtaining functional replicase complexes suggests that multiple dynamic protein-protein, protein-RNA, and protein-membrane interactions must be required for robust tombusvirus replication.Open in a separate windowFIG. 1.Binding of Rsp5p to TBSV p33 and p92 proteins in vitro. (A) Schematic representation of viral proteins and their derivatives used in the binding assay. The various domains include the transmembrane domain (TMD), arginine-proline-rich RNA-binding domain (RPR), phosphorylated serine and threonine (P), and S1 and S2 subdomains involved in p33-p33/p92 interaction. The two RNA-binding regions in p92 are shown with boxes. (B) Affinity binding (pulldown) assay to detect interaction between GST-six-His-Rsp5p and the MBP-tagged viral proteins. The MBP-tagged viral proteins and MBP produced in E. coli were immobilized on amylose affinity columns. Then, GST-six-His-tagged Rsp5p expressed in E. coli was passed through the amylose affinity columns with immobilized MBP-tagged proteins. The affinity-bound proteins were specifically eluted with maltose from the columns. The eluted proteins were analyzed by Western blotting with anti-six-His antibody to detect the amount of GST-six-His-Rsp5p specifically bound to MBP-tagged viral proteins. (C) The amounts of MBP-tagged proteins eluted from the columns were analyzed by Coomassie blue staining of SDS-PAGE gels. (D) SDS-PAGE analysis of in vitro ubiquitination of replication protein p33 by purified recombinant Rsp5p. The components in the assays are indicated at the top. The ubiquitin-MBP-p33 product, detected by anti-six-His antibody, is marked by an arrowhead. Ub, ubiquitin; +, present; −, absent.In order to identify host genes involved in tombusvirus replication and recombination, systematic genome-wide screens that covered 95% of the host genes were performed in the model host Saccharomyces cerevisiae yeast (9, 22, 34, 35). These screens led to the identification of over 150 host genes, although the functions of these genes in TBSV replication are largely unknown. In addition, proteomics analysis of the highly purified tombusvirus replicase, as well as the use of yeast protein arrays containing ∼4,100 purified proteins to identify host proteins interacting with p33 and/or p92^pol, led to the identification of ∼60 pertinent yeast proteins (12, 33). Current efforts are focused on characterizing the functions of key host proteins in TBSV replication.Most of the host factors identified facilitate tombusvirus replication, though some are inhibitory. The list of characterized host factors includes heat shock protein 70 (Hsp70), which is required for the assembly of the viral replicase in vitro, as well as for membrane insertion and intracellular targeting of the viral replication proteins in vivo (29, 43). Another important host protein is GAPDH (glyceraldehyde-3-phosphate dehydrogenase), which affects plus-strand synthesis (42). The functions of two other host factors that are also present in the replicase complex, namely, Cdc34p E2 ubiquitin-conjugating enzyme, which ubiquitinates p33 replication protein in vitro, and translation elongation factor 1A (eEF1A), which binds to a 3′ cis-acting regulatory element in the TBSV (+)RNA, are not yet characterized with respect to their roles in viral replication (12, 13). Downregulation of all four of the above-described host factors inhibited TBSV accumulation in the yeast model host and in plants (12, 13, 33, 42, 43), suggesting that they are significant players in TBSV replication.In order to further the understanding of host factor roles in viral RNA replication, this paper addresses the effect of Rsp5p E3 ubiquitin ligase on TBSV accumulation. Rsp5p was selected since we have previously found an interaction between p33 and Rsp5p, based on the yeast protein array (12). Also, p33 is mono- and biubiquitinated in yeast cells (12), and Rsp5p is known to ubiquitinate select host proteins (3). These features of Rsp5p suggest its relevance to TBSV replication. Indeed, we found that Rsp5p inhibits TBSV replication when overexpressed in yeast cells, whereas its downregulation leads to increased TBSV accumulation. The inhibition is primarily caused by Rsp5p-mediated selective degradation of p92^pol. Its negative effect on the level of p33 is substantially less. However, the inhibitory function of Rsp5p is more complex, since the purified recombinant Rsp5p also inhibited RNA replication in a cell-free TBSV replication assay, likely due to the ability of Rsp5p to bind to both p33 and p92^pol. Surprisingly, the inhibitory function of Rsp5p is not caused by the HECT domain, which is involved in protein ubiquitination, but by its WW domain, which is involved in protein interactions. The observations suggest that direct binding between Rsp5p and p33 and, more importantly, p92^pol is likely involved in the inhibition of TBSV replication.     

Quality Control of Plasma Membrane Proteins by Saccharomyces cerevisiae Nedd4-Like Ubiquitin Ligase Rsp5p under Environmental Stress Conditions

In Saccharomyces cerevisiae, when a rich nitrogen source such as ammonium is added to the culture medium, the general amino acid permease Gap1p is ubiquitinated by the yeast Nedd4-like ubiquitin ligase Rsp5p, followed by its endocytosis to the vacuole. The arrestin-like Bul1/2p adaptors for Rsp5p specifically mediate this process. In this study, to investigate the downregulation of Gap1p in response to environmental stresses, we determined the intracellular trafficking of Gap1p under various stress conditions. An increase in the extracellular ethanol concentration induced ubiquitination and trafficking of Gap1p from the plasma membrane to the vacuole in wild-type cells, whereas Gap1p remained stable on the plasma membrane under the same conditions in rsp5^A401E and Δend3 cells. A ¹⁴C-labeled citrulline uptake assay using a nonubiquitinated form of Gap1p (Gap1p^K9R/K16R) revealed that ethanol stress caused a dramatic decrease of Gap1p activity. These results suggest that Gap1p is inactivated and ubiquitinated by Rsp5p for endocytosis when S. cerevisiae cells are exposed to a high concentration of ethanol. It is noteworthy that this endocytosis occurs in a Bul1/2p-independent manner, whereas ammonium-triggered downregulation of Gap1p was almost completely inhibited in Δbul1/2 cells. We also found that other environmental stresses, such as high temperature, H₂O₂, and LiCl, also promoted endocytosis of Gap1p. Similar intracellular trafficking caused by ethanol occurred in other plasma membrane proteins (Agp1p, Tat2p, and Gnp1p). Our findings suggest that stress-induced quality control is a common process requiring Rsp5p for plasma membrane proteins in yeast.     

