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. 2003 Nov;23(21):7818-28.
doi: 10.1128/MCB.23.21.7818-7828.2003.

Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space

Ann E Frazier  1 , Agnieszka Chacinska, Kaye N Truscott, Bernard Guiard, Nikolaus Pfanner, Peter Rehling
Affiliations

Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space

Ann E Frazier et al. Mol Cell Biol. 2003 Nov.

Abstract

The mitochondrial inner membrane contains numerous multispanning integral proteins. The precursors of these hydrophobic proteins are synthesized in the cytosol and therefore have to cross the mitochondrial outer membrane and intermembrane space to reach the inner membrane. While the import pathways of noncleavable multispanning proteins, such as the metabolite carriers, have been characterized in detail by the generation of translocation intermediates, little is known about the mechanism by which cleavable preproteins of multispanning proteins, such as Oxa1, are transferred from the outer membrane to the inner membrane. We have identified a translocation intermediate of the Oxa1 preprotein in the translocase of the outer membrane (TOM) and found that there are differences from the import mechanisms of carrier proteins. The intermembrane space domain of the receptor Tom22 supports the stabilization of the Oxa1 intermediate. Transfer of the Oxa1 preprotein to the inner membrane is not affected by inactivation of the soluble TIM complexes. Both the inner membrane potential and matrix heat shock protein 70 are essential to release the preprotein from the TOM complex, suggesting a close functional cooperation of the TOM complex and the presequence translocase of the inner membrane. We conclude that mitochondria employ different mechanisms for translocation of multispanning proteins across the aqueous intermembrane space.

