RESUMEN
Loss-of-function variants in AP3D1 have been linked to Hermansky-Pudlak syndrome (HPS) 10, a severe multisystem disorder characterized by oculocutaneous albinism, immunodeficiency, neurodevelopmental delay, hearing loss (HL), and neurological abnormalities, fatal in early childhood. Here, we report a consanguineous family who presented with presumably isolated autosomal recessive (AR) HL. Whole-exome sequencing was performed on all core family members, and selected patients were screened using array-based copy-number analysis and karyotyping. Candidate variants were validated by Sanger sequencing and assessed in silico. A homozygous, likely pathogenic p.V711I missense variant in AP3D1 segregated with the HL. The family was characterized by thorough medical and laboratory examination. The HL was consistent across patients and accompanied by neurological manifestations in two brothers. The sole female patient was diagnosed with premature ovarian failure. Further findings, including mild neutropenia and reduced NK-cell cytotoxicity in some as well as brain alterations in all homozygous patients, were reminiscent of HPS10, though milder and lacking the characteristic albinism. Previously unrecognized, milder, isolated HL was identified in all heterozygous carriers. A protein model indicates that the variant interferes with protein-protein interactions. These results suggest that a missense variant alters inner-ear-specific functions leading to HL with mild HPS10-like symptoms of variable penetrance. Milder HL in heterozygous carriers may point towards semi-dominant inheritance of this trait. Since all previously reported HPS10 cases were pediatric, it is unknown whether the observed primary ovarian insufficiency recapitulates the subfertility in Ap3d1-deficient mice.
Asunto(s)
Sordera , Pérdida Auditiva Sensorineural , Síndrome de Hermanski-Pudlak , Masculino , Humanos , Preescolar , Femenino , Animales , Ratones , Síndrome de Hermanski-Pudlak/diagnóstico , Síndrome de Hermanski-Pudlak/patología , Mutación Missense , Pérdida Auditiva Sensorineural/genética , Proteínas Portadoras , Homocigoto , Complejo 3 de Proteína Adaptadora , Subunidades delta de Complexo de Proteína Adaptadora , Subunidades beta de Complejo de Proteína AdaptadoraRESUMEN
Atherosclerosis has been considered as the main cause of morbidity, mortality, and disability worldwide. The first screening for antigen markers was conducted using the serological identification of antigens by recombinant cDNA expression cloning, which has identified adaptor-related protein complex 3 subunit delta 1 (AP3D1) as an antigen recognized by serum IgG antibodies of patients with atherosclerosis. Serum antibody levels were examined using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using a recombinant protein as an antigen. It was determined that the serum antibody levels against AP3D1 were higher in patients with acute ischemic stroke (AIS), transient ischemic attack, diabetes mellitus (DM), cardiovascular disease, chronic kidney disease (CKD), esophageal squamous cell carcinoma (ESCC), and colorectal carcinoma than those in the healthy donors. The area under the curve values of DM, nephrosclerosis type of CKD, and ESCC calculated using receiver operating characteristic curve analysis were higher than those of other diseases. Correlation analysis showed that the anti-AP3D1 antibody levels were highly associated with maximum intima-media thickness, which indicates that this marker reflected the development of atherosclerosis. The results of the Japan Public Health Center-based Prospective Study indicated that this antibody marker is deemed useful as risk factors for AIS.
