RESUMEN
Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.
Asunto(s)
Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitios de Unión/genética , Inmunoprecipitación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/genética , Unión Proteica/fisiología , Transporte de Proteínas/fisiología , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Saccharomyces cerevisiaeRESUMEN
Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23-Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23-Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.
Asunto(s)
Proteínas Fúngicas/fisiología , Proteínas Mitocondriales/metabolismo , Proteínas Motoras Moleculares/fisiología , Levaduras/metabolismo , Proliferación Celular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Mutagénesis Sitio-Dirigida , Mapeo de Interacción de Proteínas , Señales de Clasificación de Proteína , Transporte de Proteínas/fisiología , Levaduras/citología , Levaduras/genéticaRESUMEN
Newly synthesized mitochondrial precursor proteins have to become unfolded by the mitochondrial Hsp70 (mtHsp70) import motor to cross the mitochondrial membranes. To assess the mechanism of unfolding of precursor proteins by mtHsp70, we designed a system to measure step sizes of the mtHsp70 import motor, which are distances at which the motor system moves along polypeptide chains during a single turnover of ATP. We made a series of fusion proteins consisting of a mitochondrial presequence containing the first mtHsp70 binding site, a spacer sequence containing an Hsp70 avoidance segment followed by the second mtHsp70 binding site, and different folded mature domains. Analyses of the dependence of the import rates of those fusion proteins on the lengths of Hsp70 avoidance segments allowed us to estimate the step sizes, which differ for different mature domains and different lengths of the spacers. These results suggest that the mtHsp70 import motor functions at least as a molecular Brownian ratchet to unfold mitochondrial precursor proteins.