RESUMEN
The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify additional signaling components, we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly represented. Included in this group are members of the Skp, Cullin, F box (SCF) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCF(Cdc4) acts together with the Cdc34 ubiquitin-conjugating enzyme at the level of the G protein; promotes degradation of the G protein alpha subunit, Gpa1, in vivo; and catalyzes Gpa1 ubiquitination in vitro. These insights to the G protein signaling network reveal the essential genome as an untapped resource for identifying new components and regulators of signal transduction pathways.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/genética , Ciclosoma-Complejo Promotor de la Anafase , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Análisis por Conglomerados , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Humanos , Fenotipo , Feromonas/genética , Feromonas/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Enzimas Ubiquitina-Conjugadoras , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless (K0)Cdc34(DeltaC) is indistinguishable from Cdc34(DeltaC) in ubiquitination of the prototype SCF(Cdc4) substrate Sic1 in vitro, and replacement of the CDC34 gene with either the (K0)cdc34(DeltaC) or the cdc34(DeltaC) allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.
Asunto(s)
Regulación hacia Abajo/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Saccharomyces cerevisiae/enzimología , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Sitios de Unión , Catálisis , Lisina/metabolismo , Regiones Promotoras Genéticas/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae , Enzimas Ubiquitina-Conjugadoras , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/aislamiento & purificaciónRESUMEN
[This corrects the article DOI: 10.1186/1747-1028-6-7.].
RESUMEN
Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome, possibly by inducing oxidative stress-mediated modifications that compromise substrate delivery to proteolytic core.
Asunto(s)
Proteínas Mutantes/metabolismo , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , alfa 1-Antitripsina/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Humanos , Hígado/metabolismo , Ratones , Mitocondrias/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/química , Poliubiquitina/química , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Proteolisis , alfa 1-Antitripsina/genéticaRESUMEN
Autoimmune destruction of insulin producing pancreatic ß-cells is the hallmark of type I diabetes. One of the key molecules implicated in the disease onset is the immunoproteasome, a protease with multiple proteolytic sites that collaborates with the constitutive 19S and the inducible 11S (PA28) activators to produce immunogenic peptides for presentation by MHC class I molecules. Despite its importance, little is known about the function and regulation of the immunoproteasome in pancreatic ß-cells. Of special interest to immunoproteasome activation in ß-cells are the effects of IFNß, a type I IFN secreted by virus-infected cells and implicated in type I diabetes onset, compared to IFNγ, the classic immunoproteasome inducer secreted by cells of the immune system. By qPCR analysis, we show that mouse insulinoma MIN6 cells and mouse islets accumulate the immune proteolytic ß1(i), ß2(i) and ß5(i), and 11S mRNAs upon exposure to IFNß or IFNγ. Higher concentrations of IFNß than IFNγ are needed for similar expression, but in each case the expression is transient, with maximal mRNA accumulation in 12 hours, and depends primarily on Interferon Regulatory Factor 1. IFNs do not alter expression of regular proteasome genes, and in the time frame of IFNß-mediated response, the immune and regular proteolytic subunits co-exist in the 20S particles. In cell extracts with ATP, these particles have normal peptidase activities and degrade polyubiquitinated proteins with rates typical of the regular proteasome, implicating normal regulation by the 19S activator. However, ATP depletion rapidly stimulates the catalytic rates in a manner consistent with levels of the 11S activator. These findings suggest that stochastic combination of regular and immune proteolytic subunits may increase the probability with which unique immunogenic peptides are produced in pancreatic ß-cells exposed to IFNß, but primarily in cells with reduced ATP levels that stimulate the 11S participation in immunoproteasome function.
