RESUMEN
Cells dynamically adapt organelle size to current physiological demand. Organelle growth requires membrane biogenesis and therefore needs to be coordinated with lipid metabolism. The endoplasmic reticulum (ER) can undergo massive expansion, but the underlying regulatory mechanisms are largely unclear. Here, we describe a genetic screen for factors involved in ER membrane expansion in budding yeast and identify the ER transmembrane protein Ice2 as a strong hit. We show that Ice2 promotes ER membrane biogenesis by opposing the phosphatidic acid phosphatase Pah1, called lipin in metazoa. Specifically, Ice2 inhibits the conserved Nem1-Spo7 complex and thus suppresses the dephosphorylation and activation of Pah1. Furthermore, Ice2 cooperates with the transcriptional regulation of lipid synthesis genes and helps to maintain cell homeostasis during ER stress. These findings establish the control of the lipin phosphatase complex as an important mechanism for regulating ER membrane biogenesis.
Asunto(s)
Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidato Fosfatasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico , Regulación Fúngica de la Expresión Génica , Metabolismo de los Lípidos , Proteínas de la Membrana/genética , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Compuestos Orgánicos/metabolismo , Fosfatidato Fosfatasa/genética , Fosforilación , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Respuesta de Proteína DesplegadaRESUMEN
Autophagy is fundamental for cell and organismal health. Two types of autophagy are conserved in eukaryotes: macroautophagy and microautophagy. During macroautophagy, autophagosomes deliver cytoplasmic constituents to endosomes or lysosomes, whereas during microautophagy lytic organelles take up cytoplasm directly. While macroautophagy has been investigated extensively, microautophagy has received much less attention. Nonetheless, it has become clear that microautophagy has a broad range of functions in biosynthetic transport, metabolic adaptation, organelle remodeling and quality control. This Review discusses the selective and non-selective microautophagic processes known in yeast, plants and animals. Based on the molecular mechanisms for the uptake of microautophagic cargo into lytic organelles, I propose to distinguish between fission-type microautophagy, which depends on ESCRT proteins, and fusion-type microautophagy, which requires the core autophagy machinery and SNARE proteins. Many questions remain to be explored, but the functional versatility and mechanistic diversity of microautophagy are beginning to emerge.
Asunto(s)
Lisosomas , Microautofagia , Animales , Autofagia , Endosomas , Saccharomyces cerevisiae/genéticaRESUMEN
Changing conditions necessitate cellular adaptation, which frequently entails adjustment of organelle size and shape. The endoplasmic reticulum (ER) is an organelle of exceptional morphological plasticity. In budding yeast, ER stress triggers the de novo formation of ER subdomains called ER whorls. These whorls are selectively degraded by a poorly defined type of microautophagy. We recently showed that ESCRT proteins are essential for microautophagic uptake of ER whorls into lysosomes, likely by mediating the final scission of the lysosomal membrane. Furthermore, ER-selective microautophagy acts in parallel with ER-selective macroautophagy. The molecular machineries for these two types of autophagy are distinct and their contributions to ER turnover vary according to conditions, suggesting that they serve different functions. Our study provides evidence for a direct role of ESCRTs in microautophagy and extends our understanding of how autophagy promotes organelle homeostasis.
