RESUMO
The endoplasmic reticulum (ER) undergoes degradation by selective macroautophagy (ER-phagy) in response to starvation or the accumulation of misfolded proteins within its lumen. In yeast, actin assembly at sites of contact between the cortical ER (cER) and endocytic pits acts to displace elements of the ER from their association with the plasma membrane (PM) so they can interact with the autophagosome assembly machinery near the vacuole. A collection of proteins tether the cER to the PM. Of these, Scs2/22 and Ist2 are required for cER-phagy, most likely through their roles in lipid transport, while deletion of the tricalbins, TCB1/2/3, bypasses those requirements. An artificial ER-PM tether blocks cER-phagy in both the wild type (WT) and a strain lacking endogenous tethers, supporting the importance of cER displacement from the PM. Scs2 and Ist2 can be cross-linked to the selective cER-phagy receptor, Atg40. The COPII cargo adaptor subunit, Lst1, associates with Atg40 and is required for cER-phagy. This requirement is also bypassed by deletion of the ER-PM tethers, suggesting a role for Lst1 prior to the displacement of the cER from the PM during cER-phagy. Although pexophagy and mitophagy also require actin assembly, deletion of ER-PM tethers does not bypass those requirements. We propose that within the context of rapamycin-induced cER-phagy, Scs2/22, Ist2, and Lst1 promote the local displacement of an element of the cER from the cortex, while Tcb1/2/3 act in opposition, anchoring the cER to the plasma membrane.
Assuntos
Autofagia , Membrana Celular , Retículo Endoplasmático , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Retículo Endoplasmático/metabolismo , Autofagia/fisiologia , Membrana Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
Reticulophagy is mediated by autophagy receptors that function in one of the two domains of the ER, tubules or flat sheets. Three different conserved mammalian receptors mediate autophagy in ER tubules: RTN3L, ATL3 and CALCOCO1. Previous studies have shown that RTN3L maintains proteostasis by targeting mutant aggregation-prone proteins for autophagy at distinct foci in ER tubules that we named ERPHS (ER-reticulophagy sites). The role for ATL3 and CALCOCO1 in proteostasis has not been addressed. Here we analyzed three different misfolded disease-causing RTN3L substrates and show that ATL3 and CALCOCO1 target the same cargoes for autophagy. Colocalization and knock down studies revealed that RTN3L and ATL3 are both required for the formation of RTN3L-containing ERPHS, while CALCOCO1 is not. We propose that RTN3L, ATL3 and CALCOCO1 work in parallel to maintain proteostasis within the ER network by targeting cargoes at different sites in the tubules.Abbreviation ATL3: atlastin GTPase 3; Baf: bafilomycin A1; CALCOCO1: calcium binding and coiled-coil domain 1; Epr1: ER-phagy receptor 1; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERPHS: ER-reticulophagy sites; LAMP1: lysosomal associated membrane protein 1; PGRMC1: progesterone receptor membrane component 1; POMC: proopiomelanocortin; Pro-AVP: pro-arginine vasopressin; RETREG1: reticulophagy regulator 1; reticulophagy: endoplasmic reticulum selective autophagy; RTN3L: reticulon 3 long isoform; VAPA: VAMP associated protein A.
Assuntos
Autofagia , Retículo Endoplasmático , Proteínas de Membrana , Proteostase , Humanos , Autofagia/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas NogoRESUMO
A recent screen of the Saccharomyces cerevisiae deletion library implicated End3 in autophagy of the endoplasmic reticulum (ER). Together with Pan1, End3 coordinates endocytic site initiation with the localized assembly of branching actin filaments that promotes invagination of endocytic pits. Oxysterol binding proteins function as an inter-organelle bridge by interacting with VAP proteins on the cortical ER and type I myosins on the endocytic pit. These proteins not only promote localized actin assembly at contact sites, they are required for ER autophagy as well. We propose that localized actin polymerization can push the edge of an ER sheet from the cell cortex toward the site of autophagosome assembly near the vacuole.
