RESUMEN
2-deoxyglucose is a glucose analog that impacts many aspects of cellular physiology. After its uptake and its phosphorylation into 2-deoxyglucose-6-phosphate (2DG6P), it interferes with several metabolic pathways including glycolysis and protein N-glycosylation. Despite this systemic effect, resistance can arise through strategies that are only partially understood. In yeast, 2DG resistance is often associated with mutations causing increased activity of the yeast 5'-AMP activated protein kinase (AMPK), Snf1. Here we focus on the contribution of a Snf1 substrate in 2DG resistance, namely the alpha-arrestin Rod1 involved in nutrient transporter endocytosis. We report that 2DG triggers the endocytosis of many plasma membrane proteins, mostly in a Rod1-dependent manner. Rod1 participates in 2DG-induced endocytosis because 2DG, following its phosphorylation by hexokinase Hxk2, triggers changes in Rod1 post-translational modifications and promotes its function in endocytosis. Mechanistically, this is explained by a transient, 2DG-induced inactivation of Snf1/AMPK by protein phosphatase 1 (PP1). We show that 2DG-induced endocytosis is detrimental to cells, and the lack of Rod1 counteracts this process by stabilizing glucose transporters at the plasma membrane. This facilitates glucose uptake, which may help override the metabolic blockade caused by 2DG, and 2DG export-thus terminating the process of 2DG detoxification. Altogether, these results shed a new light on the regulation of AMPK signaling in yeast and highlight a remarkable strategy to bypass 2DG toxicity involving glucose transporter regulation.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Proteínas de Saccharomyces cerevisiae , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Desoxiglucosa/farmacología , Endocitosis/genética , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Humanos , Redes y Vías Metabólicas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Anti-cancer strategies that target the glycolytic metabolism of tumors have been proposed. The glucose analog 2-deoxyglucose (2DG) is imported into cells and, after phosphorylation, becomes 2DG-6-phosphate, a toxic by-product that inhibits glycolysis. Using yeast as a model, we performed an unbiased mass spectrometry-based approach to probe the cellular effects of 2DG on the proteome and study resistance mechanisms to 2DG. We found that two phosphatases that target 2DG-6-phosphate were induced upon exposure to 2DG and participated in 2DG detoxification. Dog1 and Dog2 are HAD (haloacid dehalogenase)-like phosphatases, which are evolutionarily conserved. 2DG induced Dog2 by activating several signaling pathways, such as the stress response pathway mediated by the p38 MAPK ortholog Hog1, the unfolded protein response (UPR) triggered by 2DG-induced ER stress, and the cell wall integrity (CWI) pathway mediated by the MAPK Slt2. Loss of the UPR or CWI pathways led to 2DG hypersensitivity. In contrast, mutants impaired in the glucose-mediated repression of genes were 2DG resistant because glucose availability transcriptionally repressed DOG2 by inhibiting signaling mediated by the AMPK ortholog Snf1. The characterization and genome resequencing of spontaneous 2DG-resistant mutants revealed that DOG2 overexpression was a common strategy underlying 2DG resistance. The human Dog2 homolog HDHD1 displayed phosphatase activity toward 2DG-6-phosphate in vitro and its overexpression conferred 2DG resistance in HeLa cells, suggesting that this 2DG phosphatase could interfere with 2DG-based chemotherapies. These results show that HAD-like phosphatases are evolutionarily conserved regulators of 2DG resistance.
