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[This corrects the article DOI: 10.1038/celldisc.2015.18.].
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The modular adaptor protein ALIX is critically involved in endosomal sorting complexes required for transport (ESCRT)-mediated multivesicular body (MVB) sorting of activated epidermal growth factor receptor (EGFR); however, ALIX contains a default intramolecular interaction that renders ALIX unable to perform this ESCRT function. The ALIX partner protein ALG-2 is a calcium-binding protein that belongs to the calmodulin superfamily. Prompted by a defined biological function of calmodulin, we determined the role of ALG-2 in regulating ALIX involvement in MVB sorting of activated EGFR. Our results show that calcium-dependent ALG-2 interaction with ALIX completely relieves the intramolecular interaction of ALIX and promotes CHMP4-dependent ALIX association with the membrane. EGFR activation induces increased ALG-2 interaction with ALIX, and this increased interaction is responsible for increased ALIX association with the membrane. Functionally, inhibition of ALIX activation by ALG-2 inhibits MVB sorting of activated EGFR as effectively as inhibition of ALIX interaction with CHMP4 does; however, inhibition of ALIX activation by ALG-2 does not affect cytokinetic abscission or equine infectious anemia virus (EIAV) budding. These findings indicate that calcium-dependent ALG-2 interaction with ALIX is specifically responsible for generating functional ALIX that supports MVB sorting of ubiquitinated membrane receptors.
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The adaptor protein ALIX [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] links retroviruses to ESCRT (endosomal sorting complex required for transport) machinery during retroviral budding. This function of ALIX requires its interaction with the ESCRT-III component CHMP4 (charged multivesicular body protein 4) at the N-terminal Bro1 domain and retroviral Gag proteins at the middle V domain. Since cytoplasmic or recombinant ALIX is unable to interact with CHMP4 or retroviral Gag proteins under non-denaturing conditions, we constructed ALIX truncations and mutations to define the intrinsic mechanism through which ALIX interactions with these partner proteins are prohibited. Our results demonstrate that an intramolecular interaction between Patch 2 in the Bro1 domain and the TSG101 (tumour susceptibility gene 101 protein)-docking site in the proline-rich domain locks ALIX into a closed conformation that renders ALIX unable to interact with CHMP4 and retroviral Gag proteins. Relieving the intramolecular interaction of ALIX, by ectopically expressing a binding partner for one of the intramolecular interaction sites or by deleting one of these sites, promotes ALIX interaction with these partner proteins and facilitates ALIX association with the membrane. Ectopic expression of a GFP (green fluorescent protein)-ALIX mutant with a constitutively open conformation, but not the wild-type protein, increases EIAV (equine infectious anaemia virus) budding from HEK (human embryonic kidney)-293 cells. These findings predict that relieving the autoinhibitory intramolecular interaction of ALIX is a critical step for ALIX to participate in retroviral budding.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Proteínas de Ciclo Celular/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de los Retroviridae/metabolismo , Liberación del Virus/fisiología , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Membrana Celular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Virus de la Anemia Infecciosa Equina/fisiología , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteína Oncogénica pp60(v-src)/química , Proteína Oncogénica pp60(v-src)/genética , Proteína Oncogénica pp60(v-src)/metabolismo , Dominios Proteicos Ricos en Prolina/fisiología , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de los Retroviridae/química , Proteínas de los Retroviridae/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The Bro1 domain of Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], which plays important roles in endosomal sorting and multiple ESCRT (endosomal sorting complex required for transport)-linked processes, contains the docking sites for the ESCRT-III component CHMP4b (charged multivesicular body protein 4b) and the regulatory tyrosine kinase, Src. Although the structural bases for these docking sites have been defined by crystallography studies, it has not been determined whether these sites are available in the native state of Alix. In the present study, we demonstrate that these two docking sites are unavailable in recombinant Alix under native conditions and that their availabilities can be induced by detergents. In HEK (human embryonic kidney)-293 cell lysates, these two docking sites are not available in cytosolic Alix, but are available in membrane-bound Alix. These findings show that the native state of Alix does not have a functional Bro1 domain and predict that Alix's involvement in endosomal sorting and other ESCRT-linked processes requires an activation step that relieves the autoinhibition of the Bro1 domain.
