Tom22', an 8-kDa trans-Site Receptor in Plants and Protozoans, Is a Conserved Feature of the TOM Complex That Appeared Early in the Evolution of Eukaryotes

Diana Macasev*, James Whelan{dagger}, Ed Newbigin{ddagger}, Marcio C. Silva-Filho*,§, Terrence D. Mulhern* and Trevor Lithgow*

* Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Australia
{dagger} Plant Molecular Biology Group, School of Biomedical and Chemical Sciences, University of Western Australia, Crawley, Australia
{ddagger} 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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
One of the earliest events in the evolution of mitochondria was the development a means to translocate proteins made in the cytosol into the "protomitochondrion." How this was achieved remains uncertain, and the nature of the earliest version of the protein translocation machinery is not known. Comparative sequence analysis suggests three subunits, Tom40, Tom7, and Tom22 as common elements of the protein translocase in the mitochondrial outer membrane in diverse extant eukaryotes. Tom22, the 22-kDa subunit, plays a critical role in the function of this complex in fungi and animals, and we show that an 8-kDa subunit of the plant translocase is a truncated form of Tom22. It has a single transmembrane segment conforming in sequence to the same region of Tom22 from other eukaryotic lineages and a short carboxy-terminal trans domain located in the mitochondrial intermembrane space. The trans domain from the Arabidopsis thaliana protein functions in yeast lacking their own Tom22 by complementing protein import defects and restoring cell growth. Moreover, we have identified orthologs of Tom22, Tom7, and Tom40 in diverse eukaryotes such as the diatom Phaeodactylum tricornutum, the amoebic slime Dictyostelium discoideum, and the protozoan parasite Plasmodium falciparum. This finding strongly suggests these subunits as the core of the protein translocase in the earliest mitochondria.

Key Words: mitochondria • protein import • TOM complex • evolution


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
In modern eukaryotes, most mitochondrial proteins are encoded in the nucleus and synthesized in the cytosol, with defined targeting sequences that bind receptors on the mitochondrial surface that will initiate their translocation through the outer membrane via the translocase in the outer mitochondrial membrane (TOM complex). An essential translocation channel is formed from the presumed transmembrane ß-strands of Tom40 and, in fungi, its tightly bound attendant subunits: Tom6, Tom7, and Tom22 (Neupert 1997; Haucke and Lithgow 1997; Voos et al. 1999; Gabriel, Buchanan, and Lithgow 2001; Pfanner and Chacinska 2002). Yeast mitochondrial proteins are delivered to the channel by a set of receptor proteins thst include Tom20, Tom70, and Tom5 and soluble factors such as Hsp70 and MSF (Neupert 1997; Haucke and Lithgow 1997; Voos et al. 1999; Voos 2003). Homologs of these fungal proteins are encoded in the genomes of vertebrate and invertebrate animals, and functional studies done with the mammalian proteins support the view that the components of the TOM complexes from fungi and animals are conserved in structure and function (Hoogenraad, Ward, and Ryan 2002).

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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Sequence Analysis
Novel sequences were identified through iterations of Blast analysis, starting with short segments of the yeast protein sequence. Each candidate ortholog sequence was used in further rounds of Blast to confirm similarity and seek further candidates. Sequence data from the diatom Phaeodactylum tricornutum was accessed through the Diatom EST database (http://avesthagen.sznbowler.com/) and sequences from T. parva, E. tenella, and T. gondii accessed through the Sanger Centre (http://www.sanger.ac.uk/Projects/Protozoa/). Multiple sequence alignments were constructed with ClustalW. Transmembrane ß-strands were identified in Tom40 orthologs independently submitted to TBBpred (http://www.imtech.res.in/raghava/tbbpred/) and B2TMPRED (http://gpcr.biocomp.unibo.it/cgi/predictors/outer/pred_outercgi.cgi) and rationalized on a multiple sequence alignment (Casadio et al. 2002; Gentle et al. 2004). The DAS server (www.sbc.su.se/~miklos/DAS/) was used to predict transmembrane segments in Tom7 and Tom22 orthologs.