Asunto(s)
Desoxiglucosa/farmacología , Farmacorresistencia Fúngica/efectos de los fármacos , Glucólisis/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Antimetabolitos/metabolismo , Antimetabolitos/farmacología , Desoxiglucosa/metabolismo , Farmacorresistencia Fúngica/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Glucosa/metabolismo , Glucosa/farmacología , Células HeLa , Humanos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Monoéster Fosfórico Hidrolasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/genética , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genéticaRESUMEN
Proteostasis in eukaryotes is maintained by compartment-specific quality control pathways, which enable the refolding or the degradation of defective polypeptides to prevent the toxicity that may arise from their aggregation. Among these processes, translational protein quality control is performed by the Ribosome-bound Quality Control complex (RQC), which recognizes nascent peptides translated from aberrant mRNAs, polyubiquitylates these aberrant peptides, extracts them from the stalled 60S subunit and finally escorts them to the proteasome for degradation. In this review, we focus on the mechanism of action of the RQC complex from stalled 60S binding to aberrant peptide delivery to the proteasome and describe the cellular consequences of a deficiency in the RQC pathway, such as aberrant protein aggregation. In addition, this review covers the recent discoveries concerning the role of cytosolic chaperones, as well as Tom1, to prevent the accumulation of aberrant protein aggregates in case of a deficiency in the RQC pathway.
Asunto(s)
Chaperonas Moleculares/genética , Biosíntesis de Proteínas , Proteostasis/genética , Subunidades Ribosómicas Grandes de Eucariotas/genética , Saccharomyces cerevisiae/genética , Animales , Humanos , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas/genética , Proteolisis , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Nutrient availability controls the landscape of nutrient transporters present at the plasma membrane, notably by regulating their ubiquitylation and subsequent endocytosis. In yeast, this involves the Nedd4 ubiquitin ligase Rsp5 and arrestin-related trafficking adaptors (ARTs). ARTs are targeted by signaling pathways and warrant that cargo ubiquitylation and endocytosis appropriately respond to nutritional inputs. Here, we show that glucose deprivation regulates the ART protein Csr2/Art8 at multiple levels to trigger high-affinity glucose transporter endocytosis. Csr2 is transcriptionally induced in these conditions through the AMPK orthologue Snf1 and downstream transcriptional repressors. Upon synthesis, Csr2 becomes activated by ubiquitylation. In contrast, glucose replenishment induces CSR2 transcriptional shutdown and switches Csr2 to an inactive, deubiquitylated form. This glucose-induced deubiquitylation of Csr2 correlates with its phospho-dependent association with 14-3-3 proteins and involves protein kinase A. Thus, two glucose signaling pathways converge onto Csr2 to regulate hexose transporter endocytosis by glucose availability. These data illustrate novel mechanisms by which nutrients modulate ART activity and endocytosis.
Asunto(s)
Arrestina/metabolismo , Endocitosis , Glucosa/deficiencia , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas 14-3-3/metabolismo , Arrestina/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Transporte de Monosacáridos/genética , Mutación , Proteínas Nucleares/genética , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Tiempo , Transcripción Genética , UbiquitinaciónRESUMEN
Protein quality control mechanisms eliminate defective polypeptides to ensure proteostasis and to avoid the toxicity of protein aggregates. In eukaryotes, the ribosome-bound quality control (RQC) complex detects aberrant nascent peptides that remain stalled in 60S ribosomal particles due to a dysfunction in translation termination. The RQC complex polyubiquitylates aberrant polypeptides and recruits a Cdc48 hexamer to extract them from 60S particles in order to escort them to the proteasome for degradation. Whereas the steps from stalled 60S recognition to aberrant peptide polyubiquitylation by the RQC complex have been described, the mechanism leading to proteasomal degradation of these defective translation products remains unknown. We show here that the RQC complex also exists as a ribosome-unbound complex during the escort of aberrant peptides to the proteasome. In addition, we identify a new partner of this light version of the RQC complex, the E3 ubiquitin ligase Tom1. Tom1 interacts with aberrant nascent peptides and is essential to limit their accumulation and aggregation in the absence of Rqc1; however, its E3 ubiquitin ligase activity is not required. Taken together, these results reveal new roles for Tom1 in protein quality control, aggregate prevention, and, therefore, proteostasis maintenance.
