RESUMO
Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3- or BLOC-1-deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting.
Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Proteínas de Transporte/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Membrana Celular/metabolismo , Células Cultivadas , Vesículas Citoplasmáticas/metabolismo , Fibroblastos/citologia , Proteínas de Membrana Lisossomal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Fibras Musgosas Hipocampais/metabolismo , Células PC12 , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transporte Proteico , Proteínas R-SNARE/metabolismo , RatosRESUMO
[formula: see text] The syntheses of PNA oligomers containing potential ambiguous nucleobase analogues, namely 3-nitropyrrole and 5-nitroindole, have been accomplished. Hybridization properties of these PNAs with complementary oligodeoxynucleotides were evaluated by thermal denaturation experiments. Both novel residues exhibited little variation in Tm (< or = 1.5 degrees C) when positioned against any of the four nucleoside bases. The capability to incorporate degenerate sites should further expand the utility of PNA in applications where precise sequence information is not available.