RESUMEN
A broadly conserved membrane-associated protein required for the functional interaction of kinesin-I with axonal cargo was identified. Mutations in sunday driver (syd) and the axonal transport motor kinesin-I cause similar phenotypes in Drosophila, including aberrant accumulations of axonal cargoes. GFP-tagged mammalian SYD localizes to tubulovesicular structures that costain for kinesin-I and a marker of the secretory pathway. Coimmunoprecipitation analysis indicates that mouse SYD forms a complex with kinesin-I in vivo. Yeast two-hybrid analysis and in vitro interaction studies reveal that SYD directly binds kinesin-I via the tetratricopeptide repeat (TPR) domain of kinesin light chain (KLC) with K(d) congruent with 200 nM. We propose that SYD mediates the axonal transport of at least one class of vesicles by interacting directly with KLC.
Asunto(s)
Transporte Axonal , Proteínas Portadoras/metabolismo , Proteínas de Drosophila , Cinesinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Secuencia de Aminoácidos , Animales , Conducta Animal , Transporte Biológico , Proteínas Portadoras/genética , Compartimento Celular , Clonación Molecular , Drosophila/genética , Proteínas de Insectos/metabolismo , Larva/genética , Proteínas de la Membrana/genética , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Unión Proteica , Subunidades de Proteína , Técnicas del Sistema de Dos Híbridos , Red trans-Golgi/químicaRESUMEN
Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.