The C-terminal TPR Domain of Tom70 Defines a Family of Mitochondrial Protein Import Receptors Found only in Animals and Fungi Nickie C. Chan 1,2 , Vladimir A. Likic ´ 2 , Ross F. Waller 3 Terrence D. Mulhern 1,2 and Trevor Lithgow 1,2 * 1 Department of Biochemistry and Molecular Biology University of Melbourne Parkville 3010, Australia 2 Bio21 Molecular Science and Biotechnology Institute University of Melbourne Parkville 3010, Australia 3 Botany School, University of Melbourne, Parkville 3010 Australia 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. q 2006 Elsevier Ltd. All rights reserved. Keywords: protein import; tetratricopeptide repeat; Tom70; Tom71; hidden Markov models *Corresponding author Introduction Recent proteomic analyses suggest that mito- chondria 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 mitochon- drial membrane) complex, consisting of receptors Tom70 and Tom20, and the channel-forming Tom40 and its attendant subunits. Many mitochon- drial proteins are synthesised with an N-terminal presequence that provides the targeting infor- mation 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. 2–5 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. 2–4,6 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 0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. Abbreviations used: TOM, translocase in the outer mitochondrial membrane; TIM, translocase in the inner mitochondrial membrane; TPR, tetratricopeptide repeat; HMM, hidden Markov model; CCCP, m-chlorophenyl- hydrazone. E-mail address of the corresponding author: t.lithgow@unimelb.edu.au doi:10.1016/j.jmb.2006.02.062 J. Mol. Biol. (2006) 358, 1010–1022