RESUMEN
The majority of mitochondrial proteins are nuclear encoded and imported into mitochondria as precursor proteins via dedicated translocases. The translocase of the inner membrane 22 (TIM22) is a multisubunit molecular machine specialized for the translocation of hydrophobic, multi-transmembrane-spanning proteins with internal targeting signals into the inner mitochondrial membrane. Here, we undertook a crosslinking-mass spectrometry (XL-MS) approach to determine the molecular arrangement of subunits of the human TIM22 complex. Crosslinking of the isolated TIM22 complex using the BS3 crosslinker resulted in the broad generation of crosslinks across the majority of TIM22 components, including the small TIM chaperone complex. The crosslinking data uncovered several unexpected features, opening new avenues for a deeper investigation into the steps required for TIM22-mediated translocation in humans.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Complejos Multienzimáticos/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Succinimidas/química , Cromatografía Liquida , Células HEK293 , Humanos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana Mitocondrial/química , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/química , Modelos Moleculares , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Espectrometría de Masas en TándemRESUMEN
Hydrophobic inner mitochondrial membrane proteins with internal targeting signals, such as the metabolite carriers, use the carrier translocase (TIM22 complex) for transport into the inner membrane. Defects in this transport pathway have been associated with neurodegenerative disorders. While the TIM22 complex is well studied in baker's yeast, very little is known about the mammalian TIM22 complex. Using immunoprecipitation, we purified the human carrier translocase and identified a mitochondrial inner membrane protein TIM29 as a novel component, specific to metazoa. We show that TIM29 is a constituent of the 440 kDa TIM22 complex and interacts with oxidized TIM22. Our analyses demonstrate that TIM29 is required for the structural integrity of the TIM22 complex and for import of substrate proteins by the carrier translocase.