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 Tom22{Delta}trans (tom22-9) and the heterozygous yeast strain (TOM22/{Delta}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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Tom40 and Tom7 Are Widely Represented in Eukaryotes
In fungi, the approximately 40-kDa Tom40 forms a translocation pore of 20 Å through which all protein import into mitochondria occurs (Neupert 1997; Bains and Lithgow 1999; Gabriel, Buchanan, and Lithgow 2001; Pfanner and Chacinska 2002). Sequences showing homology to Tom40 were found in data derived from a range of eukaryotic species that included plants, animals, fungi, the brown alga Dunaliella salina and a related "environmental" sequence from the Sargasso Sea (accession number EAD01403 [Venter et al. 2004]), the amoebic slime Dictyostelium discoideum, as well as several species of apicomplexan parasites (fig. 1). The three-dimensional structure of Tom40 is not known, but it is widely accepted to be a membrane-embedded ß-barrel (Bains and Lithgow 1999; Buchanan 1999; Gabriel, Buchanan, and Lithgow 2001). Predictors are available to determine likely membrane-embedded ß-strands from sequence (Gromiha and Ponnuswamy 1993; Diederichs, Freigang, and Breed 1998; Jacoboni et al. 2001), and analysis of each of the Tom40 sequences discovered above predicted 16 membrane-embedded ß-strands (data not shown). These predictors are not yet reliable enough to determine a valid structural model for Tom40, but the results for the family suggests a close structural relationship that goes beyond sequence similarities (Casadio et al. 2002; Gentle et al. 2004) and verifies suggestions of an early origin for Tom40.



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FIG. 1. Tom40 has a wide spread distribution in eukaryotes. Iterative Blast was used to identify candidate Tom40 homologs from over 38 species, with representative species shown (Pf Tom40, P. falciparum; TpTom40, T. parva; TgTom40, Toxoplasma gondii; NcTom40, Neospora caninum, HaTom40, H. anthus; LeTom40, L. esculentum; AtTom40.1 and AtTom40.2, A. thaliana; OsTom40, Oryza sativa; PpTom40, P. patens; DsTom40, D. salina; DdTom40, D. discoideum; ScTom40, S. cerevisiae; EgTom40, Ashbya gossypii; SpTom40, Schizosaccharomyces pombe; DmTom40, D. melanogaster; AgTom40, Anopheles gambiae; BmTom40, Bombyx mori; CiTom40, Ciona intestinalis; XlTom40, Xenopus laevis; DrTom40, Dano rario; GgTom40, Gallus gallus; HsTom40, H. sapiens). Each sequence was independently analyzed for putative transmembrane ß-strands (shaded gray) and compiled into a multiple alignment with ClustalW. The region of the alignment corresponding to predicted strands 9, 10, and 11 is shown

 
In yeast, Tom7 is directly and tightly associated with the Tom40 barrel (Meisinger et al. 2001). A homolog of Tom7 has been identified in humans (Johnston et al. 2002), in a filamentous fungus (Dembowski et al. 2001), and in potato (Jänsch et al. 1998). With only a few, diverse Tom7 orthologs known, the sequence differences left uncertainty in how closely related the various Tom7s might be (Jänsch et al. 1998; Macasev et al. 2000). With such a small open reading frame, it remains difficult to comprehensively screen sequence data, and although no homologous sequence was detected in the Plasmodium falciparum genome, our iterative analyses eventually found Tom7 orthologs in sufficient species (i.e., the moss P. patens, the alga C. reinhardtii, the diatom Phaeodactylum tricornutum, and the amoebic slime D. discoideum) to suggest a broad presence in eukaryotes (fig. 2). The proteins have in common a predicted transmembrane segment of 16 residues that includes precise placement of several atypical residues: a polar/positive residue at +4 relative to the predicted cytosol-membrane interface, aromatics at +5 and +7, glycines at +6 and +15, and a proline at +9. The function of these unusual residues and the effect they might have on the structure of the transmembrane segment remain to be determined, but the precise placement of so many atypical residues within the transmembrane segment argues strongly that they represent homologs.



