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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34028531

RESUMO

Lipid droplets (LDs) are globular subcellular structures that store neutral lipids. LDs are closely associated with the endoplasmic reticulum (ER) and are limited by a phospholipid monolayer harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here, we address the question of whether integral membrane proteins can localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER-resident proteins. The resulting fusion proteins localized to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, was due to a redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of an outer mitochondrial membrane protein, OM14. Expression of OM14-PLIN3 induced a close apposition between LDs and mitochondria. These data indicate that the ER-LD junction constitutes a barrier for ER-resident integral membrane proteins.

2.
Autophagy ; : 1-17, 2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33111629

RESUMO

Selective degradation of the endoplasmic reticulum (ER; reticulophagy) is a type of autophagy involved in the removal of ER fragments. So far, amino acid starvation as well as ER stress have been described as inducers of reticulophagy, which in turn restores cellular energy levels and ER homeostasis. Here, we explored the autophagy-inducing mechanisms that underlie the autophagic cell death (ACD)-triggering compound loperamide (LOP) in glioblastoma cells. Interestingly, LOP triggers upregulation of the transcription factor ATF4, which is accompanied by the induction of additional ER stress markers. Notably, knockout of ATF4 significantly attenuated LOP-induced autophagy and ACD. Functionally, LOP also specifically induces the engulfment of large ER fragments within autophagosomes and lysosomes as determined by electron and fluorescence microscopy. LOP-induced reticulophagy and cell death are predominantly mediated through the reticulophagy receptor RETREG1/FAM134B and, to a lesser extent, TEX264, confirming that reticulophagy receptors can promote ACD. Strikingly, apart from triggering LOP-induced autophagy and ACD, ATF4 is also required for LOP-induced reticulophagy. These observations highlight a key role for ATF4, RETREG1 and TEX264 in response to LOP-induced ER stress, reticulophagy and ACD, and establish a novel mechanistic link between ER stress and reticulophagy, with possible implications for additional models of drug-induced ER stress. Abbreviations: ACD: autophagic cell death; ATF6: activating transcription factor 6; ATL3: atlastin 3; BafA1: bafilomycin A1; CCPG1: cell cycle progression gene 1; co-IP: co-immunoprecipitation; DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; GABARAP: GABA type A receptor-associated protein; GBM: glioblastoma multiforme; HSPA5/BiP: heat shock protein family (Hsp70) member 5; LOP: loperamide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; RETREG1/FAM134B: reticulophagy regulator 1; RTN3L: reticulon 3 long; SEC62: SEC62 homolog, protein translocation factor; TEX264: testis-expressed 264, reticulophagy receptor; UPR: unfolded protein response.

3.
EMBO J ; 39(20): e105117, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32840906

RESUMO

Heterotetrameric adapter (AP) complexes cooperate with the small GTPase Arf1 or lipids in cargo selection, vesicle formation, and budding at endomembranes in eukaryotic cells. While most AP complexes also require clathrin as the outer vesicle shell, formation of AP-3-coated vesicles involved in Golgi-to-vacuole transport in yeast has been postulated to depend on Vps41, a subunit of the vacuolar HOPS tethering complex. HOPS has also been identified as the tether of AP-3 vesicles on vacuoles. To unravel this conundrum of a dual Vps41 function, we anchored Vps41 stably to the mitochondrial outer membrane. By monitoring AP-3 recruitment, we now show that Vps41 can tether AP-3 vesicles to mitochondria, yet AP-3 vesicles can form in the absence of Vps41 or clathrin. By proximity labeling and mass spectrometry, we identify the Arf1 GTPase-activating protein (GAP) Age2 at the AP-3 coat and show that tethering, but not fusion at the vacuole can occur without complete uncoating. We conclude that AP-3 vesicles retain their coat after budding and that their complete uncoating occurs only after tethering at the vacuole.

4.
Proc Natl Acad Sci U S A ; 117(31): 18530-18539, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690699

RESUMO

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/genética
5.
Autophagy ; 16(8): 1539-1541, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32521192

RESUMO

Membrane integrity is essential for cellular survival and function. The spectrum of mechanisms protecting cellular and intracellular membranes is not fully known. Our recent work has uncovered a cellular system termed MERIT for lysosomal membrane repair, removal and replacement. Specifically, lysosomal membrane damage induces, in succession, ESCRT-dependent membrane repair, macroautophagy/autophagy-dominant removal of damaged lysosomes, and initiation of lysosomal biogenesis via transcriptional programs. The MERIT system is governed by galectins, a family of cytosolically synthesized lectins recognizing ß-galactoside glycans. We found in this study that LGALS3 (galectin 3) detects membrane damage by detecting exposed lumenal glycosyl groups, recruits and organizes ESCRT components PDCD6IP/ALIX, CHMP4A, and CHMPB at damaged sites on the lysosomes, and facilitates ESCRT-driven repair of lysosomal membrane. At later stages, LGALS3 cooperates with TRIM16, an autophagy receptor-regulator, to engage autophagy machinery in removal of excessively damaged lysosomes. In the absence of LGALS3, repair and autophagy are less efficient, whereas TFEB nuclear translocation increases to compensate lysosomal deficiency via de novo lysosomal biogenesis. The MERIT system protects endomembrane integrity against a broad spectrum of agents damaging the endolysosomal network including lysosomotropic drugs, Mycobacterium tuberculosis, or neurotoxic MAPT/tau. ABBREVIATIONS: AMPK: AMP-activated protein kinase; APEX2: engineered ascorbate peroxidase 2; ATG13: autophagy related 13; ATG16L1: autophagy related 16 like 1; BMMs: bone marrow-derived macrophages; ESCRT: endosomal sorting complexes required for transport; GPN: glycyl-L-phenylalanine 2-naphthylamide; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERIT: membrane repair, removal and replacement; MTOR: mechanistic target of rapamycin kinase; TFEB: transcription factor EB; TFRC: transferrin receptor; TRIM16: tripartite motif-containing 16.

6.
Dev Cell ; 52(1): 69-87.e8, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31813797

RESUMO

Endomembrane damage elicits homeostatic responses including ESCRT-dependent membrane repair and autophagic removal of damaged organelles. Previous studies have suggested that these systems may act separately. Here, we show that galectin-3 (Gal3), a ß-galactoside-binding cytosolic lectin, unifies and coordinates ESCRT and autophagy responses to lysosomal damage. Gal3 and its capacity to recognize damage-exposed glycans were required for efficient recruitment of the ESCRT component ALIX during lysosomal damage. Both Gal3 and ALIX were required for restoration of lysosomal function. Gal3 promoted interactions between ALIX and the downstream ESCRT-III effector CHMP4 during lysosomal repair. At later time points following lysosomal injury, Gal3 controlled autophagic responses. When this failed, as in Gal3 knockout cells, lysosomal replacement program took over through TFEB. Manifestations of this staged response, which includes membrane repair, removal, and replacement, were detected in model systems of lysosomal damage inflicted by proteopathic tau and during phagosome parasitism by Mycobacterium tuberculosis.


Assuntos
Autofagia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Galectina 3/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Tuberculose/prevenção & controle , Proteínas tau/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Glicosilação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/patogenicidade , Tuberculose/imunologia , Tuberculose/metabolismo , Tuberculose/microbiologia
7.
Autophagy ; 16(8): 1366-1379, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-31525119

RESUMO

Mitophagy is a critical process that safeguards mitochondrial quality control in order to maintain proper cellular homeostasis. Although the mitochondrial-anchored receptor Atg32-mediated cargo-recognition system has been well characterized to be essential for this process, the signaling pathway modulating its expression as a contribution of governing the mitophagy process remains largely unknown. Here, bioinformatics analyses of epigenetic or transcriptional regulators modulating gene expression allow us to identify the Paf1 complex (the polymerase-associated factor 1 complex, Paf1C) as a transcriptional repressor of ATG genes. We show that Paf1C suppresses glucose starvation-induced autophagy, but does not affect nitrogen starvation- or rapamycin-induced autophagy. Moreover, we show that Paf1C specifically regulates mitophagy through modulating ATG32 expression. Deletion of the genes encoding two core subunits of Paf1C, Paf1 and Ctr9, increases ATG32 and ATG11 expression and facilitates mitophagy activity. Although Paf1C is required for many histone modifications and gene activation, we show that Paf1C regulates mitophagy independent of its positive regulatory role in other processes. More importantly, we also demonstrate the mitophagic role of PAF1C in mammals. Overall, we conclude that Paf1C maintains mitophagy at a low level through binding the promoter of the ATG32 gene in glucose-rich conditions. Dissociation of Paf1C from ATG32 leads to the increased expression of this gene, and mitophagy induction upon glucose starvation. Thus, we uncover a new role of Paf1C in modulating the mitophagy process at the transcriptional level. ABBREVIATIONS: AMPK: AMP-activated protein kinase; ATP5F1A: ATP synthase F1 subunit alpha; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: chlorophenylhydrazone; DFP: chelator deferiprone; GFP: green fluorescent protein; H2B-Ub1: H2B monoubiquitination; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; KD: kinase dead; OPTN, optineurin; Paf1: polymerase-associated factor 1; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RT-qPCR: real-time quantitative PCR; SD-N: synthetic dropout without nitrogen base; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; WT: wild-type; YPD: yeast extract peptone dextrose; YPL: yeast extract peptone lactate.

