Tom20 and Tom22 share the common signal recognition pathway in mitochondrial protein import

Precise targeting of mitochondrial precursor proteins to mitochondria requires receptor functions of Tom20, Tom22, and Tom70 on the mitochondrial surface. Tom20 is a major import receptor that recognizes preferentially mitochondrial presequences, and Tom70 is a specialized receptor that recognizes presequence-less inner membrane proteins. The cytosolic domain of Tom22 appears to function as a receptor in cooperation with Tom20, but how its substrate specificity differs from that of Tom20 remains unclear. To reveal possible differences in substrate specificities between Tom20 and Tom22, if any, we deleted the receptor domain of Tom20 or Tom22 in mitochondria in vitro by introducing cleavage sites for a tobacco etch virus protease between the receptor domains and transmembrane segments of Tom20 and Tom22. Then mitochondria without the receptor domain of Tom20 or Tom22 were analyzed for their abilities to import various mitochondrial precursor proteins targeted to different mitochondrial subcompartments in vitro. The effects of deletion of the receptor domains on the import of different mitochondrial proteins for different import pathways were quite similar between Tom20 and Tom22. Therefore Tom20 and Tom22 are apparently involved in the same step or sequential steps along the same pathway of targeting signal recognition in import.     

Both metaxin and Tom20 together with two mitochondria-specific motifs support mitochondrial targeting of dual-targeting AtSufE1

Plant cells have two endosymbiotic organelles, chloroplasts, and mitochondria. These organelles perform specific functions that depend on organelle-specific proteins. The majority of chloroplast and mitochondrial proteins are specifically imported by the transit peptide and presequence, respectively. However, a significant number of proteins are also dually targeted to these two organelles. Currently, it is not fully understood how proteins are dually targeted to both chloroplasts and mitochondria. In this study, the mechanism underlying mitochondrial targeting of dual targeting AtSufE1 in Arabidopsis was elucidated. The N-terminal fragment containing 80 residues of AtSufE1 (AtSufE1N80) was sufficient to confer dual targeting of reporter protein, AtSufE1N80:GFP, in protoplasts. Two sequence motifs, two arginine residues at 15^th and 21^st positions, and amino acid (aa) sequence motif AKTLLLRPLK from the 31^st to 40^th aa position, were responsible for targeting to mitochondria a portion of reporter proteins amid the chloroplast targeting. The sequence motif PSEVPFRRT from the 41^st to 50^th aa position constitutes a common motif for targeting to both chloroplasts and mitochondria. For mitochondrial import of AtSufE1:N80, Metaxin played a critical role. In addition, BiFC and protein pull-down experiments showed that AtSufE1N80 specifically interacts with import receptors, Metaxin and Tom20. The interaction of AtSufE1N80 with Metaxin was required for the interaction with Tom20. Based on these results, we propose that mitochondrial targeting of dual-targeting AtSufE1 is mediated by both mitochondria-specific and common sequence motifs in the signal sequence through the interaction with import receptors, Metaxin and Tom20, in a successive manner.     

NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis?thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak β-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. DATABASE: The backbone chemical shift assignments for ThrRS-dTP(2-60) have been deposited with the Biological Magnetic Resonance Bank (BMRB) under the accession code 18248 STRUCTURED DIGITAL ABSTRACT: ThrRS-dTP?and?Tom20-4?bind?by?nuclear magnetic resonance?(View interaction).     

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.     

Tom70 Is Essential for PINK1 Import into Mitochondria

PTEN induced kinase 1 (PINK1) is a serine/threonine kinase in the outer membrane of mitochondria (OMM), and known as a responsible gene of Parkinson''s disease (PD). The precursor of PINK1 is synthesized in the cytosol and then imported into the mitochondria via the translocase of the OMM (TOM) complex. However, a large part of PINK1 import mechanism remains unclear. In this study, we examined using cell-free system the mechanism by which PINK1 is targeted to and assembled into mitochondria. Surprisingly, the main component of the import channel, Tom40 was not necessary for PINK1 import. Furthermore, we revealed that the import receptor Tom70 is essential for PINK1 import. In addition, we observed that although PINK1 has predicted mitochondrial targeting signal, it was not processed by the mitochondrial processing peptidase. Thus, our results suggest that PINK1 is imported into mitochondria by a unique pathway that is independent of the TOM core complex but crucially depends on the import receptor Tom70.     

Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70

The role of cytosolic factors in protein targeting to mitochondria is poorly understood. Here, we show that in mammals, the cytosolic chaperones Hsp90 and Hsp70 dock onto a specialized TPR domain in the import receptor Tom70 at the outer mitochondrial membrane. This interaction serves to deliver a set of preproteins to the receptor for subsequent membrane translocation dependent on the Hsp90 ATPase. Disruption of the chaperone/Tom70 recognition inhibits the import of these preproteins into mitochondria. In yeast, Hsp70 rather than Hsp90 is used in import, and Hsp70 docking is required for the formation of a productive preprotein/Tom70 complex. We outline a novel mechanism in which chaperones are recruited for a specific targeting event by a membrane-bound receptor.     

Tom34: a cytosolic cochaperone of the Hsp90/Hsp70 protein complex involved in mitochondrial protein import

Most mitochondrial membrane proteins are synthesized in the cytosol and must be delivered to the organelle in an unfolded, import competent form. In mammalian cells, the cytosolic chaperones Hsp90 and Hsp70 are part of a large cytosolic complex that deliver the membrane protein to the mitochondrion by docking with the import receptor Tom70. These two abundant chaperones have other functions in the cell suggesting that the specificity for the targeting of mitochondrial proteins requires the addition of specific factors within the targeting complex. We identify Tom34 as a cochaperone of Hsp70/Hsp90 in mitochondrial protein import. We show that Tom34 is an integral component with Hsp70 and Hsp90 in the large complex. We also demonstrate the role of Tom34 in the mitochondrial import process, as the addition of an excess of Tom34 prevents efficient mitochondrial translocation of precursor proteins that have requirements for Hsp70/Hsp90. Tom34 exhibits an affinity for mitochondrial preproteins of the Tom70 translocation pathway as demonstrated by binding assays using in vitro translated proteins as baits. In addition, we examined the specificity and the size of different complex cytosolic machines. Separation of different radiolabeled cell-free translated proteins on Native-PAGE showed the presence of a high molecular weight complex which binds hydrophobic proteins. Importantly we show that the formation of the chaperone cytosolic complex that mediates the targeting of proteins to the mitochondria contains Tom34 and assembles in the presence of a fully translated substrate protein.     

The mitochondrial import receptor Tom70: identification of a 25 kDa core domain with a specific binding site for preproteins

The mitochondrial import receptor of 70 kDa, Tom70, preferentially recognizes precursors of membrane proteins with internal targeting signals. We report the identification of a stably folded 25 kDa core domain located in the middle portion of Tom70 that contains two of the seven tetratricopeptide repeat motifs of the receptor. The core domain binds non-cleavable and cleavable preproteins carrying internal targeting signals with a specificity indistinguishable from the full-length receptor. Competition studies indicate that both types of preproteins interact with overlapping binding sites of the core domain and that at least one additional interaction site is present in the full-length receptor. We suggest a model of Tom70 function in import of membrane proteins whereby a hydrophobic preprotein concomitantly interacts with several binding sites of the receptor.     

Mitochondrial protein import: recognition of internal import signals of BCS1 by the TOM complex

BCS1, a component of the inner membrane of mitochondria, belongs to the group of proteins with internal, noncleavable import signals. Import and intramitochondrial sorting of BCS1 are encoded in the N-terminal 126 amino acid residues. Three sequence elements were identified in this region, namely, the transmembrane domain (amino acid residues 51 to 68), a presequence type helix (residues 69 to 83), and an import auxiliary region (residues 84 to 126). The transmembrane domain is not required for stable binding to the TOM complex. The Tom receptors (Tom70, Tom22 and Tom20), as determined by peptide scan analysis, interact with the presequence-like helix, yet the highest binding was to the third sequence element. We propose that the initial recognition of BCS1 precursor at the surface of the organelle mainly depends on the auxiliary region and does not require the transmembrane domain. This essential region represents a novel type of signal with targeting and sorting functions. It is recognized by all three known mitochondrial import receptors, demonstrating their capacity to decode various targeting signals. We suggest that the BCS1 precursor crosses the TOM complex as a loop structure and that once the precursor emerges from the TOM complex, all three structural elements are essential for the intramitochondrial sorting to the inner membrane.     

The intermembrane space domain of mitochondrial Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences.

