RESUMEN
Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.
Asunto(s)
Autofagosomas/metabolismo , Retículo Endoplásmico/metabolismo , Animales , Autofagia , Proteínas Relacionadas con la Autofagia/metabolismo , HumanosRESUMEN
In macroautophagy, membrane structures called autophagosomes engulf substrates and deliver them for lysosomal degradation. Autophagosomes enwrap a variety of targets with diverse sizes, from portions of cytosol to larger organelles. However, the mechanism by which autophagosome size is controlled remains elusive. We characterized a novel ER membrane protein, ERdj8, in mammalian cells. ERdj8 localizes to a meshwork-like ER subdomain along with phosphatidylinositol synthase (PIS) and autophagy-related (Atg) proteins. ERdj8 overexpression extended the size of the autophagosome through its DnaJ and TRX domains. ERdj8 ablation resulted in a defect in engulfing larger targets. C. elegans, in which the ERdj8 orthologue dnj-8 was knocked down, could perform autophagy on smaller mitochondria derived from the paternal lineage but not the somatic mitochondria. Thus, ERdj8 may play a critical role in autophagosome formation by providing the capacity to target substrates of diverse sizes for degradation.
Asunto(s)
Autofagosomas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Macroautofagia , Animales , Animales Modificados Genéticamente , Autofagosomas/genética , Autofagosomas/ultraestructura , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa/genética , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa/metabolismo , Células COS , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Chlorocebus aethiops , Retículo Endoplásmico/genética , Retículo Endoplásmico/ultraestructura , Proteínas del Choque Térmico HSP40/genética , Células HeLa , Humanos , Mitocondrias/metabolismo , Mitocondrias/ultraestructuraRESUMEN
Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca2+ dependent manner. Cytosolic Ca2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca2+ increase, thereby suppressing induction of autophagy.
Asunto(s)
Autofagia/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Calcineurina/metabolismo , Calcio/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fosforilación , Inanición/metabolismoRESUMEN
Endoplasmic reticulum (ER) stress causes the ER-resident transmembrane protein Ire1 to self-associate, leading to the formation of large oligomeric clusters. In yeast cells, this induces strong unfolded protein response (UPR) through splicing of HAC1 mRNA. Here, we demonstrate that highly ER-stressed yeast cells exhibited poor Ire1 clustering in the presence of the actin-disrupting agent latrunculin-A. Under these conditions, Ire1 may form smaller oligomers because latrunculin-A only partially diminished the Ire1-mediated splicing of HAC1 mRNA. Ire1 cluster formation was also impaired by deletion of the type-II myosin gene MYO1 or SAC6, which encodes the actin-bundling protein fimbrin. Finally, we demonstrated that Ire1 clusters are predominantly located on or near actin filaments. Therefore, we propose that actin filaments play an important role in ER stress-induced clustering of Ire1.
Asunto(s)
Actinas/metabolismo , Estrés del Retículo Endoplásmico , Glicoproteínas de Membrana/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Ditiotreitol/farmacología , Eliminación de Gen , Genes Fúngicos , Glicoproteínas de Membrana/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Familia de Multigenes , Cadenas Pesadas de Miosina/genética , Proteínas Serina-Treonina Quinasas/genética , Empalme del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tiazolidinas/farmacologíaRESUMEN
Misfolded proteins in the endoplasmic reticulum (ER) are dislocated out of the ER to the cytosol, polyubiquitinated, and degraded by the ubiquitin-proteasome system in a process collectively termed ER-associated degradation (ERAD). Recent studies have established that a mammalian ER-localized transmembrane J-protein, DNAJB12, cooperates with Hsc70, a cytosolic Hsp70 family member, to promote the ERAD of misfolded membrane proteins. Interestingly, mammalian genomes have another J-protein called DNAJB14 that shows a high sequence similarity to DNAJB12. Yet, very little was known about this protein. Here, we report the characterization of DNAJB14. Immunofluorescence study and protease protection assay showed that, like DNAJB12, DNAJB14 is an ER-localized, single membrane-spanning J-protein with its J-domain facing the cytosol. We used co-immunoprecipitation assay to find that DNAJB14 can also specifically bind Hsc70 via its J-domain to recruit this chaperone to ER membrane. Remarkably, the overexpression of DNAJB14 accelerated the degradation of misfolded membrane proteins including a mutant of cystic fibrosis transmembrane conductance regulator (CFTRΔF508), but not that of a misfolded luminal protein. Furthermore, the DNAJB14-dependent degradation of CFTRΔF508 was compromised by MG132, a proteasome inhibitor, indicating that DNAJB14 can enhance the degradation of a misfolded membrane protein using the ubiquitin-proteasome system. Thus, the mammalian ER possesses two analogous J-proteins (DNAJB14 and DNAJB12) that both can promote the ERAD of misfolded transmembrane proteins. Compared with DNAJB12 mRNA that was widely expressed in mouse tissues, DNAJB14 mRNA was expressed more weakly, being most abundant in testis, implying its specific role in this tissue.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de la Membrana/química , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/genética , Humanos , Cinética , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Chaperonas Moleculares , Datos de Secuencia Molecular , Células 3T3 NIH , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteolisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Cytosolic Hsc70/Hsp70 are known to contribute to the endoplasmic reticulum (ER)-associated degradation of membrane proteins. However, at least in mammalian cells, its partner ER-localized J-protein for this cellular event has not been identified. Here we propose that this missing protein is DNAJB12. Protease protection assay and immunofluorescence study revealed that DNAJB12 is an ER-localized single membrane-spanning protein carrying a J-domain facing the cytosol. Using co-immunoprecipitation assay, we found that DNAJB12 is able to bind Hsc70 and thus can recruit Hsc70 to the ER membrane. Remarkably, cellular overexpression of DNAJB12 accelerated the degradation of misfolded membrane proteins including cystic fibrosis transmembrane conductance regulator (CFTR), but not a misfolded luminal protein. The DNAJB12-dependent degradation of CFTR was compromised by a proteasome inhibitor, lactacystin, suggesting that this process requires the ubiquitin-proteasome system. Conversely, knockdown of DNAJB12 expression attenuated the degradation of CFTR. Thus, DNAJB12 is a novel mammalian ER-localized J-protein that plays a vital role in the quality control of membrane proteins.