Asunto(s)
Complejo 3 de Proteína Adaptadora , Subunidades delta de Complexo de Proteína Adaptadora , Aterosclerosis , Autoanticuerpos , Inmunoglobulina G , Accidente Cerebrovascular Isquémico , Complejo 3 de Proteína Adaptadora/sangre , Complejo 3 de Proteína Adaptadora/inmunología , Subunidades delta de Complexo de Proteína Adaptadora/sangre , Subunidades delta de Complexo de Proteína Adaptadora/inmunología , Anciano , Anciano de 80 o más Años , Aterosclerosis/sangre , Aterosclerosis/complicaciones , Aterosclerosis/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Accidente Cerebrovascular Isquémico/sangre , Accidente Cerebrovascular Isquémico/etiología , Accidente Cerebrovascular Isquémico/inmunología , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
Chronic obstructive pulmonary disease (COPD) is a common, complex disease and a major cause of morbidity and mortality. Although multiple genetic determinants of COPD have been implicated by genome-wide association studies (GWASs), the pathophysiological significance of these associations remains largely unknown. From a COPD protein-protein interaction network module, we selected a network path between two COPD GWAS genes for validation studies: FAM13A (family with sequence similarity 13 member A)-AP3D1-CTGF- TGFß2. We find that TGFß2, FAM13A, and AP3D1 (but not CTGF) form a cellular protein complex. Functional characterization suggests that this complex mediates the secretion of TGFß2 through an AP-3 (adaptor protein 3)-dependent pathway, with FAM13A acting as a negative regulator by targeting a late stage of this transport that involves the dissociation of coat-cargo interaction. Moreover, we find that TGFß2 is a transmembrane protein that engages the AP-3 complex for delivery to the late endosomal compartments for subsequent secretion through exosomes. These results identify a pathophysiological context that unifies the biological network role of two COPD GWAS proteins and reveal novel mechanisms of cargo transport through an intracellular pathway.
Asunto(s)
Complejo 3 de Proteína Adaptadora/metabolismo , Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Línea Celular , Exosomas/metabolismo , Proteínas Activadoras de GTPasa/genética , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Mapas de Interacción de Proteínas/genética , Transporte de Proteínas , Reproducibilidad de los Resultados , Factor de Crecimiento Transformador beta2/genéticaRESUMEN
Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1-dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3-BLOC-1 super-complex.
Asunto(s)
Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Proteínas del Tejido Nervioso/genética , Proteínas Qa-SNARE/genética , Proteínas R-SNARE/genética , Endosomas/genética , Humanos , Melanocitos/metabolismo , Melanosomas/genética , Transporte de Proteínas/genéticaRESUMEN
Several types of Hermansky-Pudlak syndromes (HPS) represent a group of immunodeficiency syndromes that feature both leukocyte defects with partial albinism of hair, skin, and eyes. These conditions share defects in genes that encode proteins involved in the biogenesis, function, and trafficking of secretory lysosomes. Mutations in AP3D1 which encode the main subunit AP-3(δ) were recently reported on one individual and led to Hermansky-Pudlak Syndrome type 10 (HPS10; OMIM 617050). HPS10 is a severe condition that manifests with symptoms of oculocutaneous albinism, neurodevelopmental delays, platelet dysfunction, and immunodeficiency. Herein we report on three affected individuals who presented with severe seizures, developmental delay, albinism, and immunodeficiency. Whole exome sequencing identified homozygosity for a deleterious sequence variant of high impact in AP3D1, c.1978delG, predicting p.Ala660Argfs*54 (NM_001261826.3). We further demonstrated an abnormal storage pathway in the platelets. The current study represents a second confirmation report and implicates AP3D1 mutations as a cause of Hermansky-Pudlak Syndrome type 10.
