Sorting switch of mitochondrial presequence translocase involves coupling of motor module to respiratory chain - PubMed (original) (raw)

Sorting switch of mitochondrial presequence translocase involves coupling of motor module to respiratory chain

Nils Wiedemann et al. J Cell Biol. 2007.

Abstract

The mitochondrial presequence translocase transports preproteins to either matrix or inner membrane. Two different translocase forms have been identified: the matrix transport form, which binds the heat-shock protein 70 (Hsp70) motor, and the inner membrane-sorting form, which lacks the motor but contains translocase of inner mitochondrial membrane 21 (Tim21). The sorting form interacts with the respiratory chain in a Tim21-dependent manner. It is unknown whether the respiratory chain-bound translocase transports preproteins and how the switch between sorting form and motor form occurs. We report that the respiratory chain-bound translocase contains preproteins in transit and, surprisingly, not only sorted but also matrix-targeted preproteins. Presequence translocase-associated motor (Pam) 16 and 18, two regulatory components of the six-subunit motor, interact with the respiratory chain independently of Tim21. Thus, the respiratory chain-bound presequence translocase is not only active in preprotein sorting to the inner membrane but also in an early stage of matrix translocation. The motor does not assemble en bloc with the translocase but apparently in a step-wise manner with the Pam16/18 module before the Hsp70 core.

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Figures

Figure 1.

Copurification of preprotein in transit with tagged respiratory chain supercomplex. (A) 35S-labeled b2(220)-DHFR was imported into isolated yeast mitochondria at different temperatures in the presence of methotrexate. Mitochondrial proteins were analyzed by SDS-PAGE and digital autoradiography. p, precursor (20%); i, intermediate; m, mature. (B) Isolated wild-type (WT) and Cor1TAP mitochondria were solubilized in digitonin and subjected to IgG chromatography. Bound complexes were eluted by tobacco etch virus protease cleavage and analyzed by blue native electrophoresis and immunodecoration. Load, 40%; elution, 100%. III2/IV and III2/IV2, respiratory chain supercomplexes. (C) b2-DHFR was imported into wild-type and Cor1TAP mitochondria at 8°C in the presence of methotrexate followed by IgG chromatography. Load and wash, 2%; elution, 100%. Samples were analyzed by SDS-PAGE and autoradiography. The yield of copurification of b2-DHFR with Cor1TAP was ∼25% of that of Tim23.

Figure 2.

TIM-accumulated preprotein interacts with respiratory chain in organello. [35S]b2-DHFR precursor was imported into wild-type and Cor1TAP mitochondria in the presence of methotrexate. After import, the mitochondria were mixed with the same amount of wild-type and Cor1TAP mitochondria as indicated and were subjected to IgG chromatography. Load and wash, 1%; elution, 100%. The samples were analyzed by SDS-PAGE and autoradiography for b2-DHFR and immunodecoration for Tim23 and Pam17.

Figure 3.

Processing of preproteins does not require Cor1 and Cor2 of complex III. (A) [35S]b2-DHFR was imported into wild-type, cor1Δ, and cor2Δ mitochondria. Samples were treated with 50 μg/ml proteinase K and analyzed by SDS-PAGE. Processed b2-DHFR was quantified using ImageQuant software (GE Healthcare). Import in wild-type mitochondria after the longest incubation time was set to 100% (control). (B) b2-DHFR was imported into wild-type and cor1Δ cor2Δ mitochondria. Samples were treated with proteinase K (Prot. K) as indicated and were analyzed by SDS-PAGE. Processing of b2-DHFR did not require Cor1/Cor2 independently of the length of the b2 part (85–167 amino acid residues). (C) [35S]Cytochrome c1 was imported into wild-type and cor1Δ cor2Δ mitochondria. Samples were treated as described for A and B.

Figure 4.

Lack of Tim21 impairs but does not block preprotein copurification with respiratory chain. (A) IgG chromatography was performed with wild-type, Cor1TAP, and Cor1TAP tim21Δ mitochondria. The samples were analyzed by SDS-PAGE and immunodecoration. (B) [35S]b2(220)-DHFR was imported into isolated mitochondria and analyzed by SDS-PAGE. (C) b2- DHFR was imported in the presence of methotrexate. IgG chromatography was performed, and samples were analyzed by autoradiography. Load and wash, 2%; elution, 100%.

Figure 5.

Pam16/18 interact with the respiratory chain. (A) [35S]b2(220)Δ-DHFR was imported into wild-type, Cor1TAP, and Cor1TAP tim21Δ mitochondria in the presence of methotrexate followed by IgG chromatography and SDS-PAGE. Load and wash, 2%; elution, 100%. [35S]F1β was imported at 2°C in the absence of added ATP. Load and wash, 1%; elution, 100%. p, precursor; i, intermediate; m, mature. (B) IgG chromatography was performed with wild-type, Cor1TAP, and Cor1TAP tim21Δ mitochondria followed by SDS-PAGE and immunodecoration. Load and flow through (FT), 2%; elution, 100%. (C) IgG chromatography was performed as described for B. Load and wash, 2.5%; elution, 100%.

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Sorting switch of mitochondrial presequence translocase involves coupling of motor module to respiratory chain - PubMed (original) (raw)