Patterns that Define the Four Domains Conserved in Known and Novel Isoforms of the Protein Import Receptor Tom20

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Tom20 is the master receptor for protein import into mitochondria. Analysis of motifs present in Tom20 sequences from fungi and animals found several highly conserved regions, including features of the transmembrane segment, the ligand-binding domain and functionally important flexible segments at the N terminus and the C terminus of the protein. Hidden Markov model searches of genome sequence data revealed novel isoforms of Tom20 in vertebrate and invertebrate animals. A three-dimensional comparative model of the novel type I Tom20, based on the structurally characterized type II isoform, shows important differences in the amino acid residues lining the ligand-binding groove, where the type I protein from animals is more similar to the fungal form of Tom20. Given that the two receptor types from mouse interact with the same set of precursor protein substrates, comparative analysis of the substrate-binding site provides unique insight into the mechanism of substrate recognition. No Tom20-related protein was found in genome sequence data from plants or protozoans, suggesting the receptor Tom20 evolved after the split of animals and fungi from the main lineage of eukaryotes.

Introduction

Genomic and proteomic studies have revealed that 5–10% of the proteins made in a eukaryotic cell are targeted to mitochondria.1, 2, 3, 4, 5, 6, 7 Specific mitochondrial targeting sequences distinguish these proteins from those that will stay in the cytosol. In some mitochondrial proteins, especially those destined for the mitochondrial matrix, the targeting sequences are cleavable basic, amphipathic helices.8, 9 However, for many mitochondrial membrane proteins, amphipathic segments serve as targeting signals, and these same amphipathic segments become the transmembrane segment or segments once the protein is embedded in the mitochondrial outer or inner membrane.10, 11

All these mitochondrial targeting sequences have in common properties of positive charge and amphipathicity but there is no consensus in primary structure. Specialized protein import receptors recognize structural aspects common to all mitochondrial targeting sequences. The import receptors are mitochondrial outer membrane proteins that can bind each of the diverse protein substrates and transfer them efficiently to the protein translocation channel, formed from the essential protein Tom40, which is the central component of the translocase in the outer mitochondrial membrane (the TOM complex).12, 13, 14, 15 While it has been shown that the import receptors dock transiently to the core TOM complex to deliver their substrate protein cargo,16, 17, 18, 19, 20 the structurally important features for interactions between the receptors and Tom40 or its attendant subunits Tom6 and Tom7 are not known.

At least three receptors, Tom20, Tom22 and Tom70, mediate protein import into mitochondria.14 The master receptor is Tom20, which binds mitochondrial targeting sequences directly, as shown with the Tom20 from the fungi Neurospora crassa and Saccharomyces cerevisiae, and from rats and humans.14, 21, 22, 23, 24 Structural analysis of the central core domain of the rat protein has shown that it includes a tetratricopeptide repeat (TPR) fold and a distal helical segment, which together form a small, globular domain with a shallow groove. The surface of the groove, which represents the substrate-binding surface in which targeting sequences sit, is formed from hydrophobic side-chains to accommodate the hydrophobic surface of the targeting sequence ligands.25 In addition to contributing to the ligand-binding groove, the TPR segment of Tom20 is needed for a productive interaction with the receptor Tom70, which facilitates recognition and binding of large hydrophobic precursor proteins.26 Other regions of Tom20 have been shown to be functionally important, but have proved intransigent to direct structural analysis.23, 25, 27, 28

In order to further understand its structure and function, we designed hidden Markov models (HMMs) to describe the Tom20 receptor, and uncovered conserved structural features in Tom20 proteins from animals and fungi. Those conserved motifs allowed us to search genome sequence data and identify new isoforms of the Tom20 receptor in animals. No Tom20-like sequence was found in plants or protozoans. Structural analysis of the novel form of Tom20 from mouse by comparative modeling, using the known structure of the classical Tom20 as a template, revealed that the two paralogs have distinguishing features in the ligand-binding groove. Furthermore, analyses in mice and worms showed that the variant Tom20 isoforms are functional, and likely provide for optimal expression of import receptors in specific cell types in metazoans.

Section snippets

Hidden Markov models define conserved sequence characteristics of Tom20 receptors from animals and fungi

HMMs can be used to describe conserved features of a family of proteins with a view to defining domain structure and for searching for proteins from distantly related species.29 To provide the sequences from which to build HMMs for Tom20, a BLAST search of GenBank was initiated with sequences of the functionally defined Tom20 from N. crassa, S. cerevisiae and Homo sapiens (see Materials and Methods). The initial set of Tom20 sequences consisted of 12 originating from animal species and six

Discussion

We collected and analyzed Tom20 sequences covering representative classes of animals and fungi for sequence motifs and used hidden Markov models to define consensus features of the import receptor and to comprehensively search the known data sets for Tom20 sequences. The search revealed a novel isoform of Tom20 in animals, and three-dimensional modeling allowed us to determine the extent of structural conservation in the Tom20 paralogs. In terms of the substrate-binding site, our analysis

Tom20 sequences used in HMM search

The initial set of Tom20 sequences consisted of 12 animal sequences (from the insects Bombyx mori and D. melanogaster, the nematodes C. elegans and Echinococcus multilocularis, the flatworms Schistosoma japonicum and Schistosoma mansoni, the coelenterate Ciona intestinalis, the mollusk Crassostrea virginica, the fish Danio rerio and Oryzias latipes, the frog Silurana tropicalis, and from H. sapiens) and six fungal sequences (Botrytis cinerea, Candida albicans, Gibberella zeae, N. crassa, S. 

Acknowledgements

We thank Daniel Bird for expert advice on Northern analysis, Paul Gleeson for expert advice and support with the mammalian cell culture experiments, and the European Bioinformatics Institute for access to CLUSTALW. This work was supported by a grant from the Australian Research Council (to T.L.), a Dora Lush Postgraduate Award (ID 310656) from the National Health and Medical Research Council (to J.H.) and an Australian Postgraduate Research Award (to A.P.).

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