Asunto(s)
Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Proteínas de Ciclo Celular/metabolismo , Terminación de la Cadena Péptídica Traduccional/fisiología , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Biosíntesis de Proteínas , Proteolisis , Proteínas de Unión al ARN/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/fisiología , Saccharomyces cerevisiae/metabolismo , UbiquitinaciónRESUMEN
Ski2-Ski3-Ski8 (Ski) is a helicase complex functioning with the RNA-degrading exosome to mediate the 3'-5' messenger RNA (mRNA) decay in turnover and quality-control pathways. We report that the Ski complex directly associates with 80S ribosomes presenting a short mRNA 3' overhang. We determined the structure of an endogenous ribosome-Ski complex using cryo-electron microscopy (EM) with a local resolution of the Ski complex ranging from 4 angstroms (Å) in the core to about 10 Å for intrinsically flexible regions. Ribosome binding displaces the autoinhibitory domain of the Ski2 helicase, positioning it in an open conformation near the ribosomal mRNA entry tunnel. We observe that the mRNA 3' overhang is threaded directly from the small ribosomal subunit to the helicase channel of Ski2, primed for ongoing exosome-mediated 3'-5' degradation.
Asunto(s)
ADN Helicasas/ultraestructura , Complejo Multienzimático de Ribonucleasas del Exosoma/ultraestructura , Proteínas Nucleares/ultraestructura , Estabilidad del ARN , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/enzimología , Microscopía por Crioelectrón , Conformación Proteica , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/enzimologíaRESUMEN
Protein homeostasis is maintained by quality control mechanisms that detect and eliminate deficient translation products. Cytosolic defective proteins can arise from translation of aberrant mRNAs lacking a termination codon (NonStop) or containing a sequence that blocks translation elongation (No-Go), which results in translational arrest. Stalled ribosomes are dissociated, aberrant mRNAs are degraded by the cytoplasmic exosome, and the nascent peptides remaining in stalled 60S exit tunnels are detected by the ribosome-bound quality control complex (RQC) composed of Ltn1, Rqc1, Rqc2, and Cdc48. Whereas Ltn1 polyubiquitylates these nascent peptides, Rqc2 directs the addition of C-terminal alanine-threonine tails (CAT-tails), and a Cdc48 hexamer is recruited to extract the nascent peptides, which are addressed to the proteasome for degradation. Although the functions of most RQC components have been described, the role of Rqc1 in this quality control process remains undetermined. In this article we show that the absence of Rqc1 or Ltn1 results in the aggregation of aberrant proteins, a phenomenon that requires CAT-tail addition to the nascent peptides by Rqc2. Our results suggest that aberrant CAT-tailed protein aggregation results from a defect in Cdc48 recruitment to stalled 60S particles, a process that requires both Rqc1 and Ltn1. These protein aggregates contain Ltn1-dependent polyubiquitin chains and are degraded by the proteasome. Finally, aggregate characterization by proteomics revealed that they contain specific chaperones including Sis1, Sgt2, Ssa1/2, and Hsp82, suggesting that these protein aggregates may be addressed to aggresome-like structures when the RQC complex fails to deliver aberrant nascent peptides to the proteasome for degradation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Alanina/química , Alanina/genética , Alanina/metabolismo , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopía Fluorescente , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas , Biosíntesis de Proteínas/genética , Proteolisis , Proteómica/métodos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Treonina/química , Treonina/genética , Treonina/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteína que Contiene ValosinaRESUMEN
Ribosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation through conserved mechanisms. For example, mRNAs lacking a stop codon are degraded by the exosome in association with its cofactor, the SKI complex, whereas the corresponding aberrant nascent polypeptides are ubiquitinated by the E3 ligases Ltn1 and Not4 and become proteasome substrates. How translation arrest is linked with polypeptide degradation is still unclear. Genetic screens with SKI and LTN1 mutants allowed us to identify translation-associated element 2 (Tae2) and ribosome quality control 1 (Rqc1), two factors that we found associated, together with Ltn1 and the AAA-ATPase Cdc48, to 60S ribosomal subunits. Translation-associated element 2 (Tae2), Rqc1, and Cdc48 were all required for degradation of polypeptides synthesized from Non-Stop mRNAs (Non-Stop protein decay; NSPD). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles. Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway, showing that the polyubiquitination of Non-Stop peptides is a progressive process. We propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S, on which Cdc48 is recruited to escort the substrate for proteasomal degradation.