* Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Australia
Plant Molecular Biology Group, School of Biomedical and Chemical Sciences, University of Western Australia, Crawley, Australia
School of Botany, University of Melbourne, Parkville, Australia
Departamento de Genetica, Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sao Paulo, Sao Paulo, Brazil
Correspondence: E-mail: lithgow{at}unimelb.edu.au
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: mitochondria protein import TOM complex evolution
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In one of the earliest steps in the evolution of eukaryotic cells, the mitochondrion was derived from an endosymbiosed bacterium. Genes transferred from the bacteria were established within the nuclear genome of the host cell, and the proteins they encoded were translated in the host cell cytoplasm (Martin and Müller 1998; Gray, Burger, and Lang 1999; Kurland and Andersson 2000; Emelyanov 2001; Gray et al. 2001; Cavalier-Smith 2002). A rudimentary TOM complex that consisted of at least the translocation channel must have been in operation in the earliest eukaryote so that these "mitochondrial" proteins could enter the protomitochondria and carry out their various functions (Karlberg et al. 2000; Herrmann 2003; Lucattini et al. 2004).
What might have been the subunit structure of that early TOM complex? Apart from fungi and animals, the only other eukaryotes from which the TOM complex has been characterized biochemically are the higher plants Arabidopsis thaliana and Solanum tuberosum (Jänsch et al. 1998; Werhahn et al. 2001). Consistent with the idea that the translocation channel was established early in evolution, the plant Tom40 shows similarity to the Tom40 from fungi and animals. However, three small subunits associated with the plant Tom40 appeared to show only limited sequence similarity with fungal and animal proteins. Furthermore, additional subunits purified from plants as protein import receptors have no sequence similarity to the animal/fungal proteins Tom5, Tom20, or Tom70, nor are orthologs of these receptors obvious from genome sequence analysis. Although a 20-kDa protein found in the plant TOM complex has been named Tom20, it is unrelated in sequence to and has a distinct topology from the Tom20 found in animals and fungi (Jänsch et al. 1998; Macasev et al. 2001; Werhahn et al. 2001). These curious data leave open questions as to when during the radiation of eukaryotes the various subunits of the TOM complex evolved, and what was the nature of the translocase early in eukaryote evolution.
Using iterative sequence searches and transmembrane strand predictors, we find Tom40, Tom7, and Tom22 in sufficient representative taxa to suggest they may be ubiquitously present in eukaryotes. Although the homologous proteins that represent Tom7 and Tom40 are well conserved through diverse species of eukaryotes, Tom22 homologs were not immediately obvious from sequence alone. However, our studies suggest that two classes of Tom22 exist. In the animal-fungal lineage, Tom22 carries an acidic amino-terminal domain that functions as a receptor on the cis (or cytosolic) face of the mitochondria. Other eukaryote groups such as green plants and the moss Physcomitrella patens, the green alga Chlamydomonas reinhardtii, and apicomplexans Plasmodium falciparum, Theileria parva, and Eimeria tenella, express a truncated version of the protein we provisionally call Tom22'. The short Tom22' carries a transmembrane domain and a trans receptor domain equivalent to Tom22, but it lacks the acidic cis receptor domain. The trans receptor domain has limited sequence conservation but is functionally conserved, and this domain from the flowering plant Arabidopsis thaliana is functional in the TOM complex of yeast.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids, Cell Biology, and Yeast Strains
The open reading frames that encode ScTom22 and AtTom22'I were amplified by PCR from yeast genomic DNA or from A. thaliana cDNA and cloned into pMET25HDEL for expression in yeast (Egan et al. 1999; Beilharz et al. 2003). To create the chimeric fusion Tom22-Attrans, the trans domain of AtTom22 was amplified with the primers 5'-GGGAGCTCAGTTGGAGCAAG-3' and 5'-GGCTCGAGTTAGAGCAGCGCACC-3' for cloning in frame behind the ScTom22 open reading frame at a position corresponding to E120.
The plasmid encoding Tom22trans (tom22-9) and the heterozygous yeast strain (TOM22/
tom22) YTJB73 have been described previously (Bolliger et al. 1995). After transformation with the appropriate plasmid, YTJB73 cells were starved on sporulation medium for up to 10 days (Lithgow et al. 1994), and the spores were dissected under a Singer MSM dissecting microscope.