Assuntos
Actinas , Proteínas de Saccharomyces cerevisiae , Actinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Autofagia , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/metabolismoRESUMO
Lipid transfer proteins mediate the exchange of lipids between closely apposed membranes at organelle contact sites and play key roles in lipid metabolism, membrane homeostasis, and cellular signaling. A recently discovered novel family of lipid transfer proteins, which includes the VPS13 proteins (VPS13A-D), adopt a rod-like bridge conformation with an extended hydrophobic groove that enables the bulk transfer of membrane lipids for membrane growth. Loss of function mutations in VPS13A and VPS13C cause chorea acanthocytosis and Parkinson's disease, respectively. VPS13A and VPS13C localize to multiple organelle contact sites, including endoplasmic reticulum (ER) - lipid droplet (LD) contact sites, but the functional roles of these proteins in LD regulation remains mostly unexplored. Here we employ CRISPR-Cas9 genome editing to generate VPS13A and VPS13C knockout cell lines in U-2 OS cells via deletion of exon 2 and introduction of an early frameshift. Analysis of LD content in these cell lines revealed that loss of either VPS13A or VPS13C results in reduced LD abundance under oleate-stimulated conditions. These data implicate two lipid transfer proteins, VPS13A and VPS13C, in LD regulation.
RESUMO
Fragments of the endoplasmic reticulum (ER) are selectively delivered to the lysosome (mammals) or vacuole (yeast) in response to starvation or the accumulation of misfolded proteins through an autophagic process known as ER-phagy. A screen of the Saccharomyces cerevisiae deletion library identified end3Δ as a candidate knockout strain that is defective in ER-phagy during starvation conditions, but not bulk autophagy. We find that loss of End3 and its stable binding partner Pan1, or inhibition of the Arp2/3 complex that is coupled by the End3-Pan1 complex to endocytic pits, blocks the association of the cortical ER autophagy receptor, Atg40, with the autophagosomal assembly scaffold protein Atg11. The membrane contact site module linking the rim of cortical ER sheets and endocytic pits, consisting of Scs2 or Scs22, Osh2 or Osh3, and Myo3 or Myo5, is also needed for ER-phagy. Both Atg40 and Scs2 are concentrated at the edges of ER sheets and can be cross-linked to each other. Our results are consistent with a model in which actin assembly at sites of contact between the cortical ER and endocytic pits contributes to ER sequestration into autophagosomes.
Assuntos
Actinas/metabolismo , Autofagossomos/metabolismo , Autofagia , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Actinas/genética , Autofagossomos/genética , Retículo Endoplasmático/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The endoplasmic reticulum (ER) forms a contiguous network of tubules and sheets. When errors in protein folding occur, misfolded proteins accumulate in the ER. Proteostasis can be restored by ER quality control pathways. Reticulophagy is an ER quality control pathway that uses resident autophagy receptors to link an ER domain to the autophagy machinery. We recently showed that the reticulophagy receptor RTN3L recruits the COPII cargo adaptor SEC24C to target disease-causing mutant proinsulin INS2Akita puncta to the lysosome for degradation. When reticulophagy is disrupted and delivery to the lysosome is blocked, large INS2Akita puncta accumulate in the ER. Photobleach analysis revealed that these puncta behave like liquid condensates and not aggregates, as previously suggested. Other reticulophagy substrates that are segregated into tubules behave like INS2Akita, whereas a substrate of the ER sheets receptor, RETREG1/FAM134B, appears to be less fluid. Large INS2Akita puncta also accumulate when ER sheets are proliferated by the loss of LNPK, or by overproduction of the sheets-producing protein, CKAP4/CLIMP63. Restoring the tubular network by overexpressing reticulons reverses this phenotype. Our findings revealed that fluid-like deleterious cargoes are segregated into tubules to prevent them from expanding and affecting cell health while they are waiting to undergo reticulophagy.
