RESUMEN
Rab GTPases are representative targets of manipulation by intracellular bacterial pathogens for hijacking membrane trafficking. Legionella pneumophila recruits many Rab GTPases to its vacuole and exploits their activities. Here, we found that infection-associated regulation of Rab10 dynamics involves ubiquitin signaling cascades mediated by the SidE and SidC families of Legionella ubiquitin ligases. Phosphoribosyl-ubiquitination of Rab10 catalyzed by the SidE ligases is crucial for its recruitment to the bacterial vacuole. SdcB, the previously uncharacterized SidC-family effector, resides on the vacuole and contributes to retention of Rab10 at the late stages of infection. We further identified MavC as a negative regulator of SdcB. By the transglutaminase activity, MavC crosslinks ubiquitin to SdcB and suppresses its function, resulting in elimination of Rab10 from the vacuole. These results demonstrate that the orchestrated actions of many L. pneumophila effectors fine-tune the dynamics of Rab10 during infection.
Asunto(s)
Proteínas Bacterianas , Legionella pneumophila , Vacuolas , Proteínas de Unión al GTP rab , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Legionella pneumophila/metabolismo , Legionella pneumophila/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Humanos , Vacuolas/metabolismo , Vacuolas/microbiología , Interacciones Huésped-Patógeno , Ubiquitinación , Animales , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Upon entry into host cells, the facultative intracellular bacterium Legionella pneumophila ( L.p .) uses its type IV secretion system, Dot/Icm, to secrete ~330 bacterial effector proteins into the host cell. Some of these effectors hijack endoplasmic reticulum (ER)-derived vesicles to form the Legionella -containing vacuole (LCV). Despite extensive investigation over decades, the fundamental question persists: Is the LCV membrane distinct from or contiguous with the host ER network? Here, we employ advanced photobleaching techniques, revealing a temporal acquisition of both smooth and rough ER (sER and rER) markers on the LCV. In the early stages of infection, the sER intimately associates with the LCV. Remarkably, as the infection progresses, the LCV evolves into a distinct niche comprising an rER membrane that is independent of the host ER network. We discover that the L.p. effector LidA binds to and recruits two host proteins of the Rab superfamily, Rab10, and Rab4, that play significant roles in acquiring sER and rER membranes, respectively. Additionally, we identify the pivotal role of a host ER-resident protein, BAP31, in orchestrating the transition from sER to rER. While previously recognized for shuttling between sER and rER, we demonstrate BAP31's role as a Rab effector, mediating communication between these ER sub-compartments. Furthermore, using genomic deletion strains, we uncover a novel L.p. effector, Lpg1152, essential for recruiting BAP31 to the LCV and facilitating its transition from sER to rER. Depletion of BAP31 or infection with an isogenic L.p. strain lacking Lpg1152 results in a growth defect. Collectively, our findings illuminate the intricate interplay between molecular players from both host and pathogen, elucidating how L.p. orchestrates the transformation of its residing vacuole membrane from a host-associated sER to a distinct rER membrane that is not contiguous with the host ER network.
RESUMEN
The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Cortactina , Proteínas Asociadas a Microtúbulos , Neoplasias de la Mama Triple Negativas , Humanos , Carcinogénesis/genética , Transformación Celular Neoplásica , Cortactina/genética , Proteínas Asociadas a Microtúbulos/genética , Neoplasias de la Mama Triple Negativas/genética , Células MDA-MB-231 , Proteínas Adaptadoras del Transporte Vesicular/genética , Microtúbulos/metabolismo , Citoesqueleto/metabolismo , Femenino , Animales , Ratones , Ratones Endogámicos BALB C , Podosomas/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
The gram-negative bacterium, Legionella pneumophila is known to manipulate the host cellular functions. L. pneumophila secretes bacterial proteins called Legionella effectors into the host cytosol that are necessary for these manipulations. The Legionella effector Lpg1137 was identified as a serine protease responsible for the degradation of syntaxin 17 (Stx17). However, how Lpg1137 specifically recognizes and degrades Stx17 remained unknown. Given that Stx17 is localized in the ER, mitochondria-associated membrane (MAM), and mitochondria, Lpg1137 likely distributes to these compartments to recognize Stx17. Here, we show that the C-terminal region of Lpg1137 binds to phosphatidic acid (PA), a MAM and mitochondria-enriched phospholipid, and that this binding is required for the correct intracellular distribution of Lpg1137. Two basic residues in the C-terminal region of Lpg1137 are required for PA binding and their mutation causes mislocalization of Lpg1137. This mutant also fails to degrade Stx17 while retaining protease activity. Taken together, our data reveal that Lpg1137 utilizes PA for its distribution to the membranous compartments in which Stx17 is localized.