Asunto(s)
Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Epilepsia/genética , Síndrome de Hermanski-Pudlak/genética , Síndromes de Inmunodeficiencia/genética , Albinismo Oculocutáneo/genética , Albinismo Oculocutáneo/fisiopatología , Alelos , Trastornos de las Plaquetas Sanguíneas/genética , Trastornos de las Plaquetas Sanguíneas/patología , Preescolar , Epilepsia/fisiopatología , Femenino , Síndrome de Hermanski-Pudlak/diagnóstico , Síndrome de Hermanski-Pudlak/fisiopatología , Humanos , Síndromes de Inmunodeficiencia/fisiopatología , Lactante , Masculino , Mutación , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/fisiopatología , Linaje , Hermanos , Gemelos/genética , Secuenciación del ExomaRESUMEN
The envelope glycoprotein 51 (gp51) is essential for bovine leukaemia virus (BLV) entry to bovine B-lymphocytes. Although the bovine adaptor protein 3 complex subunit delta-1 (boAP3D1) has been proposed as the potential receptor, the specific ligand-receptor interaction has not yet been completely defined and boAP3D1 receptor and gp51 3D structures have not been determined. This study was thus aimed at a functional annotation of boAP3D1 cellular adaptor protein and BLV gp51 and, proposing a reliable model for gp51-AP3D1 interaction using bioinformatics tools. The boAP3D1 receptor interaction patterns were calculated based on models of boAP3D1 receptor and gp51 complexes' 3D structures, which were constructed using homology techniques and data-driven docking strategy. The results showed that the participation of 6 key amino acids (aa) on gp51 (Asn170, Trp127, His115, Ala97, Ser98 and Glu128) and 4 aa on AP3D1 (Lys925, Asp807, Asp695 and Arg800) was highly probable in the interaction between gp51 and BLVR domains. Three gp51 recombinant peptides were expressed and purified to validate these results: the complete domain (rgp51), the N-terminal portion (rNgp51) and the C-terminal fragment (rCgp51); and binding assays to Madin-Darby bovine kidney (MDBK) cells were then carried out with each recombinant. It was found that rNgp51 preferentially bound to MDBK cells, suggesting this domain's functional role during invasion. The rNgp51-MDBK cell interaction was sensitive to trypsin (98% reduction) and chymotrypsin treatment (80% reduction). These results highlighted that the N-terminal portion of gp51 interacted in vitro with the AP3D1 receptor and provides a plausible in silico interaction model.
Asunto(s)
Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , Simulación por Computador , Virus de la Leucemia Bovina/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Subunidades delta de Complexo de Proteína Adaptadora/química , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Anotación de Secuencia Molecular , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Termodinámica , Proteínas del Envoltorio Viral/químicaRESUMEN
Genetic disorders affecting biogenesis and transport of lysosome-related organelles are heterogeneous diseases frequently associated with albinism. We studied a patient with albinism, neutropenia, immunodeficiency, neurodevelopmental delay, generalized seizures, and impaired hearing but with no mutation in genes so far associated with albinism and immunodeficiency. Whole exome sequencing identified a homozygous mutation in AP3D1 that leads to destabilization of the adaptor protein 3 (AP3) complex. AP3 complex formation and the degranulation defect in patient T cells were restored by retroviral reconstitution. A previously described hypopigmented mouse mutant with an Ap3d1 null mutation (mocha strain) shares the neurologic phenotype with our patient and shows a platelet storage pool deficiency characteristic of Hermansky-Pudlak syndrome (HPS) that was not studied in our patient because of a lack of bleeding. HPS2 caused by mutations in AP3B1A leads to a highly overlapping phenotype without the neurologic symptoms. The AP3 complex exists in a ubiquitous and a neuronal form. AP3D1 codes for the AP3δ subunit of the complex, which is essential for both forms. In contrast, the AP3ß3A subunit, affected in HPS2 patients, is substituted by AP3ß3B in the neuron-specific heterotetramer. AP3δ deficiency thus causes a severe neurologic disorder with immunodeficiency and albinism that we propose to classify as HPS10.