Assuntos
Autofagia , Proteostase , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Lisossomos/metabolismoRESUMO
Autophagy of the cortical ER in budding yeast was unexpectedly found to require End3, a component of the endocytic machinery that promotes the assembly of actin at endocytic pits on the plasma membrane. The cortical ER transiently interacts with invaginating endocytic pits through a linkage consisting of VAP proteins, oxysterol binding proteins and type I myosins. These proteins are required for actin assembly and for autophagy of the ER. Assembly of actin at these contact sites may direct the movement of ER away from the cortex towards sites of autophagosome assembly.
RESUMO
The endoplasmic reticulum (ER) is composed of sheets and tubules. Here we report that the COPII coat subunit, SEC24C, works with the long form of the tubular ER-phagy receptor, RTN3, to target dominant-interfering mutant proinsulin Akita puncta to lysosomes. When the delivery of Akita puncta to lysosomes was disrupted, large puncta accumulated in the ER. Unexpectedly, photobleach analysis indicated that Akita puncta behaved as condensates and not aggregates, as previously suggested. Akita puncta enlarged when either RTN3 or SEC24C were depleted, or when ER sheets were proliferated by either knocking out Lunapark or overexpressing CLIMP63. Other ER-phagy substrates that are segregated into tubules behaved like Akita, while a substrate (type I procollagen) that is degraded by the ER-phagy sheets receptor, FAM134B, did not. Conversely, when ER tubules were augmented in Lunapark knock-out cells by overexpressing reticulons, ER-phagy increased and the number of large Akita puncta was reduced. Our findings imply that segregating cargoes into tubules has two beneficial roles. First, it localizes mutant misfolded proteins, the receptor, and SEC24C to the same ER domain. Second, physically restraining condensates within tubules, before they undergo ER-phagy, prevents them from enlarging and impacting cell health.
Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proinsulina/metabolismo , Animais , Autofagia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Lisossomos , Camundongos Knockout , Agregados Proteicos , Dobramento de ProteínaRESUMO
COPII coated vesicles that bud from the endoplasmic reticulum (ER) normally traffic to the Golgi. However, during starvation, COPII vesicles are redirected to the macroautophagy pathway where they become a membrane source for autophagosomes. Phosphorylation of the coat by the casein kinase 1 (CK1), Hrr25, is a prerequisite for vesicle uncoating and membrane fusion. CK1 family members were initially thought to be constitutively active kinases that are regulated through their subcellular localization. Recent studies, however, have shown that the Rab GTPase Ypt1 binds to and activates Hrr25 (CK1δ in mammals) to spatially regulate its kinase activity. Consistent with a direct role for Hrr25 in macroautophagy, hrr25and ypt1mutants are defective in autophagosome biogenesis. These studies have provided insights into how the itinerary of COPII vesicles is coordinated on two different trafficking pathways.
Assuntos
Saccharomyces cerevisiae , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Caseína Quinase I/genética , Complexo de Golgi/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Lysosomal degradation of endoplasmic reticulum (ER) fragments by autophagy, termed ER-phagy or reticulophagy, occurs under normal as well as stress conditions. The recent discovery of multiple ER-phagy receptors has stimulated studies on the roles of ER-phagy. We discuss how the ER-phagy receptors and the cellular components that work with these receptors mediate two important functions: ER homeostasis and ER quality control. We highlight that ER-phagy plays an important role in alleviating ER expansion induced by ER stress, and acts as an alternative disposal pathway for misfolded proteins. We suggest that the latter function explains the emerging connection between ER-phagy and disease. Additional ER-phagy-associated functions and important unanswered questions are also discussed.
Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Autofagia , Estresse do Retículo Endoplasmático , HomeostaseRESUMO
Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.