Mitochondrial protein import is thought to involve the sequential interaction of preproteins with binding sites on cis and trans sides of the membranes. For translocation across the outer membrane, preproteins first interact with the cytosolic domains of import receptors (cis) and then are translocated through a general import pore, in a process proposed to involve binding to a trans site on the intermembrane space (IMS) side. Controversial results have been reported for the role of the IMS domain of the essential outer membrane protein Tom22 in formation of the trans site. We show with different mutant mitochondria that a lack of the IMS domain only moderately reduces the direct import of preproteins with N-terminal targeting sequences. The dependence of import on the IMS domain of Tom22 is significantly enhanced by removing the cytosolic domains of import receptors or by performing import in two steps, i.e., accumulation of a preprotein at the outer membrane in the absence of a membrane potential (delta psi) and subsequent import after reestablishment of a delta psi. After the removal of cytosolic receptor domains, two-step import of a cleavable preprotein strictly requires the IMS domain. In contrast, preproteins with internal targeting information do not depend on the IMS domain of Tom22. We conclude that the negatively charged IMS domain of Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences, in agreement with the acid chain hypothesis of mitochondrial protein import.     

Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones.

We have reconstituted the early steps of precursor targeting to mitochondria in a defined and soluble system consisting of the cytosolic domains of the yeast mitochondrial import receptors Tom20 and Tom70, precursor to bovine adrenal adrenodoxin (which has a cleavable targeting signal) and rat liver cytosolic chaperones hsp70 and mitochondrial import-stimulating factor (MSF). The Tom70 domain only bound the precursor in the presence of MSF, yielding a precursor-MSF-Tom70 complex; ATP hydrolysis by MSF released MSF and generated a precursor-Tom70 complex whose formation was inhibited by an excess of a functional presequence peptide, but not by 150 mM NaCl. In the presence of the Tom20 domain, ATP caused transfer of the precursor from the precursor-MSF-Tom70 complex to Tom20. The Tom20 domain alone only bound the precursor in the presence of hsp70; hsp70 itself was not incorporated into the resulting complex. Formation of the Tom20-precursor complex was inhibited by excess presequence peptide or by 150 mM NaCl. Similar results were obtained with the ADP/ATP carrier and porin precursors, which both lack a cleaved targeting signal. Correct targeting of a precursor to mitochondrial import receptors thus requires cytosolic chaperones, irrespective of the presence or absence of a cleavable presequence.     

Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria.

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.     

Distribution of binding sequences for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequence-carrying preprotein and a non-cleavable preprotein.

Preproteins destined for mitochondria either are synthesized with amino-terminal signal sequences, termed presequences, or possess internal targeting information within the protein. The preprotein translocase of the outer mitochondrial membrane (designated Tom) contains specific import receptors. The cytosolic domains of three import receptors, Tom20, Tom22, and Tom70, have been shown to interact with preproteins. Little is known about the internal targeting information in preproteins and the distribution of binding sequences for the three import receptors. We have studied the binding of the purified cytosolic domains of Tom20, Tom22, and Tom70 to cellulose-bound peptide scans derived from a presequence-carrying cleavable preprotein, cytochrome c oxidase subunit IV, and a non-cleavable preprotein with internal targeting information, the phosphate carrier. All three receptor domains are able to bind efficiently to linear 13-mer peptides, yet with different specificity. Tom20 preferentially binds to presequence segments of subunit IV. Tom22 binds to segments corresponding to the carboxyl-terminal part of the presequence and the amino-terminal part of the mature protein. Tom70 does not bind efficiently to any region of subunit IV. In contrast, Tom70 and Tom20 bind to multiple segments within the phosphate carrier, yet the amino-terminal region is excluded. Both charged and uncharged peptides derived from the phosphate carrier show specific binding properties for Tom70 and Tom20, indicating that charge is not a critical determinant of internal targeting sequences. This feature contrasts with the crucial role of positively charged amino acids in presequences. Our results demonstrate that linear peptide segments of preproteins can serve as binding sites for all three receptors with differential specificity and imply different mechanisms for translocation of cleavable and non-cleavable preproteins.     

Crystal structure of yeast mitochondrial outer membrane translocon member Tom70p

A majority of the proteins targeted to the mitochondria are transported through the translocase of the outer membrane (TOM) complex. Tom70 is a major surface receptor for mitochondrial protein precursors in the TOM complex. To investigate how Tom70 receives the mitochondrial protein precursors, we have determined the crystal structure of yeast Tom70p to 3.0 A. Tom70p forms a homodimer in the crystal. Each subunit consists primarily of tetratricopeptide repeat (TPR) motifs, which are organized into a right-handed superhelix. The TPR motifs in the N-terminal domain of Tom70p form a peptide-binding groove for the C-terminal EEVD motif of Hsp70, whereas the C-terminal domain of Tom70p contains a large pocket that may be the binding site for mitochondrial precursors. The crystal structure of Tom70p provides insights into the mechanisms of precursor transport across the mitochondrion's outer membrane.     