Asunto(s)
Adenosina Trifosfato/metabolismo , Células Secretoras de Insulina/inmunología , Interferón beta/metabolismo , Proteínas Musculares/metabolismo , Complejo de la Endopetidasa Proteasomal/inmunología , Animales , Western Blotting , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Cartilla de ADN/genética , Inmunoprecipitación , Células Secretoras de Insulina/virología , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Octoxinol , Reacción en Cadena de la Polimerasa , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Type 1 diabetes results from autoimmune destruction of the insulin producing pancreatic ß-cells. The immunoproteasome, a version of the proteasome that collaborates with the 11S/PA28 activator to generate immunogenic peptides for presentation by MHC class I molecules, has long been implicated in the onset of the disease, but little is known about immunoproteasome function and regulation in pancreatic ß-cells. Interesting insight into these issues comes from a recent analysis of the immunoproteasome expressed in pancreatic ß-cells during early antiviral defenses mediated by interferon ß (IFNß), a type I IFN implicated in the induction of the diabetic state in human and animal models. Using mouse islets and the MIN6 insulinoma cell line, Freudenburg et al. found that IFNß stimulates expression of the immunoproteasome and the 11S/PA28 activator in a manner fundamentally similar to the classic immuno-inducer IFNγ, with similar timing of mRNA accumulation and decline; similar transcriptional activation mediated primarily by the IRF1 and similar mRNA and protein levels. Furthermore, neither IFNß nor IFNγ altered the expression of regular proteolytic subunits or prevented their incorporation into proteolytic cores. As a result, immunoproteasomes had stochastic combinations of immune and regular proteolytic sites, an arrangement that would likely increase the probability with which unique immunogenic peptides are produced. However, immunoproteasomes were activated by the 11S/PA28 only under conditions of ATP depletion. A mechanism that prevents the activation of immunoproteasome at high ATP levels has not been reported before and could have a major regulatory significance, as it could suppress the generation of immunogenic peptides as cell accumulate immunoproteasome and 11S/PA28, and activate antigen processing only when ATP levels drop. We discuss implications of these new findings on the link between early antiviral response and the onset of type 1 diabetes.
RESUMEN
BACKGROUND: The S73/S97/loop motif is a hallmark of the Cdc34 family of E2 ubiquitin-conjugating enzymes that together with the SCF E3 ubiquitin ligases promote degradation of proteins involved in cell cycle and growth regulation. The inability of the loop-less Δ12Cdc34 mutant to support growth was linked to its inability to catalyze polyubiquitination. However, the loop-less triple mutant (tm) Cdc34, which not only lacks the loop but also contains the S73K and S97D substitutions typical of the K73/D97/no loop motif present in other E2s, supports growth. Whether tmCdc34 supports growth despite defective polyubiquitination, or the S73K and S97D substitutions, directly or indirectly, correct the defect caused by the loop absence, are unknown. RESULTS: tmCdc34 supports yeast viability with normal cell size and cell cycle profile despite producing fewer polyubiquitin conjugates in vivo and in vitro. The in vitro defect in Sic1 substrate polyubiquitination is similar to the defect observed in reactions with Δ12Cdc34 that cannot support growth. The synthesis of free polyubiquitin by tmCdc34 is activated only modestly and in a manner dependent on substrate recruitment to SCFCdc4. Phosphorylation of C-terminal serines in tmCdc34 by Cka2 kinase prevents the synthesis of free polyubiquitin chains, likely by promoting their attachment to substrate. Nevertheless, tmCDC34 yeast are sensitive to loss of the Ubp14 C-terminal ubiquitin hydrolase and DUBs other than Ubp14 inefficiently disassemble polyubiquitin chains produced in tmCDC34 yeast extracts, suggesting that the free chains, either synthesized de novo or recycled from substrates, have an altered structure. CONCLUSIONS: The catalytic motif replacement compromises polyubiquitination activity of Cdc34 and alters its regulation in vitro and in vivo, but either motif can support Cdc34 function in yeast viability. Robust polyubiquitination mediated by the S73/S97/loop motif is thus not necessary for Cdc34 role in yeast viability, at least under typical laboratory conditions.
RESUMEN
The yeast SCFMet30 ubiquitin ligase plays a critical role in cell division by regulating the Met4 transcriptional activator of genes that control the uptake and assimilation of sulfur into methionine and S-adenosyl-methionine. The initial view on how SCFMet30 performs its function has been driven by the assumption that SCFMet30 acts exclusively as Met4 inhibitor when high levels of methionine drive an accumulation of cysteine. We revisit this model in light of the growing evidence that SCFMet30 can also activate Met4. The notion that Met4 can be inhibited or activated depending on the sulfur metabolite context is not new, but for the first time both aspects have been linked to SCFMet30, creating an interesting regulatory paradigm in which polyubiquitination and proteolysis of a single transcriptional activator can play different roles depending on context. We discuss the emerging molecular basis and the implications of this new regulatory phenomenon.