Asunto(s)
Autofagia/fisiología , Retículo Endoplásmico/metabolismo , Lisosomas/metabolismo , Microautofagia/fisiología , Estrés del Retículo Endoplásmico/fisiología , Homeostasis/fisiología , Humanos , Membranas IntracelularesRESUMEN
ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and the ESCRT machinery as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that the ESCRT machinery directly functions in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte , Membranas Intracelulares/metabolismo , Microautofagia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Homeostasis , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Misfolded proteins in the endoplasmic reticulum (ER) activate the unfolded protein response (UPR), which enhances protein folding to restore homeostasis. Additional pathways respond to ER stress, but how they help counteract protein misfolding is incompletely understood. Here, we develop a titratable system for the induction of ER stress in yeast to enable a genetic screen for factors that augment stress resistance independently of the UPR. We identify the proteasome biogenesis regulator Rpn4 and show that it cooperates with the UPR. Rpn4 abundance increases during ER stress, first by a post-transcriptional, then by a transcriptional mechanism. Induction of RPN4 transcription is triggered by cytosolic mislocalization of secretory proteins, is mediated by multiple signaling pathways and accelerates clearance of misfolded proteins from the cytosol. Thus, Rpn4 and the UPR are complementary elements of a modular cross-compartment response to ER stress.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada , Retículo Endoplásmico/enzimología , Biogénesis de OrganelosRESUMEN
Pyrophosphate (PPi), a byproduct of macromolecule biosynthesis is maintained at low levels by soluble inorganic pyrophosphatases (sPPase) found in all eukaryotes. In plants, H+-pumping pyrophosphatases (H+-PPase) convert the substantial energy present in PPi into an electrochemical gradient. We show here, that both cold- and heat stress sensitivity of fugu5 mutants lacking the major H+-PPase isoform AVP1 is correlated with reduced SUMOylation. In addition, we show that increased PPi concentrations interfere with SUMOylation in yeast and we provide evidence that SUMO activating E1-enzymes are inhibited by micromolar concentrations of PPi in a non-competitive manner. Taken together, our results do not only provide a mechanistic explanation for the beneficial effects of AVP1 overexpression in plants but they also highlight PPi as an important integrator of metabolism and stress tolerance.
Asunto(s)
Arabidopsis/fisiología , Difosfatos/metabolismo , Estrés Fisiológico , Sumoilación , Aclimatación , Arabidopsis/enzimología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Frío , Calor , Pirofosfatasa Inorgánica/metabolismo , Isoenzimas/metabolismoRESUMEN
When faced with proteotoxic stress, cells mount adaptive responses to eliminate aberrant proteins. Adaptive responses increase the expression of protein folding and degradation factors to enhance the cellular quality control machinery. However, it is unclear whether and how this augmented machinery acquires new activities during stress. Here, we uncover a regulatory cascade in budding yeast that consists of the hydrophilin protein Roq1/Yjl144w, the HtrA-type protease Ynm3/Nma111, and the ubiquitin ligase Ubr1. Various stresses stimulate ROQ1 transcription. The Roq1 protein is cleaved by Ynm3. Cleaved Roq1 interacts with Ubr1, transforming its substrate specificity. Altered substrate recognition by Ubr1 accelerates proteasomal degradation of misfolded as well as native proteins at the endoplasmic reticulum membrane and in the cytosol. We term this pathway stress-induced homeostatically regulated protein degradation (SHRED) and propose that it promotes physiological adaptation by reprogramming a key component of the quality control machinery.
Asunto(s)
Adaptación Fisiológica/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Proteolisis , Saccharomyces cerevisiae/enzimología , Serina Endopeptidasas/metabolismo , Estrés Fisiológico/fisiología , Especificidad por Sustrato , Ubiquitina/metabolismoRESUMEN
The endoplasmic reticulum (ER) is the largest membrane-bound organelle in cells, and its size needs to be carefully controlled. Downsizing the ER by autophagy is now shown to involve Sec62, a protein that also helps to build up the organelle. This link suggests a molecular switch for ER size control.