Miscellaneous
Previously published methods were used for pulse-chase analysis (Egan et al. 1999), fluorescence microscopy, alkali extraction and trypsin treatment of membranes (Beilharz et al. 2003), mitochondrial preparation, protein import assays, and Western blotting (Bolliger et al. 1995; Gentle et al. 2004).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The trans Domain of Tom22 and Tom22' Is Functionally Equivalent
Sequence analysis of the Tom22/Tom22' family of proteins shows that a trans domain, which would be located in the intermembrane space, is always present. Although the amino acid composition of the domain is conserved (i.e., rich in glutamate, glutamine, and proline residues), the pairwise sequence similarity is low. It is becoming increasingly common to find protein domains that are structurally similar and functionally equivalent, despite sparing sequence similarity. To test whether the trans domains are functionally equivalent, the chimeric construct Tom22-Attrans was made. It contained the coding sequences for the N-terminal domain and transmembrane segment of Tom22 from S. cerevisiae fused to the coding sequence for the C-terminal trans domain of AtTom22' (fig. 4A). Both pulse-chase and immunoblotting experiments revealed that the chimeric Tom22-Attrans protein was stable when expressed in yeast cells (fig. 4B), and fluorescence microscopy showed that the GFP-tagged version of Tom22-Attrans localizes to mitochondria, as determined by costaining with the red fluorescent dye Mitotracker (fig. 4C).
|
When heterozygous TOM22/tom22 diploid yeast cells are sporulated, the two spores lacking Tom22 fail to germinate (Lithgow et al. 1994). Heterozygous TOM22/
tom22 diploid yeast cells that expressed Tom22, Tom22-
trans (a version of Tom22 that lacks the C-terminal trans domain), or Tom22-Attrans were sporulated and the haploid progeny dissected. Consistent with earlier results, the two spores that lacked Tom22 failed to germinate. Similarly, spores that lacked Tom22 germinated but grew poorly if they expressed Tom22-
trans (fig. 5A). By contrast, spores that lacked Tom22 but expressed Tom22-Attrans germinated and had near wild-type rates of growth (fig. 5A).
|
In all cases, mitochondria isolated from yeast strains expressing Tom22-Attrans imported the precursor proteins at near wild-type rates (fig. 5B). Although it is still not known what structural features allow the trans domain to bind precursor proteins and interact with Tim50 (Chacinska et al. 2003), the function is conserved in the trans domain of AtTom22'.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tom22 and Tom7 Bind Tom40 Tightly Through Unusual Transmembrane Domains
A large number of integral membrane ß-barrel structures are now known (Buchanan 1999; Delcour 2002; Schulz 2002). In none of these bacterial outer membrane proteins is an additional transmembrane subunit known to bind tightly to the ß-barrel, which gives no precedent for how Tom7 and Tom22 interact with Tom40. It is known that the interaction is mediated through the transmembrane domains (van Wilpe et al. 1999; Allen et al. 2001), and the strikingly conserved hydrophilic residues at position +4 in the Tom7 (arginine, glutamine, or histidine, depending on the species) and at +4 and +5 in the transmembrane segment of Tom22 (serine and/or threonine residues), might complement each other through hydrogen bonding if a suitably hydrophilic patch were available on the surface of the Tom40 barrel. These mutually accommodated hydrophilic interactions within the hydrophobic plane of the bilayer could serve to prevent Tom7 and Tom22 from dissociating from Tom40.
The proline residue conserved in the transmembrane segments of Tom7 and Tom22 is particularly curious (Allen et al. 2001). Statistical analysis shows proline is rarely found in transmembrane segments (Senes, Gerstein, and Engelman 2000), but a common theme is emerging where, in a variety of transmembrane proteins, a proline residue can function as a conformational switch (Sansom and Weinstein 2000; Tieleman et al. 2001; Cordes, Bright, and Sansom 2002, Curran and Engelman 2003). Either through reversible twisting about the axis of the helix or by allowing a reversible kink in the line of the helix, proline residues can transmit changes in positioning of extracellular domains, with respect to the membrane, or provide for changes in positioning of distinct segments of the transmembrane span, with respect to neighboring proteins in the membrane. Such movements might underpin the dynamics of protein translocation and, we suggest, were a mechanistic feature of the early TOM complex.
