Journal of Molecular Biology
Volume 376, Issue 3, 22 February 2008, Pages 694-704
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The Transmembrane Segment of Tom20 Is Recognized by Mim1 for Docking to the Mitochondrial TOM Complex

https://doi.org/10.1016/j.jmb.2007.12.021Get rights and content

Abstract

Mitochondria cannot be made de novo. Mitochondrial biogenesis requires that up to 1000 proteins are imported into mitochondria, and the protein import pathway relies on hetero-oligomeric translocase complexes in both the inner and outer mitochondrial membranes. The translocase in the outer membrane, the TOM complex, is composed of a core complex formed from the β-barrel channel Tom40 and additional subunits each with single, α-helical transmembrane segments. How α-helical transmembrane segments might be assembled onto a transmembrane β-barrel in the context of a membrane environment is a question of fundamental importance. The master receptor subunit of the TOM complex, Tom20, recognizes the targeting sequence on incoming mitochondrial precursor proteins, binds these protein ligands, and then transfers them to the core complex for translocation across the outer membrane. Here we show that the transmembrane segment of Tom20 contains critical residues essential for docking the Tom20 receptor into its correct environment within the TOM complex. This crucial docking reaction is catalyzed by the unique assembly factor Mim1/Tom13. Mutations in the transmembrane segment that destabilize Tom20, or deletion of Mim1, prevent Tom20 from functioning as a receptor for protein import into mitochondria.

Introduction

Mitochondria are essential organelles characteristic of eukaryotic cells. Derived from intracellular bacteria, mitochondria retain many bacterial characteristics including the presence of β-barrel proteins in the outer membrane. Since most of the mitochondrial proteins are now encoded on genes in the cell nucleus, mitochondrial proteins have to be imported from the cytosol across the mitochondrial membranes. Multisubunit protein translocases situated in the inner and outer mitochondrial membranes have evolved to perform this critical operation.1, 2, 3, 4 The translocase in the outer membrane of mitochondria, the TOM complex, is composed of a core of five subunits. The channel subunit, Tom40, has a β-barrel topology, while each of its partners has a single α-helical transmembrane segment. This combination is unique, a structure not yet observed for any membrane protein complex. While β-barrel proteins are common in the outer membranes of bacteria, α-helical transmembrane proteins are restricted instead to the bacterial inner membrane. How an α-helical transmembrane segment can be packed against a β-barrel in the context of a lipid bilayer remains unknown and cannot be interpreted from the 100 or so high-resolution structures of membrane proteins currently known, all of which have either β-barrel or α-helical transmembrane domains.5

In bacterial outer membranes, β-barrel proteins are assembled by membrane protein complexes containing a member of the Omp85 family of proteins.6, 7 Mitochondria contain an equivalent complex referred to as the SAM complex.8, 9 The SAM complex is required in order to insert the Tom40 polypeptide into the plane of the outer mitochondrial membrane10, 11 and might also catalyze formation of the barrel formed from anti-parallel β-strands. When Tom40 is released from the SAM complex as “assembly intermediate II,” it has already associated with at least one of the α-helical transmembrane proteins of the TOM complex.9 Since kinetic analysis of the assembly reveals no other intermediates, intermediate II must rapidly recruit all the additional α-helical transmembrane subunits (Tom5, Tom6, Tom7, Tom22 and Tom20) to form the mature TOM complex. At least some of these must be packed against the β-barrel surface.

Treatment of the TOM complex with urea has shown the core complex to be extremely stable: even 5 M urea is insufficient to disrupt the interaction between Tom5, Tom6, Tom7, Tom22 and the Tom40 β-barrel.12 Tom5, Tom6, Tom7 and Tom22 each have a conserved proline residue in their transmembrane segments, as well as a preponderance of glycine and hydroxylated residues: these residues are uncommon in transmembrane segments and their conservation in these small TOM proteins suggests they mediate protein–protein interactions.13, 14 The bends and curves in transmembrane helices that can be facilitated by proline and glycine residues are becoming increasingly common as means to add functional complexity to membrane protein structures,5 and a complicated architecture of protein–protein interactions is anticipated for the core TOM complex.

Tom20 is found in all species of animal and fungi.15 It is removed from the core TOM complex more readily than Tom22 and the small TOM proteins,12 and this and other evidence16, 17, 18 has been interpreted to suggest Tom20 docks only transiently and reversibly to the TOM complex, only to deliver substrate proteins. An alternative is that Tom20 is a constant component of the TOM complex, but that its interaction is more tenuous. The transmembrane segment of Tom20 contains a motif of residues,15 and the conserved series of aromatic and small side-chain residues suggests a classic knobs-and-holes19, 20 method of packing the α-helical transmembrane segment of Tom20 against the surface of the TOM complex.

Here we have established assays to measure docking of Tom20 to the TOM complex to address three questions: Is the transmembrane segment of Tom20 critical for its function as a protein import receptor? Is the Tom20 subunit in a restrained location within the TOM complex, with the conserved residues packed in the vicinity of defined residues on other proteins? Is the SAM complex, or some other component of the outer membrane, responsible for catalyzing docking of Tom20 into the mature TOM complex? We suggest that Tom20 is a stable component of the TOM complex and that the transmembrane domain of Tom20 is specifically packed in an environment where it contacts other components of the TOM complex.

Section snippets

The transmembrane segment of Tom20 is highly conserved and is essential for receptor function

Transmembrane segments formed from α-helices are hydrophobic, are characteristically of low sequence complexity and show low sequence identity across species.5 This reflects the few constraints on the sequence, other than for overall hydrophobicity, where the purpose of a transmembrane segment is to anchor a protein in a phospholipid environment. Comparative sequence analysis of the import receptor Tom20 from various species of fungi and animals showed a motif in the primary structure of the

TOM20 and MIM1 are not essential for viability

In a genome-wide survey of yeast strains derived from the type strain S288c two of the genes that were classified as essential for cell viability were YGR082W and YOL026C, and these genes encode Tom20 and Mim1, respectively.38, 39 Strains derived from the S. cerevisiae type strain S288c are known to have weak mitochondrial function 40, 41 and only in these S288c-derived strains do the TOM20 gene and MIM1 gene appear essential; in most yeast strains, Tom20 loss-of-function mutants have defects

Sequence searches and motif detection

A hidden Markov model describing Mim1 was constructed as previously described for other protein families.15 UniProt (Release 10.0 of March 2007, consisting of Swiss-Prot Release 52.0 and TrEMBL Release 35.0) was searched and sequences matching the Mim1 model were returned, totaling 27 species of fungi and representing a diversity of the kingdom (S. cerevisiae, Saccharomyces mikatae, Naumovia castellii, Kluveromyces lactis, Candida albicans, Gibberelae zea, Neurospora crassa, Schizosaccharomyces

Acknowledgements

We thank Ian Gentle for critical advice and comments on the manuscript, Peter Walsh for comments on the manuscript and Bruce Kemp for critical discussions. J.M.H. was supported by a Dora Lush Postgraduate Research Scholarship from the National Health and Medical Research Council (ID310656); N.C.C. is supported by an Australian Postgraduate Award. This work was supported by grants from the Victorian Partnership for Advanced Computing (to V.A.L. and T.L.) and from the Australian Research Council

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