Asunto(s)
Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Síndrome de Hermanski-Pudlak/clasificación , Síndrome de Hermanski-Pudlak/genética , Síndromes de Inmunodeficiencia/genética , Convulsiones/genética , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Humanos , Mutación , TransfecciónAsunto(s)
Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Cromosomas de Insectos/genética , Intercambio Genético , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Ojo/genética , Heterogeneidad Genética , Factores de Transcripción/genética , Cromosoma X/genética , AnimalesRESUMEN
Despite considerable progress in preventive and therapeutic strategies, myocardial infarction (MI) is one of the leading causes of death throughout the world. A total of 55 susceptibility genes have been identified mostly in European genome-wide association studies (GWAS). Nevertheless, large-scale GWAS from other population could possibly find additional susceptibility loci. To identify as many MI susceptibility loci as possible, we performed a large-scale genomic analysis in Japanese population. To identify MI susceptibility loci in Japanese, we conducted a GWAS using 1666 cases and 3198 controls using the Illumina Human610-Quad BeadChip and HumanHap550v3 Genotyping BeadChip. We performed replication studies using a total of 11,412 cases and 28,397 controls in the Japanese population. Our study identified two novel susceptibility loci for MI: PLCL2 on chromosome 3p24.3 (rs4618210:A>G, P = 2.60 × 10(-9), odds ratio (OR) = 0.91) and AP3D1-DOT1L-SF3A2 on chromosome 19p13.3 (rs3803915:A>C, P = 3.84 × 10(-9), OR = 0.89). Besides, a total of 14 previously reported MI susceptibility loci were replicated in our study. In particular, we validated a strong association on chromosome 12q24 (rs3782886:A>G: P = 1.14 × 10(-14), OR = 1.46). Following pathway analysis using 265 genes related to MI or coronary artery disease, we found that these loci might be involved in the pathogenesis of MI via the promotion of atherosclerosis. In the present large-scale genomic analysis, we identified PLCL2 and AP3D1-DOT1L-SF3A2 as new susceptibility loci for MI in the Japanese population. Our findings will add novel findings for MI susceptibility loci.
Asunto(s)
Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Pueblo Asiatico/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Péptidos y Proteínas de Señalización Intracelular/genética , Metiltransferasas/genética , Infarto del Miocardio/genética , Proteínas de Unión al ARN/genética , Adulto , Anciano , Alelos , Estudios de Casos y Controles , Cromosomas Humanos Par 12 , Femenino , Redes Reguladoras de Genes , Genotipo , N-Metiltransferasa de Histona-Lisina , Humanos , Japón , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Polimorfismo de Nucleótido Simple , Factores de Empalme de ARNRESUMEN
An article by Singh and colleagues in this issue of GENETICS quantifies variation in recombination rate across a small region of the Drosophila melanogaster genome, providing an opportunity for instructors of genetics to introduce or reinforce important concepts such as recombination and recombination rate variation, genome sequencing, and sequence features of the genome. Additional background information, a detailed explanation of the methods used in this study, and discussion questions are provided.
Asunto(s)
Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Cromosomas de Insectos/genética , Intercambio Genético , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Ojo/genética , Heterogeneidad Genética , Factores de Transcripción/genética , Cromosoma X/genética , AnimalesRESUMEN
Homologous recombination affects myriad aspects of genome evolution, from standing levels of nucleotide diversity to the efficacy of natural selection. Rates of crossing over show marked variability at all scales surveyed, including species-, population-, and individual-level differences. Even within genomes, crossovers are nonrandomly distributed in a wide diversity of taxa. Although intra- and intergenomic heterogeneities in crossover distribution have been documented in Drosophila, the scale and degree of crossover rate heterogeneity remain unclear. In addition, the genetic features mediating this heterogeneity are unknown. Here we quantify fine-scale heterogeneity in crossover distribution in a 2.1-Mb region of the Drosophila melanogaster X chromosome by localizing crossover breakpoints in 2500 individuals, each containing a single crossover in this specific X chromosome region. We show 90-fold variation in rates of crossing over at a 5-kb scale, place this variation in the context of several aspects of genome evolution, and identify several genetic features associated with crossover rates. Our results shed new light on the scale and magnitude of crossover rate heterogeneity in D. melanogaster and highlight potential features mediating this heterogeneity.