Asunto(s)
Legionella pneumophila , Legionella , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Legionella/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Ácidos Fosfatidicos/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismoRESUMEN
Mammalian syntaxin 17 (Stx17) has several roles in processes other than membrane fusion, including in mitochondrial division, autophagosome formation and lipid droplet expansion. In contrast to conventional syntaxins, Stx17 has a long C-terminal hydrophobic region with a hairpin-like structure flanked by a basic amino acid-enriched C-terminal tail. Although Stx17 is one of the six ancient SNAREs and is present in diverse eukaryotic organisms, it has been lost in multiple lineages during evolution. In the present study, we compared the localization and function of fly and nematode Stx17s expressed in HeLa cells with those of human Stx17. We found that fly Stx17 predominantly localizes to the cytosol and mediates autophagy, but not mitochondrial division. Nematode Stx17, on the other hand, is predominantly present in mitochondria and facilitates mitochondrial division, but is irrelevant to autophagy. These differences are likely due to different structures in the C-terminal tail. Non-participation of fly Stx17 and nematode Stx17 in mitochondrial division and autophagy, respectively, was demonstrated in individual organisms. Our results provide an insight into the evolution of Stx17 in metazoa. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Fusión de Membrana , Proteínas SNARE , Animales , Autofagia , Células HeLa , Humanos , Proteínas Qa-SNARE/genéticaRESUMEN
The intracellular bacterial pathogen Legionella pneumophila exploits host cellular systems using approximately 300 effector proteins to establish a replicative niche known as the Legionella-containing vacuole (LCV). During infection, both host and bacterial proteins interactively function on the LCVs. Here, we describe a detailed step-by-step protocol to visualize proteins associated with LCVs in host cells. This protocol can aid in analyzing whether a protein of interest influences the subcellular localization of LCV-associated proteins during infection. For complete details on the use and execution of this protocol, please refer to Kitao et al. (2020).
Asunto(s)
Proteínas Bacterianas/análisis , Técnicas Bacteriológicas/métodos , Técnica del Anticuerpo Fluorescente/métodos , Legionella pneumophila/química , Vacuolas , Proteínas Bacterianas/química , Técnicas de Cultivo de Célula , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/fisiología , Humanos , Plásmidos/genética , Plásmidos/metabolismo , Transfección , Vacuolas/química , Vacuolas/microbiologíaRESUMEN
Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6A, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6A to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6A cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Legionella pneumophila/patogenicidad , Enfermedad de los Legionarios/metabolismo , Vacuolas/microbiología , Proteínas Bacterianas/metabolismo , Retículo Endoplásmico/microbiología , Aparato de Golgi/microbiología , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/metabolismo , Transporte de Proteínas/fisiología , Vacuolas/metabolismoRESUMEN
In response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here, we show that in cholesterol-fed cells, ER-localized SCAP interacts through Sac1 phosphatidylinositol 4-phosphate (PI4P) phosphatase with a VAP-OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER-Golgi membrane contact sites (MCSs). SCAP knockdown inhibited the turnover of PI4P, perhaps due to a cholesterol transport defect, and altered the subcellular distribution of the VAP-OSBP complex. As in the case of perturbation of lipid transfer complexes at ER-Golgi MCSs, SCAP knockdown inhibited the biogenesis of the trans-Golgi network-derived transport carriers CARTS, which was reversed by expression of wild-type SCAP or a Golgi transport-defective mutant, but not of cholesterol sensing-defective mutants. Altogether, our findings reveal a new role for SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis via ER-Golgi MCSs, depending on the ER cholesterol.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Red trans-Golgi/metabolismo , Colesterol/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Transporte de Proteínas , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