Asunto(s)
Autofagia/fisiología , Retículo Endoplásmico/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Orgánulos/metabolismo , Animales , Humanos , Transporte de ProteínasRESUMEN
Flagellar length control in Chlamydomonas reinhardtii provides a simple model system in which to investigate the general question of how cells regulate organelle size. Previous work demonstrated that Chlamydomonas cytoplasm contains a pool of flagellar precursor proteins sufficient to assemble a half-length flagellum and that assembly of full-length flagella requires synthesis of additional precursors to augment the preexisting pool. The regulatory systems that control the synthesis and regeneration of this pool are not known, although transcriptional regulation clearly plays a role. We used quantitative analysis of length distributions to identify candidate genes controlling pool regeneration and found that a mutation in the p80 regulatory subunit of katanin, encoded by the PF15 gene in Chlamydomonas, alters flagellar length by changing the kinetics of precursor pool utilization. This finding suggests a model in which flagella compete with cytoplasmic microtubules for a fixed pool of tubulin, with katanin-mediated severing allowing easier access to this pool during flagellar assembly. We tested this model using a stochastic simulation that confirms that cytoplasmic microtubules can compete with flagella for a limited tubulin pool, showing that alteration of cytoplasmic microtubule severing could be sufficient to explain the effect of the pf15 mutations on flagellar length.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas Algáceas/genética , Chlamydomonas reinhardtii/genética , Flagelos/genética , Modelos Estadísticos , Precursores de Proteínas/genética , Subunidades de Proteína/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestructura , Simulación por Computador , Flagelos/metabolismo , Flagelos/ultraestructura , Regulación de la Expresión Génica , Katanina , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Tamaño de los Orgánulos , Precursores de Proteínas/metabolismo , Subunidades de Proteína/metabolismo , Transducción de Señal , Procesos Estocásticos , Transcripción Genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Selective autophagy of damaged or redundant organelles is an important mechanism for maintaining cell homeostasis. We found previously that endoplasmic reticulum (ER) stress in the yeast Saccharomyces cerevisiae causes massive ER expansion and triggers the formation of large ER whorls. Here, we show that stress-induced ER whorls are selectively taken up into the vacuole, the yeast lysosome, by a process termed ER-phagy. Import into the vacuole does not involve autophagosomes but occurs through invagination of the vacuolar membrane, indicating that ER-phagy is topologically equivalent to microautophagy. Even so, ER-phagy requires neither the core autophagy machinery nor several other proteins specifically implicated in microautophagy. Thus, autophagy of ER whorls represents a distinct type of organelle-selective autophagy. Finally, we provide evidence that ER-phagy degrades excess ER membrane, suggesting that it contributes to cell homeostasis by controlling organelle size.
Asunto(s)
Autofagia , Retículo Endoplásmico/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Estrés del Retículo Endoplásmico , Lisosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismoRESUMEN
Eisosomes are stable domains at the plasma membrane of the budding yeast Saccharomyces cerevisiae and have been proposed to function in endocytosis. Eisosomes are composed of two main cytoplasmic proteins, Pil1 and Lsp1, that form a scaffold around furrow-like plasma membrane invaginations. We show here that the poorly characterized eisosome protein Seg1/Ymr086w is important for eisosome biogenesis and architecture. Seg1 was required for efficient incorporation of Pil1 into eisosomes and the generation of normal plasma membrane furrows. Seg1 preceded Pil1 during eisosome formation and established a platform for the assembly of other eisosome components. This platform was further shaped and stabilized upon the arrival of Pil1 and Lsp1. Moreover, Seg1 abundance controlled the shape of eisosomes by determining their length. Similarly, the Schizosaccharomyces pombe Seg1-like protein Sle1 was necessary to generate the filamentous eisosomes present in fission yeast. The function of Seg1 in the stepwise biogenesis of eisosomes reveals striking architectural similarities between eisosomes in yeast and caveolae in mammals.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas de la Membrana/genética , Fosfoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Animales , Membrana Celular/metabolismo , Citoplasma/metabolismo , Endocitosis , Proteínas Fluorescentes Verdes/química , Inmunohistoquímica , Liposomas/química , Proteínas de la Membrana/fisiología , Microscopía Confocal/métodos , Microscopía Electrónica/métodos , Estructura Terciaria de Proteína , Proteómica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Schizosaccharomyces/metabolismoRESUMEN
Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.