RESUMEN
It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys(48), but not through Lys(63), target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys(48), Lys(63), and Lys(11) linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys(6) + Lys(11), Lys(27) + Lys(29), or Lys(29) + Lys(33) on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys(48) chains (E6AP) or Lys(63) chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys(63) chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys(48) chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys(48)-Ub chain or, surprisingly, to a Lys(63)-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys(63) chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys(63)-Ub chains from proteasomal degradation.
Asunto(s)
Poliubiquitina , Conformación Proteica , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Dimerización , Lisina/química , Lisina/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Poliubiquitina/química , Poliubiquitina/genética , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Troponina I/química , Troponina I/metabolismoRESUMEN
The Met4 transcriptional activator of methionine biosynthesis is negatively regulated by the SCFMet30 ubiquitin ligase in response to accumulation of methionine. This mechanism requires polyubiquitination, but not proteolysis. We report that a previously unappreciated mechanism involving growth control regulates Met4. Unless methionine is present in the growth medium, polyubiquitinated Met4 is stabilized in late exponential cultures, correlating with transcriptional repression. Polyubiquitinated Met4 becomes destabilized in a proteasome-dependent manner upon reentry into exponential growth, correlating with transcriptional activation. Met4 stabilization is regulated at the level of SCFMet30 binding and requires transcriptional cofactors. These lock Met4 and SCFMet30 into a tight complex active in ubiquitination but incapable of binding the proteasome. Release of polyubiquitinated Met4 from SCFMet30 is sufficient for degradation, and specific sulfur amino acids can promote the degradation by destabilizing Met4 binding to cofactors and SCFMet30. Thus, destabilization of cofactors and SCFMet30 binding is the rate-limiting regulatory step in Met4 proteolysis.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Coenzimas/metabolismo , Poliubiquitina/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Cisteína/metabolismo , Proteínas F-Box , Metionina/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
ATP hydrolysis is required for degradation of polyubiquitinated proteins by the 26S proteasome but is thought to play no role in proteasomal stability during the catalytic cycle. In contrast to this view, we report that ATP hydrolysis triggers rapid dissociation of the 19S regulatory particles from immunopurified 26S complexes in a manner coincident with release of the bulk of proteasome-interacting proteins. Strikingly, this mechanism leads to quantitative disassembly of the 19S into subcomplexes and free Rpn10, the polyubiquitin binding subunit. Biochemical reconstitution with purified Sic1, a prototype substrate of the Cdc34/SCF ubiquitin ligase, suggests that substrate degradation is essential for triggering the ATP hydrolysis-dependent dissociation and disassembly of the 19S and that this mechanism leads to release of degradation products. This is the first demonstration that a controlled dissociation of the 19S regulatory particles from the 26S proteasome is part of the mechanism of protein degradation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Endopeptidasas/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/ultraestructura , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Portadoras , Catálisis , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Endopeptidasas/ultraestructura , Hidrólisis , Microscopía Electrónica , Complejo de la Endopetidasa Proteasomal/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
The S. cerevisiae SCF(Cdc4) is a prototype of RING-type SCF E3s, which recruit substrates for polyubiquitination by the Cdc34 ubiquitin-conjugating enzyme. Current models propose that Cdc34 ubiquitinates the substrate while remaining bound to the RING domain. In contrast, we found that the formation of a ubiquitin thiol ester regulates the Cdc34/SCF(Cdc4) binding equilibrium by increasing the dissociation rate constant, with only a minor effect on the association rate. By using a F72VCdc34 mutant with increased affinity for the RING domain, we demonstrate that release of ubiquitin-charged Cdc34-S - Ub from the RING is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1. Release of ubiquitin-charged E2 from E3 prior to ubiquitin transfer is a previously unrecognized step in ubiquitination, which can explain both the modification of multiple lysines on the recruited substrate and the extension of polyubiquitin chains. We discuss implications of this finding for function of other ubiquitin ligases.