Coupling of Mitochondrial Import and Export Translocases by Receptor-Mediated Supercomplex Formation

Abstract

The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of β-barrel proteins use both translocases-TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the β-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring β-barrel formation in vivo and in organello and demonstrated that the β-barrel was formed and membrane inserted while the precursor was bound to SAM. β-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling.

Evidence Supporting the 19 β-Strand Model for Tom40 from Cysteine Scanning and Protease Site Accessibility Studies

“…These data provide good experimental support for the existence of the predicted ␤-strand 19 and for the approach of Cys scanning to detect membrane spanning ␤-strands. The results are also in agreement with cross-linking data suggesting the existence of ␤-strand 19 in S. cerevisiae (28).…”

“…To establish a known anchor point for the topology of the predicted structure of NcTom40 we wished to obtain experimental evidence for the existence of the most C-terminal predicted ␤-strand (strand 19 of the three-dimensional model), which has been shown to contain the ␤-signal for insertion of the protein into the mitochondrial outer membrane by the sorting and assembly machinery) complex (28,53), and also verify that the C terminus of the protein exists in the IMS. Establishing these positions would allow firm predictions for the location of loops between strands as being in either the cytosol or IMS in any model of the protein.…”

“…Comparative studies have revealed a phylogenetic relationship between Tom40 and porin (10 -13, 25). The relationship between the two proteins, coupled with extensive bioinformatic analysis and other experimental approaches, has led to the development of 19 ␤-strand models of Tom40 based on the known structure of porin (11)(12)(13)28). Tom40 is also thought to have an ␣-helical region near the N terminus, preceding the ␤-strands.…”

Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40

“…A model for such a Tom40 dimer is shown schematically in Structural analysis of Tom40 has proven difficult. Recent cross-linking data are consistent with a predicted location for the second N-terminal ␣-helix inside the Tom40 lumen (56). The location of the first ␣-helix (removed in the Tom40ca-332 and Tom40ca-319 constructs) has not been characterized.…”

Retro-translocation of mitochondrial intermembrane space proteins

“…S5B). Next, to test the involvement of Tom40 in the release process, the Tom40 C130/C138 variant was used, in which cysteine residues were placed in positions 130 and 138 (28) (Fig. S5C).…”