Asunto(s)
Autofagia/genética , Endorribonucleasas/metabolismo , Proteínas de la Membrana/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Respuesta de Proteína Desplegada/genética , Ácidos/metabolismo , Animales , Supervivencia Celular/genética , Células Cultivadas , Drosophila/genética , Endorribonucleasas/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Organismos Modificados Genéticamente , Fagosomas/genética , Fagosomas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Saccharomyces cerevisiae/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Inanición/metabolismo , Vesículas Transportadoras/metabolismo , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). Ire1, an ER-resident transmembrane kinase/RNase, senses the protein folding status inside the ER. When activated, Ire1 oligomerizes and trans-autophosphorylates, activating its RNase and initiating a nonconventional mRNA splicing reaction. Splicing results in production of the transcription factor Hac1 that induces UPR target genes; expression of these genes restores ER homeostasis by increasing its protein folding capacity and allows abatement of UPR signaling. Here, we uncouple Ire1's RNase from its kinase activity and find that cells expressing kinase-inactive Ire1 can regulate Ire1's RNase, splice HAC1 mRNA, produce Hac1 protein, and induce UPR target genes. Unlike wild-type IRE1, kinase-inactive Ire1 cells display defects in Ire1 deactivation. Failure to properly inactivate Ire1 causes chronic ER stress and reduces cell survival under UPR-inducing conditions. Thus, Ire1-catalyzed phosphoryl-transfer aids disassembly of Ire1 signaling complexes and is a critical component of the UPR homeostatic feedback loop.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Supervivencia Celular , Células Cultivadas , Homeostasis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/aislamiento & purificación , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Empalme del ARN/genética , ARN Mensajero/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Respuesta de Proteína DesplegadaRESUMEN
Many proteins mature within the secretory pathway by the acquisition of glycans. Failure to maintain the proper distribution of the glycosylation machinery might lead to disease. High expression levels of the ubiquitous Golgi protein estrogen receptor-binding fragment-associated gene 9 (EBAG9) in human tumors correlate with poor clinical prognosis, and EBAG9 overexpression in epithelial cell lines induces truncated glycans, typical of many carcinomas. Here, we addressed the pathogenetic link between EBAG9 expression and the alteration of the cellular glycome. We applied confocal microscopy, live imaging, pulse-chase labeling in conjunction with immunoprecipitation, and enzymatic activity assays in a variety of EBAG9-overexpressing or depleted epithelial tumor cell lines. EBAG9 shuttles between the ER-Golgi intermediate compartment and the cis-Golgi, and we demonstrate association of EBAG9 with coat protein complex I (COPI)-coated transport vesicles. EBAG9 overexpression imposes delay of endoplasmic reticulum-to-Golgi transport and mislocalizes components of the ER quality control and glycosylation machinery. Conversely, EBAG9 down-regulation accelerates glycoprotein transport through the Golgi and enhances mannosidase activity. Thus, EBAG9 acts as a negative regulator of a COPI-dependent ER-to-Golgi transport pathway in epithelial cells and represents a novel pathogenetic principle in which interference with intracellular membrane trafficking results in the emergence of a tumor-associated glycome.
Asunto(s)
Antígenos de Neoplasias/fisiología , Proteína Coat de Complejo I/fisiología , Glicoproteínas/metabolismo , Western Blotting , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , HumanosRESUMEN
Cells constantly adjust the sizes and shapes of their organelles according to need. In this study, we examine endoplasmic reticulum (ER) membrane expansion during the unfolded protein response (UPR) in the yeast Saccharomyces cerevisiae. We find that membrane expansion occurs through the generation of ER sheets, requires UPR signaling, and is driven by lipid biosynthesis. Uncoupling ER size control and the UPR reveals that membrane expansion alleviates ER stress independently of an increase in ER chaperone levels. Converting the sheets of the expanded ER into tubules by reticulon overexpression does not affect the ability of cells to cope with ER stress, showing that ER size rather than shape is the key factor. Thus, increasing ER size through membrane synthesis is an integral yet distinct part of the cellular program to overcome ER stress.
Asunto(s)
Retículo Endoplásmico/fisiología , Membranas Intracelulares/fisiología , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/citología , Estrés Fisiológico/fisiología , Tamaño de la Célula , Retículo Endoplásmico/ultraestructura , Membranas Intracelulares/ultraestructura , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Transducción de Señal/fisiologíaRESUMEN
With cellular organelles coming in all shapes and sizes, the principle 'form follows function' is readily discernible through the cytologist's lens. Architecturally, one might ask whether there is feedback in this organization. Does a cell 'know' when it has constructed membrane into the stacks of the Golgi, the cisternae of the mitochondria or the tubules of the endoplasmic reticulum? Proofreading can occur in vivo as both errors in nucleic acids and misfolds in proteins are recognized by the cell. Are there analogous systems which maintain/regulate the architectural integrity of organelles? Our recent paper entitled Generation of cubic membranes from controlled homotypic interactions of membrane proteins in the endoplasmic reticulum suggests that autophagy may play such a role.