The C-terminal TPR Domain of Tom70 Defines a Family of Mitochondrial Protein Import Receptors Found only in Animals and Fungi

https://doi.org/10.1016/j.jmb.2006.02.062Get rights and content

In fungi and animals the translocase in the outer mitochondrial membrane (TOM complex) consists of multiple components including the receptor subunit Tom70. Genome sequence analyses suggest no Tom70 receptor subunit exists in plants or protozoans, raising questions about its ancestry, function and the importance of its activity. Here we characterise the relationships within the Tom70 family of proteins. We find that in both fungi and animals, a conserved domain structure exists within the Tom70 family, with a transmembrane segment followed by 11 tetratricopeptide repeat motifs organised in three distinct domains. The C-terminal domain of Tom70 is highly conserved, and crucial for the import of hydrophobic substrate proteins, including those with and those without N-terminal presequences. Tom70 likely arose after fungi and animals diverged from other eukaryote lineages including plants, and subsequent gene duplication gave rise to a paralogue specific to the Saccharomyces group of yeasts. In animals and in fungi, Tom70 plays a fundamental role in the import of precursor proteins, by assisting relatively hydrophobic regions of substrate proteins into the translocation channel in the outer mitochondrial membrane. Proteins that function equivalently to Tom70 may have arisen independently in plants and protists.

Introduction

Recent proteomic analyses suggest that mitochondria in any given cell type probably contain 800–1000 different proteins.1 Ninety-nine per cent of these mitochondrial proteins are coded by nuclear genes, made in the cytosol and imported into the organelle. Their initial recognition and import through the outer mitochondrial membrane is facilitated by the multi-subunit translocase called the TOM (translocase in the outer mitochondrial membrane) complex, consisting of receptors Tom70 and Tom20, and the channel-forming Tom40 and its attendant subunits. Many mitochondrial proteins are synthesised with an N-terminal presequence that provides the targeting information needed for mitochondrial location. After passage through the TOM complex, N-terminal presequences direct the precursor to interact with the TIM23 complex in the inner membrane, and as a result the protein enters the mitochondrial matrix space where a processing peptidase removes the targeting sequence.2, 3, 4, 5 Many of the proteins destined for mitochondria do not have an N-terminal extension and instead rely on targeting information embedded internally, often at or overlapping their transmembrane domains.2, 3, 4, 6 Some of the proteins carrying these internal targeting sequences, notably members of the carrier protein family,7 will be translocated through the outer membrane and passed on to the TIM22 complex that drives the insertion of substrate proteins into the mitochondrial inner membrane.2, 4, 6

How does the TOM complex recognize and bind both N-terminal and internal targeting sequences? Although two parallel pathways are often discussed, with Tom20 and Tom70 mediating each pathway independently, a model whereby a single, hetero-oligomeric receptor was responsible for recognition of all substrates was previously proposed.8 According to this model, each molecule of precursor protein interacts through its targeting portion with Tom20, while other parts of the substrate interact with Tom70. Evidence that the Tom70 receptor binds at least some precursor proteins directly came with peptide scans of substrate proteins: recombinant Tom70 selectively binds peptides derived from discrete segments of substrates and a proteolytically stable “core” domain of ∼25 kDa is also capable of binding substrates, albeit with reduced avidity.9 In addition, a segment of Tom70 was recently identified with features similar to the “clamp” domain of the chaperone HOP, and a point mutation in this region of Tom70 prevents the binding of molecular chaperones like Hsp70.10 Several molecular chaperones in the cytosol transfer a range of substrate proteins to Tom70, suggesting that this receptor subunit plays a fundamental role in protein import into mitochondria.10, 11

Tom20 and Tom70 can interact with each other by virtue of tetratricopeptide repeats (TPRs), with a site-specific mutation in the TPR segment of Tom20 having no effect on the function of Tom20 but blocking instead the function of Tom70.12 The TPR motif is degenerate in primary structure, composed of 34 amino acid signatures that reflect structural elements of tight helix-turn-helix packing. No residue within the motif is absolutely conserved and until recently prediction of TPR segments has yielded ambiguous results.13, 14 Structural analysis of Tom20 from animals and fungi showed that a single TPR segment is found in Tom20.15, 16, 17, 18 In the case of Tom70, previous analyses suggested seven TPR motifs in Tom70 from yeast and humans,9, 10, 13, 19 but at least ten TPR motifs in the Tom70 from rats.20 With structures recently determined for a range of proteins composed of multiple TPR segments, it is now possible to confidently predict these elements from sequence alone and it is becoming clear that specific features conserved within the TPR segments can assist in defining distinct protein families and clans.

Here we evaluate structural aspects of the Tom70 family of proteins and suggest that they are characterised by 11 TPR motifs. These 11 motifs are organised with three TPRs in an N-terminal “clamp” domain, five in a “core” domain and three in a previously uncharacterised C-terminal domain. Analysis of motifs in the Tom70 family revealed contiguous motifs in the C-terminal domain with a highly conserved sequence signature centred on residues that would interact with substrate. The signature motif provides a diagnostic search tool to find members of the Tom70 family, and shows that the family arose early in the evolution of fungi and animals. A series of Tom70 mutants, designed on the predicted TPR segment structure, shows that Tom70 plays a role in facilitating the import of most, if not all, precursor proteins into mitochondria. This is true for substrate proteins with N-terminal targeting signals as well as for proteins with internal targeting signals. The C-terminal domain is crucial for binding these substrate proteins.

Section snippets

Ancestry of the Tom70 family

In a comprehensive search for Tom70 orthologues, we started with the sequences for the functionally defined Tom70 from Saccharomyces cerevisiae, Neurospora crassa and rat, retrieving an initial set of 20 sequences related to Tom70 (Supplementary Data, Figure 1). The initial set of sequences was subjected to a pattern search algorithm21 to elucidate Tom70 motifs. Hidden Markov models (HMMs) were built and proved an effective search tool, able to discriminate a family of 41 Tom70 proteins from

Discussion

With crystal structures now available for diverse proteins composed of TPR sequence elements, it becomes possible to critically analyse TPR-containing proteins like Tom70, for which little structural data is available. For most species of Tom70, 11 TPR motifs are predicted. We show that even for a protein like Tom70 from the yeast S. cerevisiae, where only nine clear TPR signatures fit completely with the consensus, secondary structure predictions together with sequence similarity to other Tom70

Motif analysis and HMM search of UniProt

Initial BLAST searches with the sequences corresponding to the functionally defined Tom70 from S. cerevisiae, N. crassa and Rattus norvegicus retrieved an initial set of 20 Tom70 sequences: seven animal sequences (from Anopheles gambiae, Danio rerio, Drosophila melanogaster, Homo sapiens, Mus musculus, R. norvegicus and Xenopus laevis) and 13 fungal sequences (from Saccharomyces bayanus, N. castellii, S. cerevisiae (Tom70) and S. cerevisiae (Tom71), Schizosaccharomyces pombe, Yarrowia lipolytica, E. 

Acknowledgements

We thank Jim Whelan and Ian Gentle for comments on the manuscript and Andreas Lennartsson and Loes Parma for their early contributions to the project. This work was supported by a grant from the Australian Research Council (to T.L. and T.D.M.) and an Australian Postgraduate Award (to N.C.C.).

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