Assuntos
Autofagossomos/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Autofagia , Linhagem Celular , Retículo Endoplasmático/genética , Endossomos/genética , Endossomos/metabolismo , Humanos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Endoplasmic reticulum (ER) homeostasis is maintained by the removal of misfolded ER proteins via different quality control pathways. Aggregation-prone proteins, including certain disease-linked proteins, are resistant to conventional ER degradation pathways and require other disposal mechanisms. Reticulophagy is a disposal pathway that uses resident autophagy receptors. How these receptors, which are dispersed throughout the ER network, target a specific ER domain for degradation is unknown. We recently showed in budding yeast, that ER stress upregulates the reticulophagy receptor, triggering its association with the COPII cargo adaptor complex, Sfb3/Lst1-Sec23 (SEC24C-SEC23 in mammals), to discrete sites on the ER. These domains are packaged into phagophores for degradation to prevent the accumulation of protein aggregates in the ER. This unconventional role for Sfb3/Lst1 is conserved in mammals and is independent of its role as a cargo adaptor on the secretory pathway. Our findings may have important therapeutic implications in protein-aggregation linked neurodegenerative disorders.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/metabolismoRESUMO
The COPII-cargo adaptor complex Lst1-Sec23 selectively sorts proteins into vesicles that bud from the endoplasmic reticulum (ER) and traffic to the Golgi. Improperly folded proteins are prevented from exiting the ER and are degraded. ER-phagy is an autophagic degradation pathway that uses ER-resident receptors. Working in yeast, we found an unexpected role for Lst1-Sec23 in ER-phagy that was independent from its function in secretion. Up-regulation of the stress-inducible ER-phagy receptor Atg40 induced the association of Lst1-Sec23 with Atg40 at distinct ER domains to package ER into autophagosomes. Lst1-mediated ER-phagy played a vital role in maintaining cellular homeostasis by preventing the accumulation of an aggregation-prone protein in the ER. Lst1 function appears to be conserved because its mammalian homolog, SEC24C, was also required for ER-phagy.
Assuntos
Autofagia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Estresse do Retículo Endoplasmático , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Resposta a Proteínas não DobradasRESUMO
The endoplasmic reticulum (ER) forms a contiguous network of tubules and sheets that is predominantly associated with the cell cortex in yeast. Upon treatment with rapamycin, the ER undergoes degradation by selective autophagy. This process, termed ER-phagy, requires Atg40, a selective autophagy receptor that localizes to the cortical ER. Here we report that ER-phagy also requires Lnp1, an ER membrane protein that normally resides at the three-way junctions of the ER network, where it serves to stabilize the network as it is continually remodeled. Rapamycin treatment increases the expression of Atg40, driving ER domains marked by Atg40 puncta to associate with Atg11, a scaffold protein needed to form autophagosomes. Although Atg40 largely localizes to the cortical ER, the autophagy machinery resides in the cell interior. The localization of Atg40 to sites of autophagosome formation is blocked in an lnp1Δ mutant or upon treatment of wild-type cells with the actin-depolymerizing drug Latrunculin A. This prevents the association of Atg40 with Atg11 and the packaging of the ER into autophagosomes. We propose that Lnp1 is needed to stabilize the actin-dependent remodeling of the ER that is essential for ER-phagy.
Assuntos
Autofagossomos/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Retículo Endoplasmático/genética , Proteínas de Membrana/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tiazolidinas/farmacologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Ypt1 and Sec4 are essential Rab GTPases that control the early and late stages of the yeast secretory pathway, respectively. A chimera consisting of Ypt1 with the switch I domain of Sec4, Ypt1-SW1Sec4, is efficiently activated in vitro by the Sec4 exchange factor, Sec2. This should lead to its ectopic activation in vivo and thereby disrupt membrane traffic. Nonetheless early studies found that yeast expressing Ypt1-SW1Sec4 as the sole copy of YPT1 exhibit no growth defect. To resolve this conundrum, we have analyzed yeast expressing various levels of Ypt1-SW1Sec4 We show that even normal expression of Ypt1-SW1Sec4 leads to kinetic transport defects at a late stage of the pathway, with secretory vesicles accumulating near exocytic sites. Higher levels are toxic. Toxicity is suppressed by truncation of Uso1, a vesicle tether required for endoplasmic reticulum-Golgi traffic. The globular head of Uso1 binds to Ypt1 and its coiled-coil tail binds to the Golgi-associated SNARE, Sed5. We propose that when Uso1 is inappropriately recruited to secretory vesicles by Ypt1-SW1Sec4, the extended coiled-coil tail blocks docking to the plasma membrane. This putative inhibitory function could serve to increase the fidelity of vesicle docking.
