RESUMO
The movement of proteins between cellular compartments requires the orchestrated actions of many factors including Rab family GTPases, Soluble NSF Attachment protein REceptors (SNAREs) and so-called tethering factors. One such tethering factor is called TRAnsport Protein Particle (TRAPP), and in humans, TRAPP proteins are distributed into two related complexes called TRAPP II and III. Although thought to act as a single unit within the complex, in the past few years it has become evident that some TRAPP proteins function independently of the complex. Consistent with this, variations in the genes encoding these proteins result in a spectrum of human diseases with diverse, but partially overlapping, phenotypes. This contrasts with other tethering factors such as COG, where variations in the genes that encode its subunits all result in an identical phenotype. In this review, we present an up-to-date summary of all the known disease-related variations of genes encoding TRAPP-associated proteins and the disorders linked to these variations which we now call TRAPPopathies.
Assuntos
Deficiências do Desenvolvimento/genética , Osteocondrodisplasias/genética , Fenótipo , Polimorfismo Genético , Proteínas de Transporte Vesicular/genética , Animais , Deficiências do Desenvolvimento/patologia , Humanos , Osteocondrodisplasias/patologia , Síndrome , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismoRESUMO
Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consanguineous Syrian family presenting with LGMD and in five individuals of Hutterite descent presenting with myopathy, infantile hyperkinetic movements, ataxia, and intellectual disability. By using a combination of whole-exome or genome sequencing with homozygosity mapping, we identified the homozygous c.2938G>A (p.Gly980Arg) missense mutation within the gryzun domain of TRAPPC11 in the Syrian LGMD family and the homozygous c.1287+5G>A splice-site mutation resulting in a 58 amino acid in-frame deletion (p.Ala372_Ser429del) in the foie gras domain of TRAPPC11 in the Hutterite families. TRAPPC11 encodes a component of the multiprotein TRAPP complex involved in membrane trafficking. We demonstrate that both mutations impair the binding ability of TRAPPC11 to other TRAPP complex components and disrupt the Golgi apparatus architecture. Marker trafficking experiments for the p.Ala372_Ser429del deletion indicated normal ER-to-Golgi trafficking but dramatically delayed exit from the Golgi to the cell surface. Moreover, we observed alterations of the lysosomal membrane glycoproteins lysosome-associated membrane protein 1 (LAMP1) and LAMP2 as a consequence of TRAPPC11 dysfunction supporting a defect in the transport of secretory proteins as the underlying pathomechanism.
Assuntos
Deficiência Intelectual/genética , Transtornos dos Movimentos/genética , Doenças Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Deleção de Sequência , Proteínas de Transporte Vesicular/metabolismo , Adolescente , Adulto , Ataxia/genética , Mapeamento Cromossômico , Consanguinidade , Creatina Quinase/sangue , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Exoma , Feminino , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Homozigoto , Humanos , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Masculino , Transtornos dos Movimentos/patologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Doenças Musculares/patologia , Distrofia Muscular do Cíngulo dos Membros/patologia , Linhagem , Ligação Proteica , Transporte Proteico , Sítios de Splice de RNA , Síria , Proteínas de Transporte Vesicular/genética , Adulto JovemRESUMO
TRAPP is a multisubunit complex that functions in membrane traffic. Mutations in the mammalian TRAPP protein C2 are linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT) that is thought to arise from an inability to secrete procollagen from the endoplasmic reticulum. Here, we show that C2 binds to the SNARE protein Syntaxin 5 and this interaction is weakened by an SEDT-causing missense mutation (D47Y). Interestingly, the equivalent mutation (D46Y) in the yeast C2 homolog Trs20p does not block anterograde traffic but did affect endocytosis. The trs20D46Y mutation interfered with the interaction between Trs20p and Trs85p (TRAPP III-specific subunit), Trs120p and Trs130p (TRAPP II-specific subunits). Size exclusion chromatography suggested that this yeast mutation destabilized the TRAPP III complex that is involved in autophagy. We further show that this mutation blocks both the selective cytosol-to-vacuole (cvt) pathway as well as non-selective autophagy. We demonstrate that the apparent molecular size of the TRAPP III complex is dependent upon membranes, and that the presence of TRAPP III is dependent upon Atg9p. Finally, we demonstrate that lipidated Bet3p is enriched in TRAPP III and that lipidation increases the efficiency of autophagy. Our study suggests that Trs20p acts as an adaptor for Trs85p and Trs120p and reveals complexities in TRAPP III assembly and function. The implications of C2D47Y in SEDT are discussed.