Comparative Genomics and Functional Features of the Tom22 Family
The sequence of the transmembrane segment of Tom22' was conserved in all the organisms examined, and a small C-terminal trans domain was always present. The function of this trans domain in ScTom22 is to assist presequence-containing proteins as they emerge from the translocation channel across the outer membrane of yeast mitochondria (Lithgow et al. 1994; Bolliger et al. 1995; Moczko et al. 1996; Kanamori et al. 1999). Although yeast cells that lack the trans domain are viable, they show growth defects that are accentuated under stressful growth conditions such as on nonfermentable carbon sources and in high-temperature incubations, or in the first rounds of mitosis needed for yeast spores to resume vegetative growth (Bolliger et al. 1995; Moczko et al. 1996; Kanamori et al. 1999; this study). In maggie mutants of D. melanogaster, an allele that lacks the trans domain (Mgeb10) shows 100% lethality for the first larval instar of homozygous flies (Vaskova et al. 2000). It now appears that this trans domain is a widespread feature of the TOM complex.
The acidic receptor domain found at the amino-terminus of Tom22 is an important feature for maintaining efficient protein targeting to the mitochondrial surface in both yeast (Bolliger et al. 1995; Egan et al. 1999) and animals (Saeki et al. 2000; Yano et al. 2000). However, it currently appears that this domain is restricted to the opisthokonts, the eukaryotic lineage that includes fungi and animals, and we tentatively suggest it is a derived condition with the Tom22' form being the ancestral component in the TOM complex. Either way, it seems likely early eukaryotes had a TOM complex consisting of Tom40 with Tom7 and a member of the Tom22/Tom22' family.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allen, R., B. Egan, K. Gabriel, T. Beilharz, and T. Lithgow. 2002. A conserved proline residue is present in the transmembrane-spanning domain of Tom7 and other tail-anchored protein subunits of the TOM translocase. FEBS Lett. 514:347-350.[CrossRef][ISI][Medline]
Bains, G., and T. Lithgow. 1999. The Tom channel in the mitochondrial outer membrane: alive and kicking. Bioessays 21:1-4.[CrossRef][ISI][Medline]
Beilharz, T., B. Egan, P. A. Silver, K. Hofmann, and T. Lithgow. 2003. Bipartite signals mediate subcellular targeting of tail-anchored membrane proteins in Saccharomyces cerevisiae. J. Biol. Chem. 278:8219-8223.
Bolliger, L., T. Junne, G. Schatz, and T. Lithgow. 1995. Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria. EMBO J. 14:6318-6326.[Abstract]
Buchanan, S. K. 1999. Beta-barrel proteins from bacterial outer membranes: structure, function and refolding. Curr. Opin. Struct. Biol. 9:455-461.[CrossRef][ISI][Medline]
Casadio, R., I. Jacoboni, A. Messina, and V. De Pinto. 2002. A 3D model of the voltage-dependent anion channel (VDAC). FEBS Lett. 520:1-7.[CrossRef][ISI][Medline]
Cavalier-Smith, T. 2002. The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa. Int. J. Syst. Evol. Microbiol. 52:297-354.
Chacinska A., P. Rehling, B. Guiard, A. E. Frazier, A. Schulze-Specking, N. Pfanner, W. Voos, and C. Meisinger. 2003. Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex. EMBO J. 22:5370-5381.
Cordes, F. S., J. N. Bright, and M. S. Sansom. 2002. Proline-induced distortions of transmembrane helices. J. Mol. Biol. 323:951-960.[CrossRef][ISI][Medline]
Curran, A. R., and D. M. Engelman. 2003. Sequence motifs, polar interactions and conformational changes in helical membrane proteins. Curr. Opin. Struct. Biol. 4:412-417.[CrossRef]
Delcour, A. H. 2002. Structure and function of pore-forming beta-barrels from bacteria. J. Mol. Microbiol. Biotechnol. 4:1-10.[ISI][Medline]
Dembowski, M., K. P. Kunkele, F. E. Nargang, W. Neupert, and D. Rapaport. 2001. Assembly of Tom6 and Tom7 into the TOM core complex of Neurospora crassa. J. Biol. Chem. 276:17679-17685.
Diederichs, K., J. Freigang, and J. Breed. 1998. Prediction by a neural network of outer membrane beta-strand protein topology. Protein Sci. 7:2413-2420.
