RESUMEN
Rim1 was previously identified as a Rab3 effector localized to the presynaptic active zone in vertebrates. Here we demonstrate that C. elegans unc-10 mutants lacking Rim are viable, but exhibit behavioral and physiological defects that are more severe than those of Rab3 mutants. Rim is localized to synaptic sites in C. elegans, but the ultrastructure of the presynaptic densities is normal in Rim mutants. Moreover, normal levels of docked synaptic vesicles were observed in mutants, suggesting that Rim is not involved in the docking process. The level of fusion competent vesicles at release sites was reduced fivefold in Rim mutants, but calcium sensitivity of release events was unchanged. Furthermore, expression of a constitutively open form of syntaxin suppressed the physiological defects of Rim mutants, suggesting Rim normally acts to regulate conformational changes in syntaxin. These data suggest Rim acts after vesicle docking likely via regulating priming.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Proteínas Portadoras/metabolismo , Genes de Helminto , Proteínas del Helminto/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Electrofisiología , Genes Reporteros , Factores de Intercambio de Guanina Nucleótido , Proteínas del Helminto/genética , Locomoción/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/fisiología , Unión Neuromuscular/ultraestructura , Estructura Terciaria de Proteína , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular , Dedos de Zinc , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Rabfilina-3ARESUMEN
The priming step of synaptic vesicle exocytosis is thought to require the formation of the SNARE complex, which comprises the proteins synaptobrevin, SNAP-25 and syntaxin. In solution syntaxin adopts a default, closed configuration that is incompatible with formation of the SNARE complex. Specifically, the amino terminus of syntaxin binds the SNARE motif and occludes interactions with the other SNARE proteins. The N terminus of syntaxin also binds the presynaptic protein UNC-13 (ref. 5). Studies in mouse, Drosophila and Caenorhabditis elegans suggest that UNC-13 functions at a post-docking step of exocytosis, most likely during synaptic vesicle priming. Therefore, UNC-13 binding to the N terminus of syntaxin may promote the open configuration of syntaxin. To test this model, we engineered mutations into C. elegans syntaxin that cause the protein to adopt the open configuration constitutively. Here we demonstrate that the open form of syntaxin can bypass the requirement for UNC-13 in synaptic vesicle priming. Thus, it is likely that UNC-13 primes synaptic vesicles for fusion by promoting the open configuration of syntaxin.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas del Helminto/metabolismo , Proteínas de la Membrana/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Animales , Caenorhabditis elegans , Calcio/metabolismo , Proteínas Portadoras , Espectroscopía de Resonancia Magnética , Fusión de Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mutagénesis , Unión Proteica , Conformación Proteica , Proteínas Qa-SNARE , Proteínas SNARERESUMEN
Three independent Tn5-lac insertions in the S1 locus of Myxococcus xanthus inactivate the sglK gene, which is nonessential for growth but required for social motility and multicellular development. The sequence of sglK reveals that it encodes a homologue of the chaperone HSP70 (DnaK). The sglK gene is cotranscribed with the upstream grpS gene, which encodes a GrpE homologue. Unlike sglK, grpS is not required for social motility or development. Wild-type M. xanthus is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein. Mutations in sglK inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that sglK mutants do not make fibrils. The fibR gene, located immediately upstream of the grpS-sglK operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida. Inactivation of fibR leads to the overproduction of fibrillin, suggesting that M. xanthus fibril production and Pseudomonas alginate production are regulated in analogous ways. M. xanthus and Pseudomonas exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria.