RESUMO
Now that whole genome sequences are available for many eukaryotic organisms from yeast to man, we can form broad hypotheses on the basis of the relative expansion of protein families. To investigate the molecular mechanisms responsible for the organization of membrane compartments, we identified members of the SNARE, coat complex, Rab and Sec1 protein families in four eukaryotic genomes. Of these families only the Rab family expanded from the unicellular yeast to the multicellular fly and worm. All families were expanded in humans, where we find 35 SNAREs, 60 Rabs and 53 coat complex subunits. In addition, we were able to resolve the SNARE class of proteins into four distinct subfamilies.
Assuntos
Membrana Celular/fisiologia , Complexo I de Proteína do Envoltório/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Compartimento Celular , Membrana Celular/genética , Complexo I de Proteína do Envoltório/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Genoma , Genoma Humano , Humanos , Proteínas de Membrana/genética , Proteínas Munc18 , Proteínas do Tecido Nervoso/genética , Proteínas SNARE , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae , Vesículas Transportadoras/genética , Vesículas Transportadoras/fisiologia , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins of the vesicle-associated membrane protein (VAMP) and syntaxin families play a central role in vesicular trafficking through the formation of complexes between proteins present on vesicle and target membranes. Formation of these complexes is proposed to mediate aspects of the specificity of vesicle trafficking and to promote fusion of the lipid bilayers. In order to further understand the molecular mechanisms that organize membrane compartments, we have characterized seven new mammalian proteins of the VAMP and syntaxin families. The proteins are broadly expressed; however, syntaxin 13 is enriched in brain and VAMP 8 in kidney. The seven novel SNAREs localize in distinct patterns overlapping with Golgi, endosomal, or lysosomal markers. Our studies support the hypothesis that evolutionary radiation of these two gene families gave rise to sets of proteins whose differential expression and combinatorial associations define and organize the membrane compartments of cells.
Assuntos
Compartimento Celular , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Transporte Biológico , Membrana Celular/metabolismo , Humanos , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Organelas/metabolismo , Ratos , Proteínas SNARE , Homologia de Sequência de AminoácidosRESUMO
The specific transfer of vesicles between organelles is critical in generating and maintaining the organization of membrane compartments within cells. Syntaxin 6 is a recently discovered member of the syntaxin family, whose constituents are required components of several vesicle trafficking pathways. To better understand the function of syntaxin 6, we generated a panel of monoclonal antibodies that specifically recognize different epitopes of the protein. Immunoelectron microscopy shows syntaxin 6 primarily on the trans-Golgi network (TGN), where is partially colocalizes with the TGN adapter protein AP-1 on clathrin-coated membranes. Additional label is present on small vesicles in the vicinity of endosome-like structures. Immunoprecipitation of syntaxin 6 revealed that it is present in a complex or complexes with alpha-soluble NSF attachment protein, vesicle-associated membrane protein 2, or cellubrevin and a mammalian homologue of VPS45, which is a member of the sec1 family implicated in Golgi to prevacuolar compartment trafficking in yeast. We show that mammalian VPS45 is found in multiple tissues, is partially membrane associated, and is enriched in the Golgi region. Converging lines of evidence suggest that syntaxin 6 mediates a TGN trafficking event, perhaps targeting to endosomes in mammalian cells.
Assuntos
Complexo de Golgi/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Transporte Biológico , Proteínas de Transporte/metabolismo , Ensaio de Imunoadsorção Enzimática , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos , Células PC12 , Proteínas Qa-SNARE , Ratos , Frações Subcelulares/metabolismoAssuntos
Transporte Biológico , Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Fusão de Membrana , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Sensíveis a N-Etilmaleimida , Organelas/metabolismo , Proteínas SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Vacúolos/metabolismoRESUMO
Despite the central role vesicular trafficking occupies in protein targeting, the molecular coding of the trafficking signals and the mechanism of vesicle docking and fusion are just beginning to be understood. We report here the cloning and initial characterization of a new member of the syntaxin family of vesicular transport receptors. Syntaxin 6 is a 255-amino acid protein with two domains predicted to form coiled-coils, as well as a carboxyl-terminal membrane anchor. Syntaxin 6 is broadly expressed and localizes in the region of the Golgi apparatus. In vitro binding studies established that syntaxin 6 binds to alpha-soluble NSF attachment protein (alpha-SNAP). The sequence homology, topology, localization, and alpha-SNAP binding suggest that syntaxin 6 is involved in intracellular vesicle trafficking.