The intracellular bacterial pathogen Legionella pneumophila uses many effector proteins delivered by the bacterial type IV secretion system (T4SS) to hijack the early secretory pathway to establish its replicative niche, known as the Legionella-containing vacuole (LCV). On LCV biogenesis, the endoplasmic reticulum (ER) vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNARE) Sec22b is recruited to the bacterial phagosome and forms non-canonical pairings with target membrane SNAREs (t-SNAREs) from the plasma membrane. Here, we identify a Legionella deubiquitinase (DUB), LotB, that can modulate the early secretory pathway by interacting with coatomer protein complex I (COPI) vesicles when ectopically expressed. We show that Sec22b is ubiquitinated upon L. pneumophila infection in a T4SS-dependent manner and that, subsequently, LotB deconjugates K63-linked ubiquitins from Sec22b. The DUB activity of LotB stimulates dissociation of the t-SNARE syntaxin 3 (Stx3) from Sec22b, which resides on the LCV. Our study highlights a bacterial strategy manipulating the dynamics of infection-induced SNARE pairing using a bacterial DUB.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Legionella pneumophila/patogenicidad , Proteínas Bacterianas/metabolismo , TransfecciónRESUMEN
Metastasis is a major cause of cancer-related death. Membrane type 1-matrix metalloproteinase (MT1-MMP) is a critical protease for local invasion and metastasis. MT1-MMP is synthesized in the endoplasmic reticulum (ER) and transported in vesicles to invadopodia, specialized subdomains of the plasma membrane, through secretory and endocytic recycling pathways. The molecular mechanism underlying intracellular transport of MT1-MMP has been extensively studied, but is not fully understood. We show that MT1-MMP diverts the SNARE Bet1 from its function in ER-Golgi transport, to promote MT1-MMP trafficking to the cell surface, likely to invadopodia. In invasive cells, Bet1 is localized in MT1-MMP-positive endosomes in addition to the Golgi apparatus, and forms a novel SNARE complex with syntaxin 4 and endosomal SNAREs. MT1-MMP may also use Bet1 for its export from raft-like structures in the ER. Our results suggest the recruitment of Bet1 at an early stage after MT1-MMP expression promotes the exit of MT1-MMP from the ER and its efficient transport to invadopodia.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas SNARE/metabolismo , Humanos , Transporte de Proteínas , Células Tumorales CultivadasRESUMEN
During the initial stage of infection, Legionella pneumophila secretes effectors that promote the fusion of endoplasmic reticulum (ER)-derived vesicles with the Legionella-containing vacuole (LCV). This fusion leads to a remodeling of the plasma membrane (PM)-derived LCV into a specialized ER-like compartment that supports bacterial replication. Although the effector DrrA has been shown to activate the small GTPase Rab1, it remains unclear how DrrA promotes the tethering of host vesicles with the LCV. Here, we show that Sec5, Sec15, and perhaps Sec6, which are subunits of the exocyst that functions in the tethering of exocytic vesicles with the PM, are required for DrrA-mediated, ER-derived vesicle recruitment to the PM-derived LCV. These exocyst components were found to interact specifically with a complex containing DrrA, and the loss of Sec5 or Sec15 significantly suppressed the recruitment of ER-derived vesicles to the LCV and inhibited intracellular replication of Legionella Importantly, Sec15 is recruited to the LCV, and Rab1 activation is necessary for this recruitment.
