Relationship between the Peptide-sensitive Channel and the Mitochondrial Outer Membrane Protein Translocation Machinery

Philippe Juin,Michel Thieffry,Jean-Pierre Henry,François M Vallette

doi:10.1074/jbc.272.9.6044

Abstract

The peptide-sensitive channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by synthetic mitochondrial presequences and by nonmitochondrial basic peptides such as dynorphin B(1-13). Both types of peptides are imported into mitochondria. However, the import of dynorphin B(1-13) had to be further characterized since its properties differed from those of the general import pathway used by mitochondrial peptides. Cross-linking experiments with iodinated dynorphin B(1-13) led to the labeling of TOM 40/ISP 42, a component of the protein import machinery of the outer membrane. Accordingly, dynorphin B(1-13) could also be used as a presequence to direct the import of a cytosolic protein into the mitochondria. Pretreatment of intact mitochondria by trypsin removed components capable of discriminating between true mitochondrial presequences and other basic peptides active on the PSC. After proteolysis, both types of peptides appeared to cross the outer membrane through the same pathway. Involvement of the PSC in the translocation complex was shown by immunoprecipitation of the PSC activity by anti-ISP 42 antibodies. Taken together, the present data reinforce the hypothesis that the PSC is the pore responsible for the translocation of protein through the outer membrane.

Highlights

Many proteins have to fully or partially cross biological membranes to reach their final destination and to achieve their biological function
Cross-linking of Dynorphin B(1–13) with Yeast Mitochondrial Membranes—Porin-deficient yeast mitochondria were incubated with 1 ␮M 125I-labeled dynorphin B(1–13) for 5 min at 30 °C under conditions that allowed its import into the organelle (11)
A mild trypsin treatment of the labeled membranes decreased the radioactive 44-kDa band without affecting the 30-kDa band. These results showed that dynorphin B(1–13) could be cross-linked to a protein of 42 kDa of the outer membrane and that this binding was inhibited by pCytOX IV(1–12)Y

Summary

Introduction

Many proteins have to fully or partially cross biological membranes to reach their final destination and to achieve their biological function. The outer membrane contains a protein translocation complex that includes different receptors capable of discriminating between different subsets of mitochondrial proteins. Dynorphin B(1–13) failed to inhibit the import of a chimeric protein, cytochrome b2-dihydrofolate reductase (DHFR), into the matrix of yeast mitochondria (11). Two import mechanisms could be distinguished: the mitochondrial sequence was translocated into the matrix in a temperature- and potential-sensitive manner and probably along the general import pathway, whereas dynorphin B(1–13) was imported into the intermembrane space by a process that was neither temperature- nor potential-sensitive This second pathway is likely to involve the PSC (12), and the aim of this work was to study its relationship with the general import pathway

Objectives

Results

Conclusion