Conserved mechanism of Oxa1 insertion into the mitochondrial inner membrane.

Oxa1 is the mitochondrial representative of a family of related proteins that mediate the insertion of substrate proteins into the membranes of bacteria, chloroplasts, and mitochondria. Several studies have demonstrated that the bacterial homologue YidC participates both in the direct uptake of proteins from the bacterial cytosol, and in the uptake of nascent proteins from the Sec translocase. Studies on the biogenesis of membrane proteins in mitochondria established that Oxa1 has the capability to receive substrates at the inner surface of the inner membrane. In this study, we asked if Oxa1 may similarly cooperate with a protein translocase within the membrane. Since Oxa1 is involved in its own biogenesis, we used the precursor of Oxa1 as a model protein and investigated its import pathway. We found that immediately after import into mitochondria, Oxa1 initially accumulates at Tim23 that forms the inner membrane protein translocase. Cleavage of the Oxa1 presequence is dependent on mtHsp70, a heat shock protein of the mitochondrial matrix. However, mutant mtHsp70 showing a defect in the release of bound substrate proteins does not interfere with subsequent membrane insertion, indicating that membrane insertion of the mature protein is essentially mtHsp70-independent. We conclude that Oxa1 has the ability to accept preproteins within the membrane.

Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore.

Tom40 is the central pore-forming component of the translocase of the outer mitochondrial membrane (TOM complex). Different views exist about the secondary structure and electrophysiological characteristics of Tom40 from Saccharomyces cerevisiae and Neurospora crassa. We have directly compared expressed and renatured Tom40 from both species and find a high content of beta-structure in circular dichroism measurements in agreement with refined secondary structure predictions. The electrophysiological characterization of renatured Tom40 reveals the same characteristics as the purified TOM complex or mitochondrial outer membrane vesicles, with two exceptions. The total conductance of the TOM complex and outer membrane vesicles is twofold higher than the total conductance of renatured Tom40, consistent with the presence of two TOM pores. TOM complex and outer membrane vesicles possess a strongly enhanced sensitivity to a mitochondrial presequence compared to Tom40 alone, in agreement with the presence of several presequence binding sites in the TOM complex, suggesting a role of the non-channel Tom proteins in regulating channel activity.

Bcl-2 and porin follow different pathways of TOM-dependent insertion into the mitochondrial outer membrane.

The bcl-2 gene encodes a 26kDa protein which functions as a central regulator of apoptosis. Here we investigated the pathway of Bcl-2alpha into the mitochondrial outer membrane using the yeast Saccharomyces cerevisiae as a model organism. We found that interactions of Bcl-2alpha with the mitochondrial import receptor Tom20 are dependent on two positively charged lysine residues in the immediate vicinity of the carboxy-terminal hydrophobic membrane anchor. The targeting function of these residues is independent of Tom22. Subsequent insertion of Bcl-2alpha into the mitochondrial outer membrane does not require Tom5 or Tom40, indicating that Bcl-2alpha bypasses the general import pore (GIP). Bcl-2alpha shows a unique pattern of interactions with the components of the mitochondrial TOM complex, demonstrating that at least two different pathways lead from the import receptor Tom20 into the mitochondrial outer membrane.

Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex.

The mitochondrial outer membrane contains a multi-subunit machinery responsible for the specific recognition and translocation of precursor proteins. This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins. Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-filled centers resembling channels. Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, we have discovered the molecular reason for the different number of channel-like structures. The TOM complex from wild-type yeast contains up to three stain-filled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures. From mutant mitochondria lacking Tom22, native electrophoresis separates an approximately 80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein. We conclude that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures.