Identification of Ψ3Tom20, a novel processed pseudogene of the human Tom20 gene, and complete characterization of Ψ1Tom20 and Ψ2Tom20

We report the identification and characterization of Ψ3Tom20, a novel processed pseudogene of the human Tom20 (hTom20) gene, which is 96.2% similarity with the hTom20 cDNA and is 5′ and 3′ truncated. In addition, we present the complete characterization of Ψ1Tom20 and Ψ2Tom20, the two other recently reported members of this pseudogene family. Comparison of the sequences of Ψ3Tom20 with that of the previously reported Ψ2Tom20 revealed and corrected an error in the previously determined sequence of Ψ2Tom20. A detailed analysis of these three pseudogenes, including their flanking regions, is presented. It suggests they probably arose from mRNAs that were polyadenylated at different sites. Possible mechanisms involved in their integration as retroposons are also discussed. Received: 29 October 1998 / Accepted: 7 May 1999     

Identification of Tom5 and Tom6 in the preprotein translocase complex of human mitochondrial outer membrane

The fungal preprotein translocase of the mitochondrial outer membrane (TOM complex) comprises import receptors Tom70, Tom20, and Tom22, import channel Tom40, and small Tom proteins Tom5, Tom6, and Tom7, which regulate TOM complex assembly. These components are conserved in mammals; unlike the other components, however, Tom5 and Tom6 remain unidentified in mammals. We immuno-isolated the TOM complex from HeLa cells expressing hTom22-FLAG and identified the human counterparts of Tom5 and Tom6, together with the other components including Tom7. These small Tom proteins are associated with Tom40 in the TOM complex. Knockdown of Tom7, but not Tom5 and Tom6, strongly compromised stability of the TOM complex. Conversely, knockdown of hTom40 decreased the level of all small Tom proteins. Matrix import of preprotein was affected by double knockdown of any combination of small Tom proteins. These results indicate that human small Tom proteins maintain the structural integrity of the TOM complex.     

Tom34 unlike Tom20 does not interact with the leader sequences of mitochondrial precursor proteins

Tom20 and Tom34 are mammalian liver proteins previously identified by others to be components of the mitochondrial import translocation apparatus. It has been shown that Tom20 interacts with the leader sequence of nuclear coded matrix space precursor proteins. Here we show with recombinantly expressed Tom proteins that Tom34 binds the mature portion of the precursor and not the leader. Both these Tom proteins inhibited the import of newly translated precursor of aldehyde dehydrogenase in an in vitro assay. Only Tom20 inhibited the import of a fusion protein of the leader of aldehyde dehydrogenase attached to dihydrofolate reductase. Antibodies against Tom20 coprecipitated both the precursor of aldehyde dehydrogenase (pALDH) and of dihydrofolate reductase (pA-DHFR). Antibodies against Tom34 interacted only when the mature portion of aldehyde dehydrogenase was present. Similar import inhibition patterns were found when other precursor and chimeric constructs we investigated. When Tom34-green fluorescence protein was transfected to HeLa cells it was observed that Tom34 was found through out the cell. It is concluded from our observation that Tom34 is a cytosolic protein, whose role appeared to be to interact with mature portion of some preproteins and may keep them in an unfolded, import compatible state.     

Binding of mitochondrial leader sequences to Tom20 assessed using a bacterial two-hybrid system shows that hydrophobic interactions are essential and that some mutated leaders that do not bind Tom20 can still be imported

Previous studies pointed to the importance of leucine residues in the binding of mitochondrial leader sequences to Tom20, an outer membrane protein translocator that initially binds the leader during import. A bacteria two-hybrid assay was here employed to determine if this could be an alternative way to investigate the binding of leader to the receptor. Leucine to alanine and arginine to glutamine mutations were made in the leader sequence from rat liver aldehyde dehydrogenase (pALDH). The leucine residues in the C-terminal of pALDH leader were found to be essential for TOM20 binding. The hydrophobic residues of another mitochondrial leader F1beta-ATPase that were important for Tom20 binding were found at the C-terminus of the leader. In contrast, it was the leucines in the N-terminus of the leader of ornithine transcarbamylase that were essential for binding. Modeling the peptides to the structure of Tom20 showed that the hydrophobic residues from the three proteins could all fit into the hydrophobic binding pocket. The mutants of pALDH that did not bind to Tom20 were still imported in vivo in transformed HeLa cells or in vitro into isolated mitochondria. In contrast, the mutant from pOTC was imported less well ( approximately 50%) while the mutant from F1beta-ATPase was not imported to any measurable extent. Binding to Tom20 might not be a prerequisite for import; however, it also is possible that import can occur even if binding to a receptor component is poor, so long as the leader binds tightly to another component of the translocator.     