Assuntos
Autofagia/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação de Sentido Incorreto/genética , Osteocondrodisplasias/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Endocitose/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Osteocondrodisplasias/metabolismo , Ligação Proteica/genética , Transporte Proteico , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vacúolos/genética , Vacúolos/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
Mutations in the trafficking protein particle complex C2 protein (TRAPPC2), a mammalian ortholog of yeast Trs20p and a component of the trafficking protein particle (TRAPP) vesicle tethering complex, have been linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT). Intriguingly, the X-linked TRAPPC2 is just one of a complement of Trs20-related genes in humans. Here we characterize TRAPPC2L, a novel, highly conserved TRAPP-interacting protein related to TRAPPC2 and the uncharacterized yeast open reading frame YEL048c. TRAPPC2L and TRAPPC2 genes are found in pairs across species and show broad and overlapping expression, suggesting they are functionally distinct, a notion supported by yeast complementation studies and biochemical characterization. RNA interference-mediated knockdown of either TRAPPC2L or TRAPPC2 in HeLa cells leads to fragmentation of the Golgi, implicating both proteins in Golgi dynamics. Gradient fractionation of cellular membranes indicates that TRAPPC2L is found with a portion of cellular TRAPP on very low-density membranes whereas the remainder of TRAPP, but not TRAPPC2L, is found associated with Golgi markers. YEL048c displays genetic interactions with TRAPP II-encoding genes and the gene product co-fractionates with and interacts with yeast TRAPP II. Taken together these results indicate that TRAPPC2L and its yeast ortholog YEL048c are novel TRAPP-interacting proteins that may modulate the function of the TRAPP II complex.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Regulação Fúngica da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Saccharomyces cerevisiae transport protein particle (TRAPP) is a family of related multisubunit complexes required for endoplasmic reticulum-to-Golgi transport (TRAPP I), endosome-to-Golgi transport (TRAPP II) or cytosol to vacuole targeting (TRAPP III). To gain insight into the relationship between these complexes, we generated random and targeted mutations in the Trs23p core subunit. Remarkably, at physiological salt concentrations only two peaks (TRAPP I and a high molecular weight peak) are detected in wild-type cells. As the salt was raised, the high molecular weight peak resolved into TRAPP II and III peaks. Deletion of a Saccharomycotina-specific domain of Trs23p resulted in destabilization of TRAPP I but had no effect on TRAPP II or III. This mutation had no observable growth phenotype, normal levels of Ypt1p-directed guanine nucleotide exchange factor activity in vivo and did not display any in vivo nor in vitro blocks in membrane traffic. Biochemical analysis indicated that TRAPP I could be produced from the TRAPP II/III peak in vitro by increasing the salt concentration. Our data suggest that the SMS domain of Trs23p is responsible for the in vitro appearance of TRAPP I in S. cerevisiae. The implications of these findings are discussed.
RESUMO
TRAPP is a multisubunit tethering complex implicated in multiple vesicle trafficking steps in Saccharomyces cerevisiae and conserved throughout eukarya, including humans. Here we confirm the role of TRAPPC2L as a stable component of mammalian TRAPP and report the identification of four novel components of the complex: C4orf41, TTC-15, KIAA1012, and Bet3L. Two of the components, KIAA1012 and Bet3L, are mammalian homologues of Trs85p and Bet3p, respectively. The remaining two novel TRAPP components, C4orf41 and TTC-15, have no homologues in S. cerevisiae. With this work, human homologues of all the S. cerevisiae TRAPP proteins, with the exception of the Saccharomycotina-specific subunit Trs65p, have now been reported. Through a multidisciplinary approach, we demonstrate that the novel proteins are bona fide components of human TRAPP and implicate C4orf41 and TTC-15 (which we call TRAPPC11 and TRAPPC12, respectively) in ER-to-Golgi trafficking at a very early stage. We further present a binary interaction map for all known mammalian TRAPP components and evidence that TRAPP oligomerizes. Our data are consistent with the absence of a TRAPP I-equivalent complex in mammalian cells, suggesting that the fundamental unit of mammalian TRAPP is distinct from that characterized in S. cerevisiae.