The HECT ubiquitin ligase Rsp5p is required for proper nuclear export of mRNA in Saccharomyces cerevisiae

The nuclear transport of both proteins and RNAs has attracted considerable interest in recent years. However, regulation pathways of the nuclear transport machineries are still not well characterized. Previous studies indicated that ubiquitination is involved in poly(A)⁺ RNA nuclear export. For this reason, we systematically investigated ubiquitin-protein ligasess from the homologous to E6-AP carboxy terminus (HECT) family for potential individual roles in nuclear transport in Saccharomyces cerevisiae . Here we report that Rsp5, an essential yeast ubiquitin ligase involved in many cellular functions, when deleted or mutated in ligase activity, blocks the nuclear export of mRNAs. Affected messenger RNAs include both total poly(A)⁺ mRNA and heat-shock mRNAs. Mutation of Rsp5 does not affect nuclear protein import or export. Deletion of RSP5 blocks mRNA export, even under conditions where its essential role in unsaturated fatty acids biosynthesis is bypassed. Using domain mapping, we find that the ligase activity is required for proper mRNA export, indicating that ubiquitination by Rsp5 acts directly or indirectly to affect RNA export. The finding that Rsp5p ligase mutations cause a more pronounced defect at high temperatures suggests that ubiquitination of transport factors by Rsp5p may also be essential during stress conditions.     

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.     

Rsp5 ubiquitin ligase modulates translation accuracy in yeast Saccharomyces cerevisiae

Rsp5p is an essential yeast ubiquitin protein ligase that ubiquitinates multiple proteins involved in various processes. Recent studies indicate that ubiquitination also affects translation. Here, we show that the strain with the rsp5-13 mutation exhibits altered sensitivity to antibiotics and a slower rate of translation. Using a sensitive dual-gene reporter system, we demonstrate that stop codon readthrough efficiency is decreased in the rsp5-13 mutant, while both +1 and -1 frameshifting were unaffected. The effect of the rsp5-13 mutation on readthrough could be reversed by increased expression of ubiquitin and partially suppressed by overproduction of the elongation factor eEF1A. As assessed by fluorescence in situ hybridization, the rsp5-13 mutant cells accumulate tRNA nuclear pools, perhaps depleting tRNA from the cytoplasm. Nuclear accumulation of tRNA is observed only when rsp5-13 cells are grown in media with high amino acid content. This defect, also reversed by overproduction of the elongation factor eEF1A, may be the primary reason for altered translational decoding accuracy.     

Redefining the catalytic HECT domain boundaries for the HECT E3 ubiquitin ligase family

There are 28 unique human members of the homologous to E6AP C-terminus (HECT) E3 ubiquitin ligase family. Each member of the HECT E3 ubiquitin ligases contains a conserved bilobal HECT domain of approximately 350 residues found near their C-termini that is responsible for their respective ubiquitylation activities. Recent studies have begun to elucidate specific roles that each HECT E3 ubiquitin ligase has in various cancers, age-induced neurodegeneration, and neurological disorders. New structural models have been recently released for some of the HECT E3 ubiquitin ligases, but many HECT domain structures have yet to be examined due to chronic insolubility and/or protein folding issues. Building on these recently published structural studies coupled with our in-house experiments discussed in the present study, we suggest that the addition of ∼50 conserved residues preceding the N-terminal to the current UniProt defined boundaries of the HECT domain are required for isolating soluble, stable, and active HECT domains. We show using in silico bioinformatic analyses coupled with secondary structural prediction software that this predicted N-terminal α-helix found in all 28 human HECT E3 ubiquitin ligases forms an obligate amphipathic α-helix that binds to a hydrophobic pocket found within the HECT N-terminal lobe. The present study brings forth the proposal to redefine the residue boundaries of the HECT domain to include this N-terminal extension that will likely be critical for future biochemical, structural, and therapeutic studies on the HECT E3 ubiquitin ligase family.     

The ubiquitin ligase Rsp5 is required for ribosome stability in Saccharomyces cerevisiae

Rsp5p is a conserved HECT-domain ubiquitin ligase with diverse roles in cellular physiology. Here we report a previously unknown role of Rsp5p in facilitating the stability of the cytoplasmic ribosome pool in budding yeast. Yeast strains carrying temperature-sensitive mutations in RSP5 showed a progressive decline in levels of 18S and 25S rRNAs and accumulation of rRNA decay fragments when cells grown in rich medium were shifted to restrictive temperature. This was accompanied by a decreased number of translating ribosomes and the appearance of ribosomal subunits with an abnormally low sedimentation rate in polysome analysis. Abrogating Rsp5p function affected stability of other tested noncoding RNA species (tRNA and snoRNA), but to a lower extent than that of rRNA, and also inhibited processing of rRNA and tRNA precursors, in agreement with previous studies. The breakdown of cellular ribosomes was not affected by deletion of key genes involved in autophagy, previously implicated in ribosome turnover upon starvation. Our results suggest that functional Rsp5p is required to maintain the integrity of cytoplasmic ribosomes under rich nutrient conditions.     

Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants

Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP) neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.     

Ubiquitination of ERMES components by the E3 ligase Rsp5 is involved in mitophagy

Mitochondria are dynamic organelles that undergo permanent fission and fusion events. These processes play an essential role in maintaining normal cellular function. In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum-mitochondrial encounter structure (ERMES) is a marker of sites of mitochondrial division, but it is also involved in a plethora of other mitochondrial functions. However, it remains unclear how these different functions are regulated. We show here that Mdm34 and Mdm12, 2 components of ERMES, are ubiquitinated by the E3 ligase Rsp5. This ubiquitination is not involved in mitochondrial dynamics or in the distribution and turnover of ERMES. Nevertheless, the ubiquitination of Mdm34 and Mdm12 was required for efficient mitophagy. We thus report here the first identification of ubiquitinated substrates participating in yeast mitophagy.     

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.     

The ubiquitin ligase Rsp5p is required for modification and sorting of membrane proteins into multivesicular bodies

Precursor forms of vacuolar proteins with transmembrane domains, such as the carboxypeptidase S Cps1p and the polyphosphatase Phm5p, are selectively sorted in endosomal compartments to vesicles that invaginate, budding into the lumen of the late endosomes, resulting in the formation of multivesicular bodies (MVBs). These proteins are then delivered to the vacuolar lumen following fusion of the MVBs with the vacuole. The sorting of Cps1p and Phm5p to these structures is mediated by ubiquitylation, and in doa4 mutant cells, which have reduced level of free ubiquitin, these proteins are missorted to the vacuolar membrane. A RING-finger ubiquitin ligase Tul1p has been shown to participate in the ubiquitylation of Cps1p and Phm5p. We show here that the HECT-ubiquitin ligase Rsp5p is also required for the ubiquitylation of these proteins, and therefore for their sorting to MVBs. Rsp5p is an essential ubiquitin ligase containing an N-terminal C2 domain followed by three WW domains, and a C-terminal catalytic HECT domain. In cells with low levels of Rsp5p (npi1 mutant cells), vacuolar hydrolases do not reach the vacuolar lumen and are instead missorted to the vacuolar membrane. The C2 domain and both the second and third WW domains of Rsp5p are important determinants for sorting to MVBs. Ubiquitylation of Cps1p was strongly reduced in the npi1 mutant strain and ubiquitylation was completely abolished in the npi1 tul1Delta double mutant. These data demonstrate that Rsp5p plays a novel and key role in intracellular trafficking, and extend the currently very short list of substrates ubiquitylated in vivo by several different ubiquitin ligases acting cooperatively.     

Yeast ubiquitin ligase Rsp5 contains nuclear localization and export signals

The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP(2) reporter. The 687-LIGGIAEIDI-696 sequence located in the Hect domain was identified as a nuclear export signal active in a Crm1-dependent manner, and its importance for the localization of Rsp5 was documented by using fluorescence microscopy and a lacZ-based reporter system. Analysis of the cellular location of other Rsp5 fragments fused with GFP(2) indicated two independent potential nuclear localization signals, both located in the Hect domain. We also uncovered Rsp5 fragments that are important to targeting/tethering Rsp5 to various regions in the cytoplasm. The presented data indicate that Rsp5 ligase is a shuttling protein whose distribution within the cytoplasm and partitioning between cytoplasmic and nuclear locations is determined by a balance between the actions of several targeting sequences and domains.     

Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase

Ubiquitin ligases (E3) select proteins for ubiquitylation, a modification that directs altered subcellular trafficking and/or degradation of the target protein. HECT domain E3 ligases not only recognize, but also directly catalyze, ligation of ubiquitin to their protein substrates. The crystal structure of the HECT domain of the human ubiquitin ligase WWP1/AIP5 maintains a two-lobed structure like the HECT domain of the human ubiquitin ligase E6AP. While the individual N and C lobes of WWP1 possess very similar folds to those of E6AP, the organization of the two lobes relative to one another is different from E6AP due to a rotation about a polypeptide hinge linking the N and C lobes. Mutational analyses suggest that a range of conformations achieved by rotation about this hinge region is essential for catalytic activity.     