8.
Life Sci Alliance ; 2(6)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31732693

RESUMO

Lectins are glycan-binding proteins with no catalytic activity and ubiquitously expressed in nature. Numerous bacteria use lectins to efficiently bind to epithelia, thus facilitating tissue colonisation. Wounded skin is one of the preferred niches for Pseudomonas aeruginosa, which has developed diverse strategies to impair tissue repair processes and promote infection. Here, we analyse the effect of the P. aeruginosa fucose-binding lectin LecB on human keratinocytes and demonstrate that it triggers events in the host, upon binding to fucosylated residues on cell membrane receptors, which extend beyond its role as an adhesion molecule. We found that LecB associates with insulin-like growth factor-1 receptor and dampens its signalling, leading to the arrest of cell cycle. In addition, we describe a novel LecB-triggered mechanism to down-regulate host cell receptors by showing that LecB leads to insulin-like growth factor-1 receptor internalisation and subsequent missorting towards intracellular endosomal compartments, without receptor activation. Overall, these data highlight that LecB is a multitask virulence factor that, through subversion of several host pathways, has a profound impact on keratinocyte proliferation and survival.


Assuntos
Queratinócitos/metabolismo , Lectinas/metabolismo , Biofilmes/efeitos dos fármacos , Glicosilação , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lectinas/química , Lectinas/fisiologia , Ligação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais/fisiologia
9.
J Cell Sci ; 132(22)2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31649143

RESUMO

Autophagy is initiated by the formation of a phagophore assembly site (PAS), the precursor of autophagosomes. In mammals, autophagosome formation sites form throughout the cytosol in specialized subdomains of the endoplasmic reticulum (ER). In yeast, the PAS is also generated close to the ER, but always in the vicinity of the vacuole. How the PAS is anchored to the vacuole and the functional significance of this localization are unknown. Here, we investigated the role of the PAS-vacuole connection for bulk autophagy in the yeast Saccharomyces cerevisiae We show that Vac8 constitutes a vacuolar tether that stably anchors the PAS to the vacuole throughout autophagosome biogenesis via the PAS component Atg13. S. cerevisiae lacking Vac8 show inefficient autophagosome-vacuole fusion, and form fewer and smaller autophagosomes that often localize away from the vacuole. Thus, the stable PAS-vacuole connection established by Vac8 creates a confined space for autophagosome biogenesis between the ER and the vacuole, and allows spatial coordination of autophagosome formation and autophagosome-vacuole fusion. These findings reveal that the spatial regulation of autophagosome formation at the vacuole is required for efficient bulk autophagy.


Assuntos
Autofagossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Autofagia , Saccharomyces cerevisiae/citologia
10.
Dev Cell ; 50(6): 755-766.e6, 2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31422915

RESUMO

Cells dynamically adjust organelle organization in response to growth and environmental cues. This requires regulation of synthesis of phospholipids, the building blocks of organelle membranes, or remodeling of their fatty-acyl (FA) composition. FAs are also the main components of triacyglycerols (TGs), which enable energy storage in lipid droplets. How cells coordinate FA metabolism with organelle biogenesis during cell growth remains unclear. Here, we show that Lro1, an acyltransferase that generates TGs from phospholipid-derived FAs in yeast, relocates from the endoplasmic reticulum to a subdomain of the inner nuclear membrane. Lro1 nuclear targeting is regulated by cell cycle and nutrient starvation signals and is inhibited when the nucleus expands. Lro1 is active at this nuclear subdomain, and its compartmentalization is critical for nuclear integrity. These data suggest that Lro1 nuclear targeting provides a site of TG synthesis, which is coupled with nuclear membrane remodeling.