Computational investigation of the conformational dynamics in Tom20-mitochondrial presequence tethered complexes

The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes.     

Peptide library approach with a disulfide tether to refine the Tom20 recognition motif in mitochondrial presequences

Many mitochondrial matrix and inner-membrane proteins are synthesized in the cytosol as precursor proteins with an N-terminal presequence, and are imported into the mitochondria. Although no distinct sequence homology has been found among mitochondrial presequences, Tom20, a general import receptor in the outer mitohcondrial membrane, binds to presequences, and distinguishes mitochondrial proteins from non-mitochonrial proteins. The recently determined structure of the cytosolic domain of Tom20 (DeltaTom20) in a complex with the presequence of rat aldehyde dehydrogenase (ALDH) showed that a short stretch of the presequence forms an amphiphilic helix, and its hydrophobic surface interacts with the hydrophobic-binding groove of Tom20. The following NMR analyses revealed a common five-residue pattern for Tom20 binding in five different presequences. To refine the common amino acid motif for the recognition by Tom20, we introduced a new peptide library approach in this study: we prepared a mixture of ALDH presequence variants, tethered these peptides to DeltaTom20 in a competitive manner by an intermolecular disulfide bond, and determined the relative affinities by MALDI-TOF mass spectrometry. We successfully deduced a refined, common motif for the recognition by Tom20, and found that the segment consisting of residues 14-20 of the ALDH presequence was locally optimized in the sequence space, with respect to Tom20 binding.     

Gene structure of the human mitochondrial outer membrane receptor Tom20 and evolutionary study of its family of processed pseudogenes.

The structure of the human gene encoding the mitochondrial outer membrane receptor Tom20 has been determined from overlapping clones obtained using PCR-based techniques. The 20kb human Tom20 gene (hTom20) consists of five exons separated by four introns. The 5' flanking region presents features common with other nuclear genes encoding mitochondrial proteins. Comparison with its homologs and putative homologs in other species has revealed common features in their TPR motifs and other relevant protein domains. Aspects concerning evolutionary origins of the family of processed pseudogenes of hTom20 are also discussed.     

pANT: a method for the pairwise assessment of nonfunctionalization times of processed pseudogenes

We present a method for pairwise Assessment of Nonfunctionalization Times (pANT) in processed pseudogenes. Contrary to existing methods for estimating nonfunctionalization times, pANT utilizes previously calculated probabilities of nucleotide substitution as explicit rate measurements, rather than assume that the substitution rates are the same for all nucleotides. Thus, the method allows a more accurate computation of the time that has elapsed since the nonfunctionalization of a pseudogene. Whereas existing methods require the sequence of an orthologous functional gene, which is not always at hand, pANT only uses the pairwise alignment of the gene/pseudogene pair, thus expanding the range of problems that can be tackled. To estimate evolutionary times in nonfunctional sequences, pANT measures the differences in the pairwise alignment of a gene and its paralogous processed pseudogene, using only the first and second codon positions. It assumes that, because of functional constraints, these positions in the sequence of the functional homolog have not changed since the time of nonfunctionalization of the pseudogene. Hence, the sequence of the gene may be used as the ancestor of the pseudogene. We show that the method's reliance on a detailed substitution matrix, which is derived separately for each species, makes it more accurate than existing methods. We applied pANT to the case of the unitary alpha-1,3-galactosyltransferase human pseudogene and found that our estimate of the nonfunctionalization time was in agreement with that obtained by taxonomic and paleontological considerations pertaining to the divergence between platyrrhines (New World monkeys) and cattarhines (Old World monkeys).     

Characterization of the human cyclophilin gene and of related processed pseudogenes

The human cyclophilin gene was isolated from a genomic library derived from leucocyte DNA and sequenced. The gene contains five exons and four introns. The amino acid sequence deduced from the exons matches perfectly the one previously determined from the T-cell cyclophilin cDNA. A TATA box is visible in the promoter region and putative Sp1 binding sites are also found there as well as in the first intron. Six members of the middle repetitive Alu gene family are present in one or other orientation in the non-coding regions of the cyclophilin gene. Hybridisation of genomic DNA to probes derived from the promoter region or the first intron indicates that the cyclophilin gene is present as a single copy in the human haploid genome. Seven other cyclophilin-related DNA clones isolated from the same library were also characterized. They show a high degree of similarity to the cyclophilin cDNA and are colinear to it. However, multiple genetic lesions, often including deletion and/or insertion events which modify the reading frame, are found in these clones which are therefore likely to represent processed pseudogenes.     