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Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Sitios de Unión de Anticuerpos , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/inmunología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/inmunología , Células Cultivadas , Regulación hacia Abajo/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte , Epítopos/inmunología , Homeostasis/fisiología , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfoproteínas/inmunología , Desnaturalización Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Homología de Secuencia de Aminoácido , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/inmunologíaRESUMEN
Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to extracellular Alix retards WI38 cell adhesion and spreading on fibronectin and vitronectin. Alix knockdown in WI38 cells reduces spreading and fibronectin assembly, and the effect is partially complemented by coating recombinant Alix on the cell substratum. Immortalized NIH/3T3 fibroblasts deposit less Alix on the substratum and have defects in alpha5beta1-integrin functions. Coating recombinant Alix on the culture substratum for NIH/3T3 cells promotes alpha5beta1-integrin-mediated cell adhesions and fibronectin assembly, and these effects require the aa 605-709 region of Alix. These findings demonstrate that a sub-population of Alix localizes extracellularly and regulates integrin-mediated cell adhesions and fibronectin matrix assembly.
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Proteínas de Unión al Calcio/fisiología , Adhesión Celular/fisiología , Matriz Extracelular/fisiología , Integrina alfa5beta1/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Línea Celular , Forma de la Célula/fisiología , Fibroblastos/fisiología , Fibronectinas/metabolismo , Ratones , Proteínas Recombinantes/metabolismo , Vitronectina/metabolismoRESUMEN
Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], a component of the endosomal sorting machinery, contains a three-dimensional docking site for HIV-1 p6(Gag) or EIAV (equine infectious anaemia virus) p9(Gag), and binding of the viral protein to this docking site allows the virus to hijack the host endosomal sorting machinery for budding from the plasma membrane. In the present study, we identified a monoclonal antibody that specifically recognizes the docking site for p6(Gag)/p9(Gag) and we used this antibody to probe the accessibility of the docking site in Alix. Our results show that the docking site is not available in cytosolic or recombinant Alix under native conditions and becomes available upon addition of the detergent Nonidet P40 or SDS. In HEK (human embryonic kidney)-293 cell lysates, an active p6(Gag)/p9(Gag) docking site is specifically available in Alix from the membrane fraction. The findings of the present study demonstrate that formation or exposure of the p6(Gag)/p9(Gag) docking site in Alix is a regulated event and that Alix association with the membrane may play a positive role in this process.
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Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , VIH-1/metabolismo , Virus de la Anemia Infecciosa Equina/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Proteínas de Unión al Calcio/inmunología , Proteínas de Ciclo Celular/inmunología , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Productos del Gen gag/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Unión Proteica , Conformación ProteicaRESUMEN
The conserved adaptor protein Alix, also called AIP1 or Hp95, promotes flattening and alignment of cultured mammalian fibroblasts; however, the mechanism by which Alix regulates fibroblast morphology is not understood. Here we demonstrate that Alix in WI38 cells, which require Alix expression for maintaining typical fibroblast morphology, associates with filamentous actin (F-actin) and F-actin-based structures lamellipodia and stress fibers. Reducing Alix expression by small interfering RNA (siRNA) decreases F-actin content and inhibits stress fiber assembly. In cell-free systems, Alix directly interacts with F-actin at both the N-terminal Bro1 domain and the C-terminal proline-rich domain. In Alix immunoprecipitates from WI38 cell lysates, actin is the most abundant partner protein of Alix. In addition, the N-terminal half of the middle region of Alix binds cortactin, an activator of the ARP2/3 complex-mediated initiation of actin polymerization. Alix is required for lamellipodial localization of cortactin. The C-terminal half of the middle region of Alix interacts with alpha-actinin, a key factor that bundles F-actin in stress fibers. Alix knockdown decreases the amount of alpha-actinin that associates with F-actin. These findings establish crucial involvement of Alix in actin cytoskeleton assembly.