Assuntos
Compartimento Celular , Membrana Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Triglicerídeos/biossíntese , Biocatálise , Ciclo Celular , Nucléolo Celular/metabolismo , Forma do Núcleo Celular , Homeostase , Imageamento Tridimensional , Gotículas Lipídicas/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Science ; 365(6448): 53-60, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31273116

RESUMO

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 Dobradas
12.
Cell Death Dis ; 9(10): 994, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30250198

RESUMO

Autophagy is a well-described degradation mechanism that promotes cell survival upon nutrient starvation and other forms of cellular stresses. In addition, there is growing evidence showing that autophagy can exert a lethal function via autophagic cell death (ACD). As ACD has been implicated in apoptosis-resistant glioblastoma (GBM), there is a high medical need for identifying novel ACD-inducing drugs. Therefore, we screened a library containing 70 autophagy-inducing compounds to induce ATG5-dependent cell death in human MZ-54 GBM cells. Here, we identified three compounds, i.e. loperamide, pimozide, and STF-62247 that significantly induce cell death in several GBM cell lines compared to CRISPR/Cas9-generated ATG5- or ATG7-deficient cells, pointing to a death-promoting role of autophagy. Further cell death analyses conducted using pharmacological inhibitors revealed that apoptosis, ferroptosis, and necroptosis only play minor roles in loperamide-, pimozide- or STF-62247-induced cell death. Intriguingly, these three compounds induce massive lipidation of the autophagy marker protein LC3B as well as the formation of LC3B puncta, which are characteristic of autophagy. Furthermore, loperamide, pimozide, and STF-62247 enhance the autophagic flux in parental MZ-54 cells, but not in ATG5 or ATG7 knockout (KO) MZ-54 cells. In addition, loperamide- and pimozide-treated cells display a massive formation of autophagosomes and autolysosomes at the ultrastructural level. Finally, stimulation of autophagy by all three compounds is accompanied by dephosphorylation of mammalian target of rapamycin complex 1 (mTORC1), a well-known negative regulator of autophagy. In summary, our results indicate that loperamide, pimozide, and STF-62247 induce ATG5- and ATG7-dependent cell death in GBM cells, which is preceded by a massive induction of autophagy. These findings emphasize the lethal function and potential clinical relevance of hyperactivated autophagy in GBM.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Loperamida/farmacologia , Pimozida/farmacologia , Piridinas/farmacologia , Tiazóis/farmacologia , Autofagossomos/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/ultraestrutura , Linhagem Celular Tumoral , Endossomos/metabolismo , Glioblastoma/patologia , Glioblastoma/ultraestrutura , Células HT29 , Humanos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo
13.
J Cell Biol ; 217(8): 2743-2763, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29848619

RESUMO

The autophagy-related (Atg) proteins play a key role in the formation of autophagosomes, the hallmark of autophagy. The function of the cluster composed by Atg2, Atg18, and transmembrane Atg9 is completely unknown despite their importance in autophagy. In this study, we provide insights into the molecular role of these proteins by identifying and characterizing Atg2 point mutants impaired in Atg9 binding. We show that Atg2 associates to autophagosomal membranes through lipid binding and independently from Atg9. Its interaction with Atg9, however, is key for Atg2 confinement to the growing phagophore extremities and subsequent association of Atg18. Assembly of the Atg9-Atg2-Atg18 complex is important to establish phagophore-endoplasmic reticulum (ER) contact sites. In turn, disruption of the Atg2-Atg9 interaction leads to an aberrant topological distribution of both Atg2 and ER contact sites on forming phagophores, which severely impairs autophagy. Altogether, our data shed light in the interrelationship between Atg9, Atg2, and Atg18 and highlight the possible functional relevance of the phagophore-ER contact sites in phagophore expansion.


Assuntos
Proteínas Relacionadas à Autofagia/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatos de Fosfatidilinositol/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Autophagy ; 14(8): 1435-1455, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29940786

RESUMO

Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A1 (BafA1), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA1. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.


Assuntos
Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Cloroquina/farmacologia , Lisossomos/metabolismo , Fusão de Membrana/efeitos dos fármacos , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/ultraestrutura , Receptores ErbB/metabolismo , Feminino , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Hidroxicloroquina/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Macrolídeos/farmacologia , Camundongos Endogâmicos C57BL , Proteólise/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo
15.
Nat Commun ; 9(1): 1761, 2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720625

RESUMO

The understanding that organelles are not floating in the cytosol, but rather held in an organized yet dynamic interplay through membrane contact sites, is altering the way we grasp cell biological phenomena. However, we still have not identified the entire repertoire of contact sites, their tethering molecules and functions. To systematically characterize contact sites and their tethering molecules here we employ a proximity detection method based on split fluorophores and discover four potential new yeast contact sites. We then focus on a little-studied yet highly disease-relevant contact, the Peroxisome-Mitochondria (PerMit) proximity, and uncover and characterize two tether proteins: Fzo1 and Pex34. We genetically expand the PerMit contact site and demonstrate a physiological function in ß-oxidation of fatty acids. Our work showcases how systematic analysis of contact site machinery and functions can deepen our understanding of these structures in health and disease.