Assuntos
Membrana Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The secretory and autophagy pathways can be thought of as the biosynthetic (i.e., anabolic) and degradative (i.e., catabolic) branches of the endomembrane system. In analogy to anabolic and catabolic pathways in metabolism, there is mounting evidence that the secretory and autophagy pathways are intimately linked and that certain regulatory elements are shared between them. Here we highlight the parallels and points of intersection between these two evolutionarily highly conserved and fundamental endomembrane systems. The intersection of these pathways may play an important role in remodeling membranes during cellular stress.
Assuntos
Autofagia/fisiologia , Via Secretória/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Humanos , Transporte Proteico/fisiologia , ProteínasRESUMO
The induction of autophagy by nutrient deprivation leads to a rapid increase in the formation of autophagosomes, unique organelles that replenish the cellular pool of nutrients by sequestering cytoplasmic material for degradation. The urgent need for membranes to form autophagosomes during starvation to maintain homeostasis leads to a dramatic rearrangement of intracellular membranes. Here we discuss recent findings that have begun to uncover how different parts of the secretory pathway directly and indirectly contribute to autophagosome formation during starvation.
Assuntos
Autofagossomos/metabolismo , Autofagia , Via Secretória , Animais , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , Modelos BiológicosRESUMO
The upregulation of autophagosome formation in response to nutrient deprivation requires significant intracellular membrane rearrangements that are poorly understood. Recent findings have implicated COPII-coated vesicles, well known as ER-Golgi cargo transport carriers, as key players in macroautophagy. The role of COPII vesicles in macroautophagy and how they interact with autophagy-related (Atg) proteins was unknown. In our recent report, we show that during nutrient deprivation, phosphorylation of the membrane-distal surface of the COPII coat subunit Sec24 facilitates the interaction of Sec24 with the Atg machinery (specifically, Atg9) to regulate the abundance of autophagosomes during starvation. Phosphorylation of Sec24 is specifically required for macroautophagy, but not ER-Golgi transport. These findings begin to unravel the unique function of COPII vesicles during starvation-induced macroautophagy.
Assuntos
Autofagossomos/metabolismo , Autofagia/fisiologia , Transporte Biológico/fisiologia , Fagossomos/metabolismo , Animais , Complexo de Golgi/metabolismo , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Endoplasmic Reticulum (ER)-derived COPII coated vesicles constitutively transport secretory cargo to the Golgi. However, during starvation-induced stress, COPII vesicles have been implicated as a membrane source for autophagosomes, distinct organelles that engulf cellular components for degradation by macroautophagy (hereafter called autophagy). How cells regulate core trafficking machinery to fulfill dramatically different cellular roles in response to environmental cues is unknown. Here we show that phosphorylation of conserved amino acids on the membrane-distal surface of the Saccharomyces cerevisiae COPII cargo adaptor, Sec24, reprograms COPII vesicles for autophagy. We also show casein kinase 1 (Hrr25) is a key kinase that phosphorylates this regulatory surface. During autophagy, Sec24 phosphorylation regulates autophagosome number and its interaction with the C-terminus of Atg9, a component of the autophagy machinery required for autophagosome initiation. We propose that the acute need to produce autophagosomes during starvation drives the interaction of Sec24 with Atg9 to increase autophagosome abundance.
Assuntos
Autofagossomos/metabolismo , Proteínas de Membrana/metabolismo , Biogênese de Organelas , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Caseína Quinase I/metabolismo , Fosforilação , Ligação Proteica , Saccharomyces cerevisiae/metabolismoRESUMO
Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER-Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.