Asunto(s)
Membrana Celular/metabolismo , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/metabolismo , Vacuolas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/genética , Membrana Celular/microbiología , Membrana Celular/patología , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/microbiología , Retículo Endoplásmico/patología , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Humanos , Legionella pneumophila/genética , Enfermedad de los Legionarios/genética , Enfermedad de los Legionarios/patología , Vacuolas/genética , Vacuolas/microbiología , Vacuolas/patología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
PGAM5, a mitochondrial protein phosphatase that is genetically and biochemically linked to PINK1, facilitates mitochondrial division by dephosphorylating the mitochondrial fission factor Drp1. At the onset of mitophagy, PGAM5 is cleaved by PARL, a rhomboid protease that degrades PINK1 in healthy cells, and the cleaved form facilitates the engulfment of damaged mitochondria by autophagosomes by dephosphorylating the mitophagy receptor FUNDC1. Here, we show that the function and localization of PGAM5 are regulated by syntaxin 17 (Stx17), a mitochondria-associated membrane/mitochondria protein implicated in mitochondrial dynamics in fed cells and autophagy in starved cells. In healthy cells, loss of Stx17 causes PGAM5 aggregation within mitochondria and thereby failure of the dephosphorylation of Drp1, leading to mitochondrial elongation. In Parkin-mediated mitophagy, Stx17 is prerequisite for PGAM5 to interact with FUNDC1. Our results reveal that the Stx17-PGAM5 axis plays pivotal roles in mitochondrial division and PINK1/Parkin-mediated mitophagy.
Asunto(s)
Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Mitofagia , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Qa-SNARE/metabolismo , Transducción de Señal , Autofagosomas/metabolismo , Dinaminas , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metaloproteasas/genética , Metaloproteasas/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/genética , Fosfoproteínas Fosfatasas/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteolisis , Proteínas Qa-SNARE/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
In fed cells, syntaxin 17 (Stx17) is associated with microtubules at the endoplasmic reticulum-mitochondria interface and promotes mitochondrial fission by determining the localization and function of the mitochondrial fission factor Drp1. Upon starvation, Stx17 dissociates from microtubules and Drp1, and binds to Atg14L, a subunit of the phosphatidylinositol 3-kinase complex, to facilitate phosphatidylinositol 3-phosphate production and thereby autophagosome formation, but the mechanism underlying this phenomenon remains unknown. Here we identify MAP1B-LC1 (microtubule-associated protein 1B-light chain 1) as a critical regulator of Stx17 function. Depletion of MAP1B-LC1 causes Stx17-dependent autophagosome accumulation even under nutrient-rich conditions, whereas its overexpression blocks starvation-induced autophagosome formation. MAP1B-LC1 links microtubules and Stx17 in fed cells, and starvation causes the dephosphorylation of MAP1B-LC1 at Thr217, allowing Stx17 to dissociate from MAP1B-LC1 and bind to Atg14L. Our results reveal the mechanism by which Stx17 changes its binding partners in response to nutrient status.
Asunto(s)
Autofagosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Qa-SNARE/metabolismo , Autofagia , Retículo Endoplásmico/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Mitocondrias/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Tubulina (Proteína)/metabolismoRESUMEN
Lipid droplets (LDs) are ubiquitous organelles that contain neutral lipids and are surrounded by a phospholipid monolayer. How proteins specifically localize to the phospholipid monolayer of the LD surface has been a matter of extensive investigations. In the present study, we show that syntaxin 17 (Stx17), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein whose expression in the liver is regulated by diet, participates in LD biogenesis by regulating the distribution of acyl-CoA synthetase (ACSL)3, a key enzyme for LD biogenesis that redistributes from the endoplasmic reticulum (ER) to LDs during LD formation. Stx17 interacts with ACSL3, but not with LD formation-unrelated ACSL1 or ACSL4, through its SNARE domain. The interaction occurs at the ER-mitochondria interface and depends on the active site occupancy of ACSL3. Depletion of Stx17 impairs ACSL3 redistribution to nascent LDs. The defect in LD maturation due to Stx17 knockdown can be compensated for by ACSL3 overexpression, suggesting that Stx17 increases the efficiency of ACSL3 redistribution to LDs. Moreover, we show that the interaction between Stx17 and ACSL3 during LD maturation may be regulated by synaptosomal-associated protein of 23 kDa.