Egan, B., T. Beilharz, R. George, S. Isenmann, S. Gratzer, B. Wattenberg, and T. Lithgow. 1999. Targeting of tail-anchored proteins to yeast mitochondria in vivo. FEBS Lett. 451:243-248.[CrossRef][ISI][Medline]
Emelyanov, V. V. 2001. Evolutionary relationship of Rickettsiae and mitochondria. FEBS Lett. 501:11-18.[CrossRef][ISI][Medline]
Esaki, M., T. Kanamori, S. Nishikawa, I. Shin, P. G. Schultz, and T. Endo. 2003. Tom40 protein import channel binds to non-native proteins and prevents their aggregation. Nat. Struct. Biol. 10:988-994.[CrossRef][ISI][Medline]
Gabriel, K., S. K. Buchanan, and T. Lithgow. 2001. The alpha and the beta: protein translocation across mitochondrial and plastid outer membranes. Trends Biochem. Sci. 26:36-40.[CrossRef][ISI][Medline]
Gabriel, K., B. Egan, and T. Lithgow. 2003. Tom40, the import channel of the mitochondrial outer membrane, plays an active role in sorting imported proteins. Embo J. 22:2380-2386.
Gentle I., K. Gabriel, R. Waller, P. Beech, and T. Lithgow. 2004. The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria. J. Cell Biol. 164:19-24.
Gray, M. W., G. Burger, and B. F. Lang. 1999. Mitochondrial evolution. Science 283:1476-1481.
Gray, M. W., G. Burger, and B. F. Lang. 2001. The origin and early evolution of mitochondria. Genome Biol. 2:REVIEWS1018.[Medline]
Gromiha, M. M., and P. K. Ponnuswamy. 1993. Prediction of transmembrane beta-strands from hydrophobic characteristics of proteins. Int. J. Pept. Protein Res. 42:420-431.[ISI][Medline]
Haucke V., and T. Lithgow. 1997. The first steps of protein import into mitochondria. J. Bioenerg. Biomembr. 29:11-17.[CrossRef][ISI][Medline]
Herrmann, J. M. 2003. Converting bacteria to organelles: evolution of mitochondrial protein sorting. Trends Microbiol. 11:74-79.[CrossRef][ISI][Medline]
Hoogenraad, N. J., L. A. Ward, and M. T. Ryan. 2002. Import and assembly of proteins into mitochondria of mammalian cells. Biochim. Biophys. Acta 1592:97-105.[ISI][Medline]
Jacoboni, I., P. L. Martelli, P. Fariselli, V. De Pinto, and R. Casadio. 2001. Prediction of the transmembrane regions of beta-barrel membrane proteins with a neural network-based predictor. Protein Sci. 10:779-787.
Jänsch, L., V. Kruft, U. K. Schmitz, and H. P. Braun. 1998. Unique composition of the preprotein translocase of the outer mitochondrial membrane from plants. J. Biol. Chem. 273:17251-17257.
Johnston, A. J., J. Hoogenraad, D. A. Dougan, K. N. Truscott, M. Yano, M. Mori, N. J. Hoogenraad, and M. T. Ryan. 2002. Insertion and assembly of human Tom7 into the preprotein translocase complex of the outer mitochondrial membrane. J. Biol. Chem. 277:42197-42204.
Kanamori, T., S. Nishikawa, M. Nakai, I. Shin, P. G. Schultz, and T. Endo. 1999. Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane. Proc. Natl. Acad. Sci. USA 96:3634-3639.
Karlberg, O., B. Canback, C. G. Kurland, and S. G. Andersson. 2000. The dual origin of the yeast mitochondrial proteome. Yeast 17:170-187.[CrossRef][ISI][Medline]
Kiebler, M., P. Kiel, H. Schneider, I. van der Klei, N. Pfanner, and W. Neupert. 1993. The mitochondrial receptor complex: a central role of MOM22 in mediating preprotein transfer from receptors to the general insertion pore. Cell 74:483-492.[ISI][Medline]
Kurland, C. G., and S. G. Andersson. 2000. Origin and evolution of the mitochondrial proteome. Microbiol. Mol. Biol. Rev. 64:786-820.