The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division

We identify a mitochondrial E3 ubiquitin ligase, MARCH5, as a critical regulator of mitochondrial fission. MARCH5 RING mutants and MARCH5 RNA interference induce an abnormal elongation and interconnection of mitochondria indicative of an inhibition of mitochondrial division. The aberrant mitochondrial phenotypes in MARCH5 RING mutant-expressing cells are reversed by ectopic expression of Drp1, but not another mitochondrial fission protein Fis1. Moreover, as indicated by abnormal clustering and mitochondrial accumulation of Drp1, as well as decreased cellular mobility of YFP-Drp1 in cells expressing MARCH5 RING mutants, MARCH5 activity regulates the subcellular trafficking of Drp1, likely by impacting the correct assembly at scission sites or the disassembly step of fission complexes. Loss of this activity may account for the observed mitochondrial division defects. Finally, MARCH5 RING mutants and endogenous Drp1, but not wild-type MARCH5 or Fis1, co-assemble into abnormally enlarged clusters in a Drp1 GTPase-dependent manner, suggesting molecular interactions among these proteins. Collectively, our data suggest a model in which mitochondrial division is regulated by a MARCH5 ubiquitin-dependent switch.     

A high throughput screen to identify substrates for the ubiquitin ligase Rsp5

Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.     

Autoantigen Ro52 is an E3 ubiquitin ligase

Anti-Ro/SSA antibodies are classic autoantibodies commonly found in patients with Sj?gren's syndrome, a chronic autoimmune disease characterized by dryness of the eyes and mouth. The autoantibodies recognize a RING-finger protein, Ro52, whose function is still unknown. Since many RING-finger proteins have been identified as E3 ubiquitin ligases, this study was designed to determine whether Ro52 functions as an E3 ubiquitin ligase. For this purpose, recombinant Ro52 was purified from bacterial lysate and used to investigate its activity of E3 ubiquitin ligase in vitro. Its enzymatic activity was also tested in HEK293T cells using wild-type Ro52 and its RING-finger mutant. Our results indicated that Ro52 ubiquitinates itself in cooperation with E2 ubiquitin-conjugating enzyme UbcH5B, thereby validating that Ro52 is a RING-finger-type E3 ubiquitin ligase. Importantly, this ubiquitin modification is predominantly monoubiquitination, which does not target Ro52 to the proteasome for degradation.     

The inflammation-associated Salmonella SopA is a HECT-like E3 ubiquitin ligase

Salmonella translocate a group of type III effectors into the host cells to induce entry, promote survival and cause intestinal inflammation. Although the biochemical and cellular mechanisms of how bacterial effectors function inside host cells remain largely unknown, studies have indicated that a likely strategy is to exploit host cellular pathways through functional mimicry. We report here that SopA, a Salmonella type III effector, mimics the mammalian HECT E3 ubiquitin ligase. SopA preferentially uses the host UbcH5a, UbcH5c and UbcH7 as E2s, which are involved in inflammation. Both the wild-type SopA and the mutant SopAC753S were expressed and translocated at similar levels during the infection of HeLa cells. A Salmonella strain expressing a catalytically incompetent SopAC753S mutant had reduced Salmonella-induced polymorphonuclear leukocytes transepithelial migration. We speculate that SopA ubiquitinate bacterial/host proteins involved in Salmonella-induced intestinal inflammation.     

E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis

Background and Aim

Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.

Methods

The expression and localization of synoviolin in the liver were analyzed in CCl₄-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno^+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno^+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno^−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno^−/− MEF cells.

Results

In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno^+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno^+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno^−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

Conclusion

Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.     

The ubiquitin E3 ligase POSH regulates calcium homeostasis through spatial control of Herp

The ubiquitin (Ub) domain protein Herp plays a crucial role in the maintenance of calcium homeostasis during endoplasmic reticulum (ER) stress. We now show that Herp is a substrate as well as an activator of the E3 Ub ligase POSH. Herp-mediated POSH activation requires the Ubl domain and exclusively promotes lysine-63-linked polyubiquitination. Confocal microscopy demonstrates that Herp resides mostly in the trans-Golgi network, but, shortly after calcium perturbation by thapsigargin (Tpg), it appears mainly in the ER. Substitution of all lysine residues within the Ubl domain abolishes lysine-63-linked polyubiquitination of Herp in vitro and calcium-induced Herp relocalization that is also abrogated by the overexpression of a dominant-negative POSHV14A. A correlation exists between the kinetics of Tpg-induced Herp relocalization and POSH-dependent polyubiquitination. Finally, the overexpression of POSH attenuates, whereas the inhibition of POSH by the expression of POSHV14A or by RNA interference enhances Tpg-induced calcium burst. Altogether, these results establish a critical role for POSH-mediated ubiquitination in the maintenance of calcium homeostasis through the spatial control of Herp.     

The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.