The assembly pathway of the mitochondrial carrier translocase involves four preprotein translocases

Abstract

The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.

FIG. 1.

Membrane-integral components of the TIM22 complex assemble via different intermediates. ³⁵S-radiolabeled Tim22 (A), Tim54 (B), and Tim18 (C) were imported into isolated tim22-14 mitochondria at temperatures of 16°C to 25°C in the presence or absence of Δψ and subsequently treated with 50 μg/ml proteinase K (Prot. K). After solubilization in digitonin buffer, samples were subjected to blue native electrophoresis and analyzed by digital autoradiography. Import of Tim22 (D), Tim54 (E), and Tim18 (F) into isolated tim54-11 and tim54-16 mitochondria and subsequent sample analysis was carried out as described for panels A to C. Tim22 (G), Tim54 (H), and Tim18 (I) were imported into tim18Δ mitochondria as described above. Arrowheads, low-molecular-mass form of Tim54; asterisks, low-molecular-mass intermediate of Tim18; WT, wild type.

FIG. 2.

Tim22 assembly occurs through low-molecular-mass intermediates. (A) Antibody shift analysis of imported Tim22 in wild-type (WT) mitochondria. After swelling of mitochondria, complexes were shifted by incubation with increasing amounts of antisera against Tim22 and Tim18. After solubilization with digitonin buffer, protein complexes were separated by blue native electrophoresis and analyzed by digital autoradiography. (B) Pulse-chase analysis of Tim22 assembly. ³⁵S-labeled Tim22 was imported into mitochondria as indicated in the scheme, and samples were analyzed by blue native electrophoresis and digital autoradiography. (C) Radiolabeled Tim22 was imported into wild-type and tim10-2 mitochondria in the presence or absence of a Δψ and treated with 50 μg/ml proteinase K. Import was carried out at 25°C after a 15-min preincubation of mitochondria at 37°C. Samples were subjected to SDS-PAGE and analyzed by digital autoradiography. (D) Import of Tim22 precursor protein was done as described for panel C, and samples were analyzed by blue native electrophoresis and digital autoradiography (left panel). Isolated wild-type and tim10-2 mitochondria were incubated at 37°C for 15 min prior to solubilization in digitonin buffer and separation of complexes by blue native electrophoresis. Western blot analysis was performed with the indicated antiserum (right panel).

FIG. 3.

Tim54 forms a TOM-TIM intermediate. (A) Assembly of radiolabeled Tim54 in wild-type (WT) mitochondria. After import of Tim54, samples were left untreated or treated with 50 μg/ml proteinase K, solubilized in digitonin buffer, and then analyzed by blue native electrophoresis and digital autoradiography. (B) After import of radiolabeled Tim54, complexes were shifted with antisera against Tom40 and porin or incubated with BSA. As a control, Tom22 was imported and shifted as described for Tim54. Analysis was carried out by blue native electrophoresis and digital autoradiography. (C) Radiolabeled Tim54 was imported into isolated wild-type and tim50-1 mitochondria in the presence or absence of Δψ. After treatment with proteinase K, proteins were separated by SDS-PAGE and visualized by digital autoradiography. (D) ³⁵S-labeled Tim54 was imported into wild-type and tim50-1 mitochondria. After treatment with digitonin-containing buffer, complexes were separated by blue native electrophoresis and subjected to digital autoradiography. (E) Tim54 was imported into wild-type mitochondria and subjected to antibody shift/depletion analysis as described in Materials and Methods. Samples were analyzed by blue native electrophoresis and digital autoradiography. Arrowheads, low-molecular-mass form of Tim54.

FIG. 4.

Tim54 assembly is dependent on the TIM10 complex. (A) Assembly of Tim54 in tim10-2 mutant mitochondria. Radiolabeled Tim54 was imported into isolated wild-type (WT) and tim10-2 mitochondria in the presence or absence of a Δψ after a 15-min preincubation at 37°C. Where indicated, mitochondria were treated with proteinase K. Complexes were separated by blue native electrophoresis and visualized by digital autoradiography. (B) ³⁵S-labeled Tim54 was imported as described in panel A, including treatment with proteinase K. Samples were analyzed by SDS-PAGE and digital autoradiography. (C) Radiolabeled Tim54 was imported. The mitochondria were treated with proteinase K and subjected to treatment with carbonate. Total (T), pellet (P), and supernatant (S) were analyzed by SDS-PAGE and digital autoradiography ([³⁵S]Tim54) or immunolabeling (Tom70, Mge1). (D) Radiolabeled Tim18 was imported into wild-type and tim10-2 mitochondria as described in panel A. Arrowhead, low-molecular-mass form of Tim54; asterisk, low-molecular-mass intermediate of Tim18.

FIG. 5.

Tim54 and Tim10 interact at the carrier translocase. (A) Tim18_ProtA mitochondria were subjected to chemical cross-linking as described in Materials and Methods. The TIM22 complex was purified by IgG-Sepharose chromatography. After washing and elution, proteins were separated by SDS-PAGE and analyzed by immunolabeling with antibodies against Tim54 and Tim10. Circle, unspecific cross-reaction of anti-Tim10. (B) After import of radiolabeled Tim54 into Tim18_ProtA mitochondria, proteins were cross-linked as described in Materials and Methods and the TIM22 complex was purified with IgG-Sepharose. After washing and elution of the proteins, an immunoprecipitation was performed with antibodies against Tim9, Tim10, and Tim12. Samples were analyzed by SDS-PAGE and digital autoradiography.