RESUMO
SNARE proteins participate in recognition and fusion of membranes. A SNARE complex consisting of vti1b, syntaxin 8, syntaxin 7, and endobrevin/VAMP-8 which is required for fusion of late endosomes in vitro has been identified recently. Here, we generated mice deficient in vti1b to study the function of this protein in vivo. vti1b-deficient mice had reduced amounts of syntaxin 8 due to degradation of the syntaxin 8 protein, while the amounts of syntaxin 7 and endobrevin did not change. These data indicate that vti1b is specifically required for the stability of a single SNARE partner. vti1b-deficient mice were viable and fertile. Most vti1b-deficient mice were indistinguishable from wild-type mice and did not display defects in transport to the lysosome. However, 20% of the vti1b-deficient mice were smaller. Lysosomal degradation of an endocytosed protein was slightly delayed in hepatocytes derived from these mice. Multivesicular bodies and autophagic vacuoles accumulated in hepatocytes of some smaller vti1b-deficient mice. This suggests that other SNAREs can compensate for the reduction in syntaxin 8 and for the loss of vti1b in most mice even though vti1b shows only 30% amino acid identity with its closest relative.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas de Transporte Vesicular , Alelos , Animais , Northern Blotting , Western Blotting , Proteínas de Transporte/metabolismo , Células Cultivadas , DNA Complementar/metabolismo , Endossomos/metabolismo , Éxons , Biblioteca Gênica , Vetores Genéticos , Genótipo , Hepatócitos/metabolismo , Fígado/patologia , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Modelos Genéticos , Fenótipo , Testes de Precipitina , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , RNA Mensageiro/metabolismo , Recombinação Genética , Proteínas SNARE , Fatores de TempoRESUMO
Two mammalian proteins, vtila and vtilb, are homologous to the yeast Q-SNARE Vtilp which is part of several SNARE complexes in different transport steps. In vitro experiments suggest distinct functions for vtila and vtilb. Here we compared the subcellular localization of endogenous vtila and vtilb by immunofluorescence and immuno-electron microscopy. Both proteins had a distinct but overlapping localization. vtila was found predominantly on the Golgi and the TGN, vtilb mostly on tubules and vesicles in the TGN area and on endosomes. vti1a coimmunoprecipitated with VAMP-4, syntaxin 6, and syntaxin 16. These four SNAREs could assemble into a SNARE complex of conserved structure because one SNARE motif of each subgroup is present. vtila-beta, VAMP-4, syntaxin 6, and syntaxin 16 are coenriched with small synaptic vesicles and with clathrin-coated vesicles isolated from rat brain synaptosomes. Therefore, this SNARE complex may have a role in synaptic vesicle biogenesis or recycling.