Asunto(s)
Coenzima A Ligasas/metabolismo , Gotas Lipídicas/metabolismo , Proteínas Qa-SNARE/metabolismo , Células 3T3-L1 , Animales , Células Cultivadas , Femenino , Células HEK293 , Células Hep G2 , Humanos , RatonesRESUMEN
Pathogens subvert host defense systems including autophagy and apoptosis for their survival and proliferation. Legionella pneumophila is a Gram-negative bacterium that grows in alveolar macrophages and causes severe pneumonia. Early during infection Legionella secretes effector proteins that convert the plasma membrane-derived vacuole containing Legionella into an endoplasmic reticulum (ER)-like replicative vacuole. These vacuoles ultimately fuse with the ER, where the pathogen replicates. Recently, we showed that one of the effectors, Lpg1137, is a serine protease that targets the mitochondria-associated ER membrane (MAM) and degrades STX17 (syntaxin 17), a SNARE implicated in macroautophagy/autophagy as well as mitochondria dynamics and membrane trafficking in fed cells. Degradation of STX17 blocks autophagy and BAX-induced apoptosis.
Asunto(s)
Autofagia , Legionella pneumophila , Legionella , Proteínas Qa-SNARE , VacuolasRESUMEN
Mitochondria are powerhouses and central to metabolism in cells. They are highly dynamic organelles that continuously fuse, divide, and move along the cytoskeleton to form the mitochondrial network. The fusion and fission are catalyzed by four dynamin-related GTPases in mammals that are controlled by a variety of protein-protein interactions and posttranslational modifications. Mitochondrial dynamics and metabolism are linked and regulate each other. Starvation induces mitochondrial elongation, which enables the mitochondria to produce energy more efficiently and to escape from autophagic degradation. Damaged portions of mitochondria are removed from the healthy parts by division, and subsequently degraded via a specific mode of autophagy termed mitophagy. Recent studies shed light on the contribution of the endoplasmic reticulum to mitochondrial dynamics and the cooperation of the two organelles for the progression of autophagy including mitophagy. A subdomain of the endoplasmic reticulum apposed to mitochondria is called the mitochondria-associated membrane (MAM), which comprises a unique set of proteins that interact with mitochondrial proteins. Here we review our current understanding of the molecular mechanisms of mitochondrial dynamics and mitochondria-related processes in the context of the interaction with the endoplasmic reticulum.
Asunto(s)
Autofagia , Retículo Endoplásmico/patología , Mitocondrias/patología , Dinámicas Mitocondriales , Membranas Mitocondriales/patología , Transducción de Señal , Animales , Retículo Endoplásmico/metabolismo , Metabolismo Energético , Humanos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/patología , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , MitofagiaRESUMEN
During infection of macrophages, the pathogenic bacterium Legionella pneumophila secretes effector proteins that induce the conversion of the plasma membrane-derived vacuole into an endoplasmic reticulum (ER)-like replicative vacuole. These ER-like vacuoles are ultimately fused with the ER, where the pathogen replicates. Here we show that the L. pneumophila effector Lpg1137 is a serine protease that targets the mitochondria and their associated membranes. Lpg1137 binds to and cleaves syntaxin 17, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein that is known to participate in the regulation of mitochondrial dynamics through interaction with the mitochondrial fission factor Drp1 in fed cells and in autophagy through interaction with Atg14L and other SNAREs in starved cells. Cleavage of syntaxin 17 inhibits not only autophagy but also staurosporine-induced apoptosis occurring in a Bax, Drp1-dependent manner. Thus, L. pneumophila can shut down ER-mitochondria communication through cleavage of syntaxin 17.
