Journal of Molecular Biology
Patterns that Define the Four Domains Conserved in Known and Novel Isoforms of the Protein Import Receptor Tom20
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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2013, Trends in Plant ScienceCitation Excerpt :Mitochondria targeting signals, or presequences, also have diverse sequences. Interestingly, mitochondrial receptor TOM20 is encoded by a single gene in yeast (Saccharomyces cerevisiae) but by multiple genes in Drosophila, vertebrates, and Arabidopsis [61–63]. Modeling of the presequence-binding groove of two rodent TOM20 isoforms [61] has revealed significant structural differences.
Unique components of the plant mitochondrial protein import apparatus
2013, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :Multiple TOM70 dimers are then recruited to the complex and the protein is passed through the import pore in an ATP dependent manner [25]. Interestingly, whilst orthologues to TOM20 or TOM70 of animals and fungi are absent from the plant mitochondrial outer membrane, proteins of similar size and function are found (see below), suggesting that TOM20 and TOM70 in yeast and animals evolved subsequent to the divergence of the eukaryotic lineages [27]. Whilst this may explain the absence of these receptors in plants, the core, conserved components of the outer membrane import apparatus similarly demonstrate divergent structures in plants.
Recognition of mitochondrial targeting sequences by the import receptors Tom20 and Tom22
2011, Journal of Molecular BiologyCitation Excerpt :Therefore, it was concluded that the nonacidic region of the cytosolic domain of Tom22 plays the key receptor role in animals and fungi, and that hydrophobic interactions are also important for interaction between presequence and cis-ScTom22. This does not exclude the acidic region of ScTom22 recognizing positively charged groups of either free ScTom20 or substrate-bound ScTom20.20,23,31,42–44 Previous experiments undertaken with yeast have shown that the cytosolic domains of Tom20 and Tom22 interact28–30 (Fig. 8a).
The evolution of protein targeting and translocation systems
2010, Biochimica et Biophysica Acta - Molecular Cell Research