Asunto(s)
Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Cromosomas de Insectos/genética , Intercambio Genético , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Ojo/genética , Heterogeneidad Genética , Factores de Transcripción/genética , Cromosoma X/genética , Animales , Puntos de Rotura del CromosomaRESUMEN
Adaptor protein complex 3 (AP-3) is a heterotetramer that is involved in signal-mediated protein sorting to endosomal-lysosomal organelles. AP-3 deficiency in humans, induced by mutations in the AP3B1 gene, which encodes the ß3A subunit of the AP-3 complex, results in Hermansky-Pudlak syndrome 2 (HPS2), which is a rare genetic disorder with defective lysosome-related organelles. In a previous study, we identified the AP-3 complex as an important contributor to HIV-1 assembly and release. We hypothesized that cells from patients affected by HPS2 should demonstrate abnormalities of HIV-1 assembly. Here we report that HIV-1 particle assembly and release are indeed diminished in HPS2 fibroblast cultures. Transient or stable expression of the full-length wild-type ß3A subunit in HPS2 fibroblasts restored the impaired virus assembly and release. In contrast, virus-like particle release mediated by MA-deficient Gag mutants lacking the AP-3 binding site was not altered in HPS2 cells, indicating that the MA domain serves as the major viral determinant required for the recruitment of the AP-3 complex. AP-3 deficiency decreased HIV-1 Gag localization at the plasma membrane and late endosomes and increased the accumulation of HIV-1 Gag at an intermediate step between early and late endosomes. Blockage of the clathrin-mediated endocytic pathway in HPS2 cells did not reverse the inhibited virus assembly and release imposed by the AP-3 deficiency. These results demonstrate that the intact and stable AP-3 complex is required for HIV-1 assembly and release, and the involvement of the AP-3 complex in late stages of the HIV-1 replication cycle is independent of clathrin-mediated endocytosis.
Asunto(s)
Complejo 3 de Proteína Adaptadora/metabolismo , Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , VIH-1/fisiología , Síndrome de Hermanski-Pudlak , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Complejo 3 de Proteína Adaptadora/deficiencia , Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/deficiencia , Subunidades delta de Complexo de Proteína Adaptadora/genética , Membrana Celular/metabolismo , Membrana Celular/virología , Células Cultivadas , Clatrina/antagonistas & inhibidores , Endocitosis , Fibroblastos/virología , VIH-1/metabolismo , Síndrome de Hermanski-Pudlak/genética , Síndrome de Hermanski-Pudlak/metabolismo , Síndrome de Hermanski-Pudlak/virología , Humanos , Mutación , Transducción de Señal , Piel/virología , Liberación del Virus/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail-localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.
Asunto(s)
Complejo 3 de Proteína Adaptadora/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Lisosomas/metabolismo , Cuerpos Multivesiculares/metabolismo , Receptor PAR-1/metabolismo , Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células HeLa , Humanos , Immunoblotting , Microscopía Confocal , Modelos Biológicos , Unión Proteica , Transporte de Proteínas , Interferencia de ARN , Receptor PAR-1/genética , Transducción de Señal , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
During the late phase of the Human Immunodeficiency Virus Type-1 (HIV-1) replication cycle, viral Gag proteins and the intact RNA genome are trafficked to specific sub-cellular membranes where virus assembly and budding occurs. Targeting to the plasma membranes of T cells and macrophages is mediated by interactions between the N-terminal matrix (MA) domain of Gag and cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)] molecules. However, in macrophages and dendritic cells, a subset of Gag proteins appears to be targeted to tetraspanin enriched viral compartments, a process that appears to be mediated by MA interactions with the Delta subunit of the cellular Adaptor Protein AP-3 (AP-3δ). We cloned, overexpressed and purified the protein interactive domain of AP-3δ and probed for MA binding by NMR. Unexpectedly, no evidence of binding was observed in these in vitro experiments, even at relatively high protein concentrations (200µM), suggesting that AP-3δ plays an alternative role in HIV-1 assembly.