Identification of a mammalian homologue of the fungal Tom70 mitochondrial precursor protein import receptor as a thyroid hormone-regulated gene in specific brain regions

Thyroid hormone is an important regulator of mammalian brain maturation. By differential display PCR, we isolated a cDNA clone (S2) that is specifically up-regulated in the striatum of neonatal hypothyroid rats. S2 was identified as KIAA0719, the first human gene distantly homologous to the fungal Tom70, which encodes a member of the translocase mitochondrial outer membrane complex involved in the import of preproteins into the mitochondria. By northern and in situ hybridization studies, KIAA0719 was found to be up-regulated in the striatum, nucleus accumbens, and discrete cortical layers of 15-day-old hypothyroid rats. In contrast, lower expression was found in the olfactory tubercle, whereas no differences were detected in other brain regions. Significantly, treatment of hypothyroid animals with single injections of thyroxine restored the normal levels of KIAA0719 expression. Moreover, treatment of control animals with thyroxine led to a reduced expression, demonstrating a negative hormonal regulation in vivo. Thus, KIAA0719 gene expression is regulated by thyroid hormone in the neonatal rat brain in a region-specific fashion. Given the role of the homologous Tom70 gene, the alteration of KIAA0719 expression may contribute to the changes in mitochondrial morphology and physiology caused by hypothyroidism in the developing rat brain.     

Characterization of the bovine BCL2L1 gene and related pseudogenes

We have amplified and characterized partial regions of exons 2 and 3 of the bovine BCL2L1 gene, one of the anti-apoptotic members of the B-cell lymphoma 2 gene family. Cloning and sequencing of the amplified products revealed the existence of several BCL2L1-related sequences, including the bovine BCL2L1 gene and various processed pseudogenes. The bovine BCL2L1 gene revealed two polymorphic nucleotide sequences that resulted in two protein variants, with amino acid replacements at positions 60 and 69. In addition, we report three bovine BCL2L1-related sequences (BCL2L1psi) that probably correspond to intronless processed pseudogenes. These BCL2L1psi pseudogene sequences have accumulated multiple substitutions, deletions and insertions that translated into stop codons or changed the open reading frame of the functional gene. We provide evidence suggesting that the retro-transposition event that originated these processed pseudogenes took place before the divergence of the Cervidae and Bovidae families.     

Interaction between the human mitochondrial import receptors Tom20 and Tom70 in vitro suggests a chaperone displacement mechanism

The mitochondrial import receptor Tom70 contains a tetratricopeptide repeat (TPR) clamp domain, which allows the receptor to interact with the molecular chaperones, Hsc70/Hsp70 and Hsp90. Preprotein recognition by Tom70, a critical step to initiate import, is dependent on these cytosolic chaperones. Preproteins are subsequently released from the receptor for translocation across the outer membrane, yet the mechanism of this step is unknown. Here, we report that Tom20 interacts with the TPR clamp domain of Tom70 via a conserved C-terminal DDVE motif. This interaction was observed by cross-linking endogenous proteins on the outer membrane of mitochondria from HeLa cells and in co-precipitation and NMR titrations with purified proteins. Upon mutation of the TPR clamp domain or deletion of the DDVE motif, the interaction was impaired. In co-precipitation experiments, the Tom20-Tom70 interaction was inhibited by C-terminal peptides from Tom20, as well as from Hsc70 and Hsp90. The Hsp90-Tom70 interaction was measured with surface plasmon resonance, and the same peptides inhibited the interaction. Thus, Tom20 competes with the chaperones for Tom70 binding. Interestingly, antibody blocking of Tom20 did not increase the efficiency of Tom70-dependent preprotein import; instead, it impaired the Tom70 import pathway in addition to the Tom20 pathway. The functional interaction between Tom20 and Tom70 may be required at a later step of the Tom70-mediated import, after chaperone docking. We suggest a novel model in which Tom20 binds Tom70 to facilitate preprotein release from the chaperones by competition.     

Characterization of the functional gene and several processed pseudogenes in the human triosephosphate isomerase gene family.

The functional gene and three intronless pseudogenes for human triosephosphate isomerase were isolated from a recombinant DNA library and characterized in detail. The functional gene spans 3.5 kilobase pairs and is split into seven exons. Its promoter contains putative TATA and CCAAT boxes and is extremely rich in G and C residues (76%). The pseudogenes share a high degree of homology with the functional gene but contain mutations that preclude the synthesis of an active triosephosphate isomerase enzyme. Sequence divergence calculations indicate that these pseudogenes arose approximately 18 million years ago. We present evidence that there is a single functional gene in the human triosephosphate isomerase gene family.     

Identification of two active functional domains of human adenylate kinase 5

A full length cDNA that partially corresponded to human adenylate kinase 5 (AK5) was identified and shown to encode for two separate domains. The full length protein could be divided in two distinct functional domains, a previously unidentified domain of 338 amino acids and a second domain of 198 amino acids that corresponded to the protein characterized as AK5, now called AK5p2. The first domain, AK5p1, phosphorylated AMP, CMP, dAMP and dCMP with ATP or GTP as phosphate donors similarly to AK5p2. Our data demonstrate that human AK5 has two separate functional domains and that both have enzymatic activity.     

Insights into the genomic features and evolutionary impact of the genes configuring duplicated pseudogenes in human

Pseudogenes, regarded as ‘genomic fossils’, are DNA sequences resembling functional genes in perspective of sequence homology but completely non-functional. In this study, we explored the unique characteristic features of human genes, configuring classical duplicated pseudogenes. We found that progenitors of duplicated pseudogenes are characterized by a high expressivity, and ability to encode hub-proteins in association with a high evolutionary rate. Such unusual features are endorsed by longer protein length, elevated CpG content, and a high recombination rate. The non-functionalization of their duplicated copies can be attributed to the overabundance of gene paralog number in concert with functional redundancy.     

Tom1L1在细胞信号转导及受体内吞中的研究进展

Tom1L1（Tom1 like 1）参与并调节细胞信号转导及受体运输通路。在不同细胞中Tom1L1对信号转导具有不同的调节作用。Tom1L1-CHC（clathrinheavychain）复合物减少Src蛋白在小窝（caveolae）处富集,从而阻碍Src蛋白与血小板衍生因子（platelet derived growth factor,PDGF）受体的结合。抑制PDGF受体介导的有丝分裂和转化信号传导。活化的表皮生长因子受体（epidermal growth factor receptor,EGFR）通过Src家族蛋白激酶（src family kinase,SFK）磷酸化T0m1L1,磷酸化的Tom1L1通过Grb2和Shc的桥梁作用与EGFR结合,介导EGFR的内吞进程。Tom1L1和Hrs（hepatocyte growth factor regulated tyrosine kinase substrate）、TSG101（tumor susceptibility gene 101）的相互作用表明,它也可能参与了泛素化蛋白分选入多泡体的过程。该文就其在细胞信号转导通路及受体内吞／分选过程的作用作一综述。     

Nucleotide sequence of a cDNA for the dihydrolipoamide acetyltransferase component of human pyruvate dehydrogenase complex

Deoxynucleotide sequencing of a cDNA for the dihydrolipoamide acetyltransferase (PDC-E₂) component of human pyruvate dehydrogenase complex (PDC) revealed an open reading frame of 1848 base pairs corresponding to a leader sequence of 54 amino acids and a mature protein of 561 amino acids (59 551 Da). Both an amino-terminal lipoyl-bearing domain and a carboxy-terminal catalytic domain are present in the deduced amino acid sequence. The lipoyl-bearing domain contains two repeating units of 127 amino acids, each harboring one lipoic acid-binding lysine. Thus, mammalian PDC-E₂ differs as to the number of lipoic acid-binding sites from other dihydrolipoamide acyltransferases in both prokaryotic and eukaryotic organisms.     

Yeast two-hybrid screening identifies binding partners of human Tom34 that have ATPase activity and form a complex with Tom34 in the cytosol

In the accompany paper (Mukhopadhyay, A., Avramova, L. V. and Weiner, H., Arch. Biochem. Biophys.), it was shown that Tom34, a previously proposed putative translocase of the mitochondrial outer membrane, binds to the mature region of a precursor protein and appears to be a cytosol protein. Here Tom34 was used as bait in a yeast two-hybrid screening to search for its potential binding partners. Two of the identified proteins were the ATPase-related valosin-containing protein (VCP) and the lysosomal H(+)-transporting ATPase member M (ATP6M). Tom34 was found primarily in the cytosol while VCP and ATP6M were found in the cytosol as well as in nonmitochondrial organelles. Tom34 formed a approximately 400-kDa complex with them in the cytosol. Tom34 was found to possess a weak ATPase activity that did not change when associated with VCP. The tetratricopeptide repeat (TPR) motif region of Tom34 (residue 201-256) was responsible for binding to the other proteins. Tom34 appears not to be a member of the mitochondrial outer membrane translocase family but might function as a chaperone-like protein during protein translocation.