Assuntos
Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Citoplasma/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Peroxinas/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Mol Biol Cell ; 29(9): 1089-1099, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29514932

RESUMO

Macroautophagy (hereafter autophagy) is a cellular recycling pathway essential for cell survival during nutrient deprivation that culminates in the degradation of cargo within the vacuole in yeast and the lysosome in mammals, followed by efflux of the resultant macromolecules back into the cytosol. The yeast vacuole is home to many different hydrolytic proteins and while few have established roles in autophagy, the involvement of others remains unclear. The vacuolar serine carboxypeptidase Y (Prc1) has not been previously shown to have a role in vacuolar zymogen activation and has not been directly implicated in the terminal degradation steps of autophagy. Through a combination of molecular genetic, cell biological, and biochemical approaches, we have shown that Prc1 has a functional homologue, Ybr139w, and that cells deficient in both Prc1 and Ybr139w have defects in autophagy-dependent protein synthesis, vacuolar zymogen activation, and autophagic body breakdown. Thus, we have demonstrated that Ybr139w and Prc1 have important roles in proteolytic processing in the vacuole and the terminal steps of autophagy.


Assuntos
Carboxipeptidases/metabolismo , Carboxipeptidases/farmacologia , Vacúolos/metabolismo , Autofagia/fisiologia , Lisossomos/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/fisiologia
17.
J Cell Biol ; 217(1): 269-282, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29187527

RESUMO

Functional heterogeneity within the lipid droplet (LD) pool of a single cell has been observed, yet the underlying mechanisms remain enigmatic. Here, we report on identification of a specialized LD subpopulation characterized by a unique proteome and a defined geographical location at the nucleus-vacuole junction contact site. In search for factors determining identity of these LDs, we screened ∼6,000 yeast mutants for loss of targeting of the subpopulation marker Pdr16 and identified Ldo45 (LD organization protein of 45 kD) as a crucial targeting determinant. Ldo45 is the product of a splicing event connecting two adjacent genes (YMR147W and YMR148W/OSW5/LDO16). We show that Ldo proteins cooperate with the LD biogenesis component seipin and establish LD identity by defining positioning and surface-protein composition. Our studies suggest a mechanism to establish functional differentiation of organelles, opening the door to better understanding of metabolic decisions in cells.


Assuntos
Gotículas Lipídicas/metabolismo , Proteínas de Membrana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Gotículas Lipídicas/classificação , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma , Saccharomyces cerevisiae/metabolismo
18.
Viruses ; 9(9)2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28872588

RESUMO

The porcine epidemic diarrhea virus (PEDV) is a coronavirus (CoV) belonging to the α-CoV genus and it causes high mortality in infected sucking piglets, resulting in substantial losses in the farming industry. CoV trigger a drastic reorganization of host cell membranes to promote their replication and egression, but a detailed description of the intracellular remodeling induced by PEDV is still missing. In this study, we examined qualitatively and quantitatively, using electron microscopy, the intracellular membrane reorganization induced by PEDV over the course of an infection. With our ultrastructural approach, we reveal that, as most of CoV, PEDV initially forms double-membrane vesicles (DMVs) and convoluted membranes (CMs), which probably serve as replication/transcription platforms. Interestingly, we also found that viral particles start to form almost simultaneously in both the endoplasmic reticulum and the large virion-containing vacuoles (LVCVs), which are compartments originating from the Golgi, confirming that α-CoV assemble indistinguishably in two different organelles of the secretory pathway. Moreover, PEDV virons appear to have an immature and a mature form, similar to another α-CoV the transmissible gastroenteritis coronavirus (TGEV). Altogether, our study underlies the similarities and differences between the lifecycle of α-CoV and that of viruses belonging to other CoV subfamilies.


Assuntos
Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Membranas Intracelulares/ultraestrutura , Vírus da Diarreia Epidêmica Suína/fisiologia , Animais , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Membranas Intracelulares/virologia , Microscopia Eletrônica , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Vírus da Diarreia Epidêmica Suína/ultraestrutura , Suínos , Vacúolos/ultraestrutura , Vacúolos/virologia , Células Vero
19.
Elife ; 62017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28617241

RESUMO

The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.


Assuntos
Autofagia , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Linhagem Celular , Humanos , Multimerização Proteica
20.
EMBO Rep ; 18(5): 765-780, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28330855

RESUMO

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3-interacting region (LIR) at the C-terminus of the protein and a novel motif at the N-terminus. Although both sites are important for Atg4-Atg8 interaction in vivo, only the new N-terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE-bound Atg8.


Assuntos
Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Membranas/química , Membranas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Fagossomos/metabolismo , Fosfatidiletanolaminas/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
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