Asunto(s)
Complejo 3 de Proteína Adaptadora/metabolismo , Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , Antígenos VIH/metabolismo , VIH-1/fisiología , Mapeo de Interacción de Proteínas , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Clonación Molecular , Expresión Génica , Humanos , Espectroscopía de Resonancia Magnética , Unión ProteicaRESUMEN
VAMP7 is involved in the fusion of late endocytic compartments with other membranes. One possible mechanism of VAMP7 delivery to these late compartments is via the AP3 trafficking adaptor. We show that the linker of the δ-adaptin subunit of AP3 binds the VAMP7 longin domain and determines the structure of their complex. Mutation of residues on both partners abolishes the interaction in vitro and in vivo. The binding of VAMP7 to δ-adaptin requires the VAMP7 SNARE motif to be engaged in SNARE complex formation and hence AP3 must transport VAMP7 when VAMP7 is part of a cis-SNARE complex. The absence of δ-adaptin causes destabilization of the AP3 complex in mouse mocha fibroblasts and mislocalization of VAMP7. The mislocalization can be rescued by transfection with wild-type δ-adaptin but not by δ-adaptin containing mutations that abolish VAMP7 binding, despite in all cases intact AP3 being present and LAMP1 trafficking being rescued.
Asunto(s)
Complejo 3 de Proteína Adaptadora/metabolismo , Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , Transporte de Proteínas/fisiología , Proteínas R-SNARE/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Cristalografía por Rayos X , Endocitosis , Endosomas/metabolismo , Fibroblastos , Citometría de Flujo , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Coats define the composition of carriers budding from organelles. In addition, coats interact with membrane tethers required for vesicular fusion. The yeast AP-3 (Adaptor Protein Complex 3) coat and the class C Vps/HOPS (HOmotypic fusion and Protein Sorting) tether follow this model as their interaction occurs at the carrier fusion step. Here we show that mammalian Vps class C/HOPS subunits and clathrin interact and that acute perturbation of clathrin function disrupts the endosomal distribution of Vps class C/HOPS tethers in HEK293T and polarized neuronal cells. Vps class C/HOPS subunits and clathrin exist in complex with either AP-3 or hepatocyte growth factor receptor substrate (Hrs). Moreover, Vps class C/HOPS proteins cofractionate with clathrin-coated vesicles, which are devoid of Hrs. Expression of FK506 binding protein (FKBP)-clathrin light chain chimeras, to inhibit clathrin membrane association dynamics, increased Vps class C/HOPS subunit content in rab5 endosomal compartments. Additionally, Vps class C/HOPS subunits were concentrated at tips of neuronal processes, and their delivery was impaired by expression of FKBP-clathrin chimeras and AP20187 incubation. These data support a model in which Vps class C/HOPS subunits incorporate into clathrin-coated endosomal domains and carriers in mammalian cells. We propose that vesicular (AP-3) and nonvesicular (Hrs) clathrin mechanisms segregate class C Vps/HOPS tethers to organelles and domains of mammalian cells bearing complex architectures.
Asunto(s)
Polaridad Celular , Clatrina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Complejo 3 de Proteína Adaptadora/metabolismo , Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , Animales , Línea Celular , Clatrina/antagonistas & inhibidores , Clatrina/genética , Vesículas Cubiertas por Clatrina/efectos de los fármacos , Vesículas Cubiertas por Clatrina/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Humanos , Inmunoprecipitación , Complejos Multiproteicos/metabolismo , Neuronas/metabolismo , Unión Proteica , Subunidades de Proteína/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Tacrolimus/análogos & derivados , Tacrolimus/farmacología , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Adaptor protein complexes (APs) are evolutionarily conserved heterotetramers that couple cargo selection to the formation of highly curved membranes during vesicle budding. In Saccharomyces cerevisiae, AP-3 mediates vesicle traffic from the late Golgi to the vacuolar lysosome. The HOPS subunit Vps41 is one of the few proteins reported to have a specific role in AP-3 traffic, yet its function remains undefined. We now show that although the AP-3 delta subunit, Apl5, binds Vps41 directly, this interaction occurs preferentially within the context of the HOPS docking complex. Fluorescence microscopy indicates that Vps41 and other HOPS subunits do not detectably colocalize with AP-3 at the late Golgi or on post-Golgi (Sec7-negative) vesicles. Vps41 and HOPS do, however, transiently colocalize with AP-3 vesicles when these vesicles dock at the vacuole membrane. In cells with mutations in HOPS subunits or the vacuole SNARE Vam3, AP-3 shifts from the cytosol to a membrane fraction. Fluorescence microscopy suggests that this fraction consists of post-Golgi AP-3 vesicles that have failed to dock or fuse at the vacuole membrane. We propose that AP-3 remains associated with budded vesicles, interacts with Vps41 and HOPS upon vesicle docking at the vacuole, and finally dissociates during docking or fusion.