Asunto(s)
Proteínas Bacterianas/metabolismo , Retículo Endoplásmico/microbiología , Legionella pneumophila/metabolismo , Macrófagos/microbiología , Mitocondrias/metabolismo , Proteínas Qa-SNARE/metabolismo , Animales , Apoptosis , Autofagia , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Interferencia de ARN , Proteínas SNARE/metabolismo , Fracciones SubcelularesRESUMEN
Vesicle-associated membrane protein-associated protein (VAP) is an endoplasmic reticulum (ER)-resident integral membrane protein that controls a nonvesicular mode of ceramide and cholesterol transfer from the ER to the Golgi complex by interacting with ceramide transfer protein and oxysterol-binding protein (OSBP), respectively. We report that VAP and its interacting proteins are required for the processing and secretion of pancreatic adenocarcinoma up-regulated factor, whose transport from the trans-Golgi network (TGN) to the cell surface is mediated by transport carriers called "carriers of the trans-Golgi network to the cell surface" (CARTS). In VAP-depleted cells, diacylglycerol level at the TGN was decreased and CARTS formation was impaired. We found that VAP forms a complex with not only OSBP but also Sac1 phosphoinositide phosphatase at specialized ER subdomains that are closely apposed to the trans-Golgi/TGN, most likely reflecting membrane contact sites. Immobilization of ER-Golgi contacts dramatically reduced CARTS production, indicating that association-dissociation dynamics of the two membranes are important. On the basis of these findings, we propose that the ER-Golgi contacts play a pivotal role in lipid metabolism to control the biogenesis of transport carriers from the TGN.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Red trans-Golgi/metabolismo , Ceramidas/metabolismo , Colesterol/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular , Lectinas/genética , Lectinas/metabolismo , Fusión de Membrana/genética , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas R-SNARE/genética , Proteínas de Transporte Vesicular/genética , Red trans-Golgi/genéticaRESUMEN
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and its attachment protein, α-SNAP (encoded by NAPA), catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of fusion events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates the endosomal trafficking of epidermal growth factor (EGF) receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry analyses revealed that γ-SNAP interacts with a limited range of SNAREs, including endosomal ones. γ-SNAP, as well as α-SNAP, mediated the disassembly of endosomal syntaxin-7-containing SNARE complexes. Overexpression and small interfering (si)RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in a limited range of organelles - including endosomes - and their trafficking pathways.
Asunto(s)
Endocitosis/fisiología , Receptores ErbB/metabolismo , Transporte de Proteínas/fisiología , Proteínas Qa-SNARE/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/genética , Membrana Celular/metabolismo , Endosomas/metabolismo , Células Hep G2 , Humanos , Fusión de Membrana/fisiología , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño , Transferrina/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Recent evidence suggests that endoplasmic reticulum (ER) tubules mark the sites where the GTPase Drp1 promotes mitochondrial fission via a largely unknown mechanism. Here, we show that the SNARE protein syntaxin 17 (Syn17) is present on raft-like structures of ER-mitochondria contact sites and promotes mitochondrial fission by determining Drp1 localization and activity. The hairpin-like C-terminal hydrophobic domain, including Lys-254, but not the SNARE domain, is important for this regulation. Syn17 also regulates ER Ca(2+) homeostasis and interferes with Rab32-mediated regulation of mitochondrial dynamics. Starvation disrupts the Syn17-Drp1 interaction, thus favoring mitochondrial elongation during autophagy. Because we also demonstrate that Syn17 is an ancient SNARE, our findings suggest that Syn17 is one of the original key regulators for ER-mitochondria contact sites present in the last eukaryotic common ancestor. As such, Syn17 acts as a switch that responds to nutrient conditions and integrates functions for the ER and autophagosomes with mitochondrial dynamics.