Signal-anchored proteins follow a unique insertion pathway into the outer membrane of mitochondria

Signal-anchored proteins are a class of mitochondrial outer membrane proteins that expose a hydrophilic domain to the cytosol and are anchored to the membrane by a single transmembrane domain in the N-terminal region. Like the vast majority of mitochondrial proteins, signal-anchored proteins are synthesized on cytosolic ribosomes and are subsequently imported into the organelle. We have studied the mechanisms by which precursors of these proteins are recognized by the mitochondria and are inserted into the outer membrane. The import of signal-anchored proteins was found to be independent of the known import receptors, Tom20 and Tom70, but to require the major Tom component, Tom40. In contrast to precursors destined to internal compartments of mitochondria and those of outer membrane beta-barrel proteins, precursors of signal-anchored proteins appear not to be inserted via the general import pore. Taken together, we propose a novel pathway for insertion of these proteins into the outer membrane of mitochondria.     

A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments

The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component–depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail–anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs.     

The enteropathogenic Escherichia coli (EPEC) Map effector is imported into the mitochondrial matrix by the TOM/Hsp70 system and alters organelle morphology

Enteropathogenic Escherichia coli (EPEC) is a human intestinal pathogen and a major cause of diarrhoea, particularly among infants in developing countries. EPEC target the Map and EspF multifunctional effector proteins to host mitochondria - organelles that play crucial roles in regulating cellular processes such as programmed cell death (apoptosis). While both molecules interfere with the organelles ability to maintain a membrane potential, EspF plays the predominant role and is responsible for triggering cell death. To learn more about the Map-mitochondria interaction, we studied Map localization to mitochondria with purified mitochondria (from mammalian and yeast cells) and within intact yeast. This revealed that (i) Map targeting is dependent on the predicted N-terminal mitochondrial targeting sequence, (ii) the N-terminal 44 residues are sufficient to target proteins to mitochondria and (iii) Map import involves the mitochondrial outer membrane translocase (Tom22 and Tom40), the mitochondrial membrane potential, and the matrix chaperone, mtHsp70. These results are consistent with Map import into the mitochondria matrix via the classical import mechanism. As all known, Map-associated phenotypes in mammalian cells are independent of mitochondrial targeting, this may indicate that import serves as a mechanism to remove Map from the cytoplasm thereby regulating cytoplasmic function. Intriguingly, Map, but not EspF, alters mitochondrial morphology with deletion analysis revealing important roles for residues 101-152. Changes in mitochondrial morphology have been linked to alterations in the ability of these organelles to regulate cellular processes providing a possible additional role for Map import into mitochondria.     

Function of cytosolic chaperones in Tom70-mediated mitochondrial import

The great majority of mitochondrial proteins are synthesized by cytosolic ribosomes and then imported into the organelle post-translationally. The translocase of the outer membrane (TOM) is a proteinaceous machinery that contains surface receptors for preprotein recognition and also serves as the main entry gateway into mitochondria. Mitochondrial targeting requires various cytosolic factors, in particular the molecular chaperones Hsc70/Hsp70 and Hsp90. The chaperone activity of Hsc70/Hsp70 and Hsp90 occurs in coordinated cycles of ATP hydrolysis and substrate binding, and is regulated by a number of co-chaperone proteins. The import receptor Tom70 is a member of the tetratricopeptide repeat (TPR) co-chaperone family and contains a conserved TPR clamp domain for interaction with Hsc70 and Hsp90. Such interaction is essential for the initiation of the import process. This review will discuss the roles of Hsc70 and Hsp90 in mitochondrial import and summarize recent progress in understanding these pathways.     

The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria

The ADP/ATP carrier (AAC) is a major representative of mitochondrial preproteins lacking an N-terminal presequence. AAC contains targeting information in each of its three modules, which has led to a search for the dominant targeting region. An alternative, not yet tested model would be that several distinct targeting signals function simultaneously in import of the preprotein. We report that the three AAC modules cooperate in binding to the receptor Tom70 such that three Tom70 dimers are recruited to one preprotein. The modules are transferred to the import pore in a stepwise manner and cooperate again in the accumulation of AAC in the general import pore complex. AAC can cross the outer membrane with an internal segment first, i.e. in a loop formation. Each module of AAC is required for dimerization in the inner membrane. We propose a new concept for import of the hydrophobic carrier proteins into mitochondria where multiple signals cooperate in receptor recruitment, outer membrane translocation via loop formation and assembly in the inner membrane.