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FIG. 2. Tom7, like Tom40, is a fundamental component of the TOM complex. Iterative Blast analysis and ClustalW were used to assemble this collection of Tom7 family members from cDNA collections of diverse eukaryotes. The topology has been experimentally determined for the proteins from yeast (Allen et al. 2000) and human (Johnston et al. 2003). Alignment of the various Tom7 sequences shows the conserved transmembrane segment (shaded), which includes the sequence motif R/H/Q-W-G-F-X-P-X5-G (darker shading)

 
Tom22 Family Members Come in Short or Long Forms
Tom22 orthologs have been characterized in humans, flies, and two species of fungus (Kiebler et al. 1993; Lithgow et al. 1994; Saeki et al. 2000; Vaskova et al. 2000; Yano et al. 2000). Early work on the TOM complex from plants showed that a 22-kDa subunit was not present (Jänsch et al. 1998; Werhahn et al. 2001), although sequence similarity was noted between an 8-kDa subunit and Tom22 (Macasev et al. 2001). Iterative Blast analysis with partial Tom22 sequences from HsTom22 and ScTom22 revealed related sequences in the genomes of all members of the green plant lineage for which substantial sequence data are available and related sequences in the diatom P. tricornutum and in the apicomplexan parasites P. falciparum, E. tenella, and T. parva. Multiple sequence alignment (fig. 3) shows the conserved nature of the approximately 8-kDa protein from these various organisms: a short, basic N-terminal domain (net charge ranges from +4 to +9), a single segment predicted to form an {alpha}-helical transmembrane domain and a short C-terminal domain rich in Glu and Gln. The sequences from S. tuberosum and A. thaliana correspond to those determined biochemically as 8-kDa subunits of the TOM complex (Jänsch et al. 1998; Werhahn et al. 2001). We refer to the truncated form of Tom22 as Tom22'.



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FIG. 3. Tom22 and Tom22': conservation of protein sequence from animals, fungi, and plants. Iterative Blast analysis was used to assemble the sequences shown in the ClustalW alignment. A single predicted transmembrane segment was determined with the DAS algorithm (boxed). Including are highly conserved tryptophan, hydroxylated, and proline residues (gray). In the extramembrane domains, acidic (red) and basic (blue) residues are shown. Asterisks indicate incomplete cDNA sequences

 
These sequences would encode proteins substantially shorter than the animal and fungal Tom22, lacking the acidic, amino-terminal domain (fig. 3). However, several of the sequence characteristics of the transmembrane segment of Tom22 are conserved in the Tom22' proteins (fig. 3, boxed). The gray bars in figure 3 denote the absolutely conserved Pro and Trp residues (Allen et al. 2002) and the two to three consecutive hydroxylated residues that sit downstream in the transmembrane segment. Also striking at the distal end of each transmembrane segment is a Glu residue, of functional importance because in Drosophila melanogaster changes at this residue result in significantly slower rates of growth and development (Vaskova et al. 2000).

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).



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FIG. 4. The chimeric protein Tom22-Attrans is stably expressed in the mitochondrial outer membrane of yeast. (A) Domain structure of ScTom22, AtTom22', and Tom22-Attrans. TM designates the central transmembrane segment. (B) Mitochondria (75-µg protein) were isolated from yeast cells that expressed ScTom22 or Tom22-Attrans and were analyzed by SDS-PAGE and immunoblotting with antibodies that recognized the GFP epitope in the Tom22 fusion proteins or the ß-subunit of the F1F0-ATP synthetase (F1ß). (C) Yeast cells that expressed Tom22-Attrans as a GFP fusion were costained with Mitotracker Red and examined by fluorescence microscopy. (D) Mitochondria were isolated from yeast cells that expressed Tom22-Attrans and extracted with sodium carbonate (pH 11.5). After centrifugation, the membrane fraction P and supernatant S were analyzed by SDS-PAGE and immunoblotting with antibodies to the outer membrane proteins porin and Tom22, the intermembrane space protein cytochrome b2, and the peripheral inner membrane protein F1ß. (E) Mitochondria were isolated from yeast cells that expressed Tom22-Attrans and incubated with the indicated amount of trypsin. After addition of phenylmethylsulfonyfluoride, mitochondrial samples (100-µg protein) were analyzed by SDS-PAGE and immunoblotting

 
Mitochondria isolated from heterozygous diploid yeast cells expressing Tom22-Attrans were subjected to treatment with sodium carbonate at pH 11.5, and the resulting membrane fragments were reisolated. Immunoblots showed that Tom22-Attrans was an integral membrane protein, as it remained in the extracted membranes with the other integral proteins Tom20 and porin. The peripheral proteins F1ß and cytochrome b2 were recovered in the extracts (fig. 4D). When isolated mitochondria were treated with trypsin, the surface exposed protein Tom20 was degraded, but the mitochondrial membranes protected proteins in the intermembrane space (cytochrome b2) and matrix (F1ß) (fig. 4E). The N-terminal GFP epitope of Tom22-Attrans was on the mitochondrial surface, as predicted by the topology of ScTom22 (Lithgow et al. 1994).