Lithgow, T., T. Junne, K. Suda, S. Gratzer, and G. Schatz. 1994. The mitochondrial outer membrane protein Mas22p is essential for protein import and viability of yeast. Proc. Natl. Acad. Sci. USA 91:11973-11977.
Lucattini R, V. Likic, and T. Lithgow. 2003. Bacterial proteins predisposed for targeting to mitochondria. Mol. Biol. Evol. 21:652-658.[ISI]
Lucattini, R., V. Likic, and T. Lithgow. 2004. Bacterial proteins predisposed for targeting to mitochondria. Mol. Biol. Evol. 21:652-658.
Macasev D., E. Newbigin, J. Whelan, and T. Lithgow. 2000. How do plant mitochondria avoid importing chloroplast proteins? Components of the import apparatus Tom20 and Tom22 from Arabidopsis differ from their fungal counterparts. Plant Physiol. 123:1-6.
Martin, W., and M. Müller. 1998. The hydrogen hypothesis for the first eukaryote. Nature 392:37-44.[CrossRef][ISI][Medline]
Meisinger, C., M. T. Ryan, K. Hill, K. Model, J. H. Lim, A. Sickmann, H. Muller, H. E. Meyer, R. Wagner, and N. Pfanner. 2001. Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small Tom proteins, and import receptors. Mol. Cell. Biol. 21:2337-2348.
Moczko, M., U. Bömer, M. Kübrich, N. Zufall, A. Hönlinger, and N. Pfanner. 1997. The intermembrane space domain of mitochondrial Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences. Mol. Cell. Biol. 17:6574-6584.[Abstract]
Neupert, W. 1997. Protein import into mitochondria. Annu. Rev. Biochem. 66:863-917.[CrossRef][ISI][Medline]
Pfanner, N., and A. Chacinska. 2002. The mitochondrial import machinery: preprotein-conducting channels with binding sites for presequences. Biochim. Biophys. Acta 1592:15-24.[ISI][Medline]
Saeki, K., H. Suzuki, and M. Tsuneoka, (13 co-authors). 2000. Identification of mammalian TOM22 as a subunit of the preprotein translocase of the mitochondrial outer membrane. J. Biol. Chem. 275:31996-2002.
Sansom, M. S., and H. Weinstein. 2000. Hinges, swivels and switches: the role of prolines in signalling via transmembrane alpha-helices. Trends Pharmacol. Sci. 21:445-451.[CrossRef][ISI][Medline]
Schulz, G. E. 2002. The structure of bacterial outer membrane proteins. Biochim. Biophys. Acta 1565:308-317.[ISI][Medline]
Senes, A, M. Gerstein, and D. M. Engelman. 2000. Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions. J. Mol. Biol. 296:921-936.[CrossRef][ISI][Medline]
Tieleman, D. P., I. H. Shrivastava, M. R. Ulmschneider, and M. S. Sansom. 2001. Proline-induced hinges in transmembrane helices: possible roles in ion channel gating. Proteins 44:63-72.[CrossRef][ISI][Medline]
van Wilpe, S., M. T. Ryan, and K. Hill, (13 co-authors). 1999. Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase. Nature 401:485-489.[CrossRef][ISI][Medline]
Vaskova, M., A. M. Bentley, S. Marshall, P. Reid, C. S. Thummel, and A. J. Andres. 2000. Genetic analysis of the Drosophila 63F early puff: characterization of mutations in E63-1 and maggie, a putative Tom22. Genetics 156:229-244.
Venter, J. C., K. Remington, and J. F. Heidelberg, et al. (23 co-authors). 2004. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304:66-74.
Voos, W., H. Martin, T. Krimmer, and N. Pfanner. 1999. Mechanisms of protein translocation into mitochondria. Biochim. Biophys. Acta 1422:235-254.[ISI][Medline]
Voos, W. 2003. A new connection: chaperones meet a mitochondrial receptor. Mol. Cell 11:1-3.[ISI][Medline]
Werhahn, W., A. Niemeyer, L. Jansch, V. V. Kruft, U. K. Schmitz, and H. P. Braun. 2001. Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis: identification of multiple forms of TOM20. Plant Physiol. 125:943-954.
Yano, M., N. Hoogenraad, K. Terada, and M. Mori. 2000. Identification and functional analysis of human Tom22 for protein import into mitochondria. Mol. Cell. Biol. 20:7205-7213.