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Figures

FIG. 1.
FIG. 1.
Accumulation of the precursor of Oxa1 in a 500-kDa complex of yeast mitochondria. 35S-labeled Oxa1 precursor was imported into wild-type mitochondria at 25°C in the presence or absence (addition of 1 μM valinomycin, 20 μM oligomycin, and 8 μM antimycin A) of a Δψ for the indicated times. (A) After import, mitochondria were either left untreated or treated with proteinase K (Prot. K), reisolated, and subjected to SDS-PAGE. The radiolabeled precursor (p) and mature (m) form of Oxa1 were visualized by digital autoradiography. (B) Following import of Oxa1 as for panel A, mitochondria were directly reisolated, solubilized in buffer containing 1% (wt/vol) digitonin, and analyzed by BN-PAGE. (C) Radiolabeled Oxa1 precursor was imported into wild-type mitochondria in the absence of a Δψ for the indicated times. Subsequently, samples were split and either left untreated or treated with proteinase K. After reisolation, mitochondria were solubilized and analyzed as for panel B.
FIG. 2.
FIG. 2.
The precursor of Oxa1 forms a productive translocation intermediate. (A) Radiolabeled Oxa1 precursor was imported for 5 min at 25°C into mitochondria in the presence of FCCP, as indicated. The reaction mixtures were subsequently split in half and either subjected to proteinase K (Prot. K) treatment and SDS-PAGE or left untreated, solubilized in buffer containing 1% (wt/vol) digitonin, and subjected to BN-PAGE analysis. mOxa1, mature Oxa1. (B) Import of 35S-labeled Oxa1 precursor was performed either in the presence of a Δψ, in the presence of valinomycin (Val), or in the presence of 60 μM FCCP for 20 min at 25°C. After reisolation, mitochondria were resuspended in fresh import buffer and subjected to a second incubation at 10°C for the indicated times. During the second incubation, one sample remained in the presence of Δψ, one sample remained without a Δψ (Val), and samples that had previously received FCCP were now incubated in the presence of a reestablished Δψ. Samples were analyzed by BN-PAGE after solubilization or subjected to SDS-PAGE after proteinase K treatment.
FIG. 3.
FIG. 3.
Accumulation of the Oxa1 precursor in the GIP complex. (A) Radiolabeled Oxa1 precursor was accumulated in wild-type mitochondria in the absence of a Δψ. After reisolation, mitochondria were left untreated (lane 1), received preimmune IgGs (lane 2), or received IgGs directed against the indicated Tom proteins or porin. Subsequently, mitochondria were solubilized in digitonin buffer and analyzed by BN-PAGE and digital autoradiography. (B) Left panel, wild-type (WT) and tom22-2 mitochondria were solubilized in digitonin buffer, protein complexes were separated by BN-PAGE, and proteins were transferred to polyvinylidene difluoride membranes by Western blotting. The GIP complex was detected with anti-Tom40 antiserum. Right panel, the 35S-labeled Oxa1 precursor was imported into wild-type and tom22-2 mitochondria for the indicated times at 25°C in the presence of a Δψ. Mitochondria were treated with proteinase K and analyzed by SDS-PAGE, and quantification of the digital autoradiogram was performed with ImageQuant 1.2 (Molecular Dynamics). The amount of maximal import after 4 min was set to 100% (control). (C) Import of radiolabeled Oxa1 precursor into wild-type and tom22-2 mitochondria was performed for the indicated times at 25°C in the presence or absence of a Δψ. The mitochondria were then reisolated and solubilized in digitonin buffer. Samples were subsequently analyzed by BN-PAGE and digital autoradiography.
FIG. 4.
FIG. 4.
The small TIM complexes of the intermembrane space are dispensable for Oxa1 import. (A) Radiolabeled Oxa1 precursor or AAC was imported at 15°C for the times indicated into wild-type (WT) or tim10-2 mitochondria in the presence of a Δψ, unless noted otherwise. After import, the mitochondria were subjected to proteinase K digestion and analyzed by SDS-PAGE. mOxa1, mature Oxa1. (B) Import of Oxa1 precursor (upper panel) and AAC (lower panel) at 25°C was performed as for panel A, except that proteinase K treatment was omitted and samples were analyzed by BN-PAGE after solubilization of mitochondria in digitonin buffer. (C) 35S-labeled Oxa1 precursor was imported in wild-type and tim8Δ tim13Δ mitochondria at 25°C. Mitochondria were either subjected to proteinase K digestion and SDS-PAGE analysis or directly reisolated, solubilized in digitonin buffer, and assayed by BN-PAGE for complex formation.
FIG. 5.
FIG. 5.
Requirement for mtHsp70 (Ssc1) in Oxa1 import. (A) Import of the 35S-labeled Oxa1 precursor into wild-type (WT) and ssc1-3 mitochondria was performed after a 15-min temperature shift of the isolated mitochondria to 37°C (+ heat shock) or without a temperature shift (− heat shock) for the indicated times in the presence of a Δψ, unless indicated otherwise. Mitochondria were treated with proteinase K after import and analyzed by SDS-PAGE. mOxa1, mature Oxa1. (B) Imports of the Oxa1 precursor were performed after temperature shift of the isolated wild-type and ssc1-3 mitochondria to 37°C as described above. Proteinase K treatment was omitted; mitochondria were reisolated and solubilized, and complexes were assessed by BN-PAGE. (C) The experiment was performed as described for panel B except that wild-type and ssc1-3 mitochondria remained at 25°C for the entire import experiment.
FIG. 6.
FIG. 6.
Formation of a translocation intermediate by the multispanning Cox18 protein. (A) Schematic comparison of the domain organizations of Oxa1 and Cox18 (primary structures). Hatched boxes, presequences; black boxes, transmembrane segments. (B) Radiolabeled Cox18 precursor (pCox18) was imported at 25°C for the indicated times in the presence or absence of a Δψ into wild-type (WT) and ssc1-3 mitochondria after a 15-min preshift of the mitochondria to 37°C. Mitochondria were subjected to proteinase K digestion and analyzed by SDS-PAGE. mCox18, mature Cox18. (C) 35S-labeled Cox18, Oxa1, F1β, and CoxVa precursor were imported into wild-type mitochondria in the presence or absence of a Δψ at 25°C for 20 min. After the import reaction, mitochondria were reisolated, solubilized in digitonin buffer, and analyzed by BN-PAGE, followed by digital autoradiography (samples 1 lanes 8). In addition, mitochondria that had been incubated with the precursors of F1β and CoxVa were analyzed by SDS-PAGE after treatment with or without proteinase K (Prot.K) (lanes 9 to 16). A fraction of the F1β precursor associates with mitochondria in the absence of a Δψ in an unspecific, nonproductive manner (59). Bands marked by an asterisk probably represent products of internal initiation of translation and are not specifically imported into mitochondria (22, 23). (D) Cox18 was imported into wild-type and ssc1-3 mitochondria for the indicated times in the presence or absence of a Δψ. Import was performed at 25°C for 15 min after a 15-min preincubation of the mitochondria at 37°C. Mitochondria were then reisolated and solubilized in digitonin buffer before separation by BN-PAGE. (E) Radiolabeled Cox18 precursor was imported in the presence or absence of a Δψ into tim10-2 mitochondria (left panel) and tim8Δ tim13Δ mitochondria (right panel) at 25°C for the indicated times. After import, the mitochondria were solubilized in digitonin buffer and analyzed by BN-PAGE.
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