When heterozygous TOM22/{Delta}tom22 diploid yeast cells are sporulated, the two spores lacking Tom22 fail to germinate (Lithgow et al. 1994). Heterozygous TOM22/{Delta}tom22 diploid yeast cells that expressed Tom22, Tom22-{Delta}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-{Delta}trans (fig. 5A). By contrast, spores that lacked Tom22 but expressed Tom22-Attrans germinated and had near wild-type rates of growth (fig. 5A).



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FIG. 5. AtTom22' is functionally equivalent to Tom22. (A) Heterozygous (TOM22/{Delta}tom22) yeast cells were transformed with plasmids that encoded ScTom22 or Tom22-{Delta}trans or Tom22-Attrans, sporulated, and the haploid progeny dissected onto YPAD plates. The colony size after 2 to 3 days growth is an indication of initial growth rates. (B) Mitochondria were isolated from haploid {Delta}tom22 yeast cells that expressed ScTom22 or Tom22-{Delta}trans or Tom22-Attrans. Precursor forms (p) of F1ß or cytochrome b2 or Su9-DHFR were translated in vitro in the presence of 35S-methionine, and incubated with mitochondria (50-µg protein) for the indicated times before analysis by SDS-PAGE. Fluorography reveals the accumulation of the processed forms of each protein after import into mitochondria

 
The trans domain of Tom22 binds mitochondrial targeting sequences on precursors to enhance their transfer to the Tim50 and Tim23 subunits of the TIM23 complex (Bolliger et al. 1995; Moczko et al. 1997; Kanamori et al. 1999). This transfer includes precursor proteins destined for translocation through TIM23 to the mitochondrial matrix and those that will be processed at TIM23 for release into the intermembrane space. When mitochondria isolated from haploid yeast cells that expressed Tom22, Tom22-{Delta}trans, or Tom22-Attrans were tested with 35S-labeled precursor proteins (fig. 5B), mitochondria from which the trans domain of Tom22 had been deleted (Tom22-{Delta}trans) imported precursors of the ß-subunit of the F1F0-ATP synthetase and a reporter protein (Su9-DHFR) into the matrix at rates that were threefold to fivefold slower than those of wild-type (Tom22) yeast. This finding is consistent with earlier results (Bolliger et al. 1995; Moczko et al. 1997). Import of the intermembrane space protein cytochrome b2 was also affected; mutants imported the precursor at half the rate seen with wild-type mitochondria (fig. 5B).

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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
A Primitive TOM Complex in Early Eukaryotes
A crucial step in the evolution of the endosymbiotic bacterium, or "protomitochondria," which gave rise to mitochondria was providing a means for substrate protein transport across the outer membrane (Herrmann 2003; Karlberg et al. 2000; Lucattini et al. 2004). A recent model of these early steps posits the presence of a ß-barrel protein in the bacterial outer membrane that could bind, if weakly, these substrate proteins (Lucattini et al. 2004). Our sequence analysis finds the widespread distribution of the ß-barrel Tom40 in eukaryotes, but we have not established any relationship to bacterial proteins that might tell more of the ancestry of Tom40. In the course of adaptive change to facilitate binding of substrate proteins and their intramitochondrial sorting (Gabriel et al. 2003; Esaki et al. 2003), Tom40 appears to have diverged too far in sequence characteristics from any of the ß-barrel proteins found in extant bacterial species. Given its essential structural and functional role in providing the translocation channel through the outer membrane, it is reasonable to assume Tom40 as the earliest component of the protein translocase. We suggest that Tom7 and Tom22 were also derived early in the course of eukaryote evolution.

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
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank Ross Waller, Kip Gabriel, and Adrienne Clarke for critical comments on the manuscript, and we are grateful to Chris Bowler for access to the Diatom EST database. This work was supported by grants from the Australian Research Council (to T.L., T.D.M., and J.W.), a research fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil, (to M.C.S.F.) and a Melbourne Research Scholarship (to D.M.).


    Footnotes
 
Geoffrey McFadden, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 

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Accepted for publication May 11, 2004.