Rsp5 Ubiquitin-Protein Ligase Mediates DNA Damage-Induced Degradation of the Large Subunit of RNA Polymerase II in Saccharomyces cerevisiae

Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.     

Sna3 Is an Rsp5 Adaptor Protein that Relies on Ubiquitination for Its MVB Sorting

The process in which ubiquitin ( Ub ) conjugation is required for trafficking of integral membrane proteins into multivesicular bodies ( MVBs ) and eventual degradation in the lumen of lysosomes/vacuoles is well defined. However , Ub ‐independent pathways into MVBs are less understood. To better understand this process, we have further characterized the membrane protein Sna 3, the prototypical Ub ‐independent cargo protein sorted through the MVB pathway in yeast. We show that Sna 3 trafficking to the vacuole is critically dependent on Rsp 5 ligase activity and ubiquitination. We find Sna 3 undergoes Ub ‐dependent MVB sorting by either becoming ubiquitinated itself or associating with other ubiquitinated membrane protein substrates. In addition, our functional studies support a role for Sna 3 as an adaptor protein that recruits Rsp 5 to cargo such as the methionine transporter Mup 1, resulting in efficient Mup 1 delivery to the vacuole .     

Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1^K29R-K31R-GFP remained. The HPG1-1 (Rsp5^P514T) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.     

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.     

Exploring Protein Space: From Hydrolase to Ligase by Substitution

The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence–function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.     

Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

A genetic deficit mediated by SNP rs143383 that leads to reduced expression of GDF5 is strongly associated with large-joint osteoarthritis. We speculated that this deficit could be attenuated by the application of exogenous GDF5 protein and as a first step we have assessed what effect such application has on primary osteoarthritis chondrocyte gene expression. Chondrocytes harvested from cartilage of osteoarthritic patients who had undergone joint replacement were cultured with wildtype recombinant mouse and human GDF5 protein. We also studied variants of GDF5, one that has a higher affinity for the receptor BMPR-IA and one that is insensitive to the GDF5 antagonist noggin. As a positive control, chondrocytes were treated with TGF-β1. Chondrocytes were cultured in monolayer and micromass and the expression of genes coding for catabolic and anabolic proteins of cartilage were measured by quantitative PCR. The expression of the GDF5 receptor genes and the presence of their protein products was confirmed and the ability of GDF5 signal to translocate to the nucleus was demonstrated by the activation of a luciferase reporter construct. The capacity of GDF5 to elicit an intracellular signal in chondrocytes was demonstrated by the phosphorylation of intracellular Smads. Chondrocytes cultured with TGF-β1 demonstrated a consistent down regulation of MMP1, MMP13 and a consistent upregulation of TIMP1 and COL2A1 with both culture techniques. In contrast, chondrocytes cultured with wildtype GDF5, or its variants, did not show any consistent response, irrespective of the culture technique used. Our results show that osteoarthritis chondrocytes do not respond in a predictable manner to culture with exogenous GDF5. This may be a cause or a consequence of the osteoarthritis disease process and will need to be surmounted if treatment with exogenous GDF5 is to be advanced as a potential means to overcome the genetic deficit conferring osteoarthritis susceptibility at this gene.     

A Functional Analysis of the Yeast Ubiquitin Ligase Rsp5: The Involvement of the Ubiquitin-Conjugating Enzyme Ubc4 and Poly-Ubiquitination in Ethanol-Induced Down-Regulation of Targeted Proteins

Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae. We have found that the Ala401Glu rsp5 mutant is hypersensitive to various stresses, suggesting that Rsp5 is a key enzyme for yeast cell growth under stress conditions. The ubiquitination and the subsequent degradation of stress-induced misfolded proteins are indispensable for cell survival under stress conditions. In this study, we analyzed the ubiquitin-conjugating enzyme Ubc4 and the poly-ubiquitination of targeted proteins involved in the function of Rsp5 under ethanol stress conditions. Ubc4 was found to be important in yeast cell growth and poly-ubiquitination of the bulk proteins in the presence of ethanol. The general amino acid permease Gap1 is poly-ubiquitinated via Lys63 and is down-regulated after the addition of ammonium ions through a process requiring Rsp5. We found that Gap1 was removed from the plasma membrane in the presence of ethanol in a Rsp5-dependent manner, and that the disappearance of Gap1 required Ubc4 and involved the lysine residues of ubiquitin. Our results also indicate that Lys6 of ubiquitin might inhibit the disappearance of Gap1. These results suggest that Rsp5 down-regulates the ethanol-induced misfolded forms of Gap1. In addition, it appears that the substrates of Rsp5 are appropriately poly-ubiquitinated via different lysine residues of ubiquitin under various growth conditions.