Asunto(s)
Complejo 3 de Proteína Adaptadora/metabolismo , Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Transporte Biológico/fisiología , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Membranas Intracelulares/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Adaptins are subunits of the heterotetrameric (beta/mu/gamma/sigma) adaptor protein (AP) complexes that are involved in clathrin-mediated membrane trafficking. Here, we show that in Schizosaccharomyces pombe the deletion strains of each individual subunit of the AP-1 complex [Apl2 (beta), Apl4 (gamma), Apm1 (mu) and Aps1 (sigma)] caused distinct phenotypes on growth sensitivity to temperature or drugs. We also show that the Deltaapm1 and Deltaapl2 mutants displayed similar but more severe phenotypes than those of Deltaaps1 or Deltaapl4 mutants. Furthermore, the Deltaapl2Deltaaps1 and Deltaapl2Deltaapl4 double mutants displayed synthetic growth defects, whereas the Deltaaps1Deltaapl4 and Deltaapl2Deltaapm1 double mutants did not. In pull-down assay, Apm1 binds Apl2 even in the absence of Aps1 and Apl4, and Apl4 binds Aps1 even in the absence of Apm1 and Apl2. Consistently, the deletion of any subunit generally caused the disassociation of the heterotetrameric complex from endosomes, although some subunits weakly localized to endosomes. In addition, the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1. Altogether, results suggest that the four subunits are all essential for the heterotetrameric complex formation and for the AP-1 function in exit transport from endosomes.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Subunidades del Complejo de Proteínas Adaptadoras/genética , Subunidades del Complejo de Proteínas Adaptadoras/metabolismo , Eliminación de Gen , Schizosaccharomyces/crecimiento & desarrollo , Complejo 1 de Proteína Adaptadora/genética , Subunidades beta de Complejo de Proteína Adaptadora/genética , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Subunidades delta de Complexo de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , Subunidades mu de Complejo de Proteína Adaptadora/genética , Subunidades mu de Complejo de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Datos de Secuencia Molecular , Fenotipo , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Temperatura , Ácido Valproico/farmacologíaRESUMEN
Dendritic cells (DC) are crucial components of the early events of HIV infection. Dendritic cells capture and internalize HIV at mucosal surfaces and efficiently transfer the virus to CD4+ T cells in trans through infectious synapses (trans-infection pathway). Alternatively, HIV-1 replicates in DC (R5-HIV-1) (cis-infection pathway). Here, we analyzed HIV trafficking in DC during the trans-infection pathway as well as the cis-infection pathway. Confocal immunofluorescence microscopy demonstrated that after capture by DC, R5-HIV-1 and HIV-1 pseudotyped with vesicular stomatitis virus protein G colocalized in a viral compartment enriched in tetraspanins including CD81, CD82 and CD9, although at different levels, indicating a role of the viral envelope in targeting to the tetraspanin-rich compartment. Replication of R5-HIV-1 in DC (cis-infection pathway) also led to the accumulation, in an envelope-independent manner, of mature viral particles in a tetraspanin-rich compartment. A fraction of the HIV-1-containing compartments appeared directly accessible from the cell surface. In sharp contrast with the trans-infection pathway, the delta-subunit of the adaptor protein 3 (AP-3) complex was enriched on the HIV-1-containing compartment during R5-HIV-1 replication in DC (cis-infection pathway). Downregulation of AP-3 delta-adaptin reduced significantly viral particle release from HIV-1-infected DC. Together, these studies demonstrate a role for AP-3 in HIV replication in a tetraspanin-rich compartment in DC and contribute to the elucidation of the trafficking pathways required for DC-T cell transfer of HIV-1 infection, a critical step during the early events of HIV infection.