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.     

Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria.

Mitochondrial precursor proteins made in the cytosol bind to a hetero-oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This 'trans' domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N-terminal targeting sequence of a translocation-arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration-driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3- to 8-fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursor's basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.     

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.     

The Tim21 binding domain connects the preprotein translocases of both mitochondrial membranes

Proteins destined for the mitochondrial matrix are imported by the translocase of the outer membrane--the TOM complex--and the presequence translocase of the inner membrane--the TIM23 complex. At present, there is no structural information on components of the presequence translocase. Tim21, a subunit of the presequence translocase consisting of a membrane anchor and a carboxy-terminal domain exposed to the intermembrane space, directly connects the TOM and TIM23 complexes by binding to the intermembrane space domain of the Tom22 receptor. We crystallized the binding domain of Tim21 of Saccharomyces cerevisiae and determined its structure at 1.6 A resolution. The Tim21 structure represents a new alpha/beta-mixed protein fold with two alpha-helices flanked by an extended eight-stranded beta-sheet. We also identified a core sequence of Tom22 that binds to Tim21. Furthermore, negatively charged amino-acid residues of Tom22 are important for binding to Tim21. Here we suggest a mechanism for the TOM-TIM interaction.     

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.     

Mitochondrial import receptors Tom20 and Tom22 have chaperone-like activity

Mitochondrial preproteins are synthesized in the cytosol with N-terminal signal sequences (presequences) or internal targeting signals. Generally, preproteins with presequences are initially recognized by Tom20 (translocase of the outer membrane) and, subsequently, by Tom22, whereas hydrophobic preproteins with internal targeting signals are first recognized by Tom70. Recent studies suggest that Tom70 associates with molecular chaperones, thereby maintaining their substrate preproteins in an import-competent state. However, such a function has not been reported for other Tom component(s). Here, we investigated a role for Tom20 in preventing substrate preproteins from aggregating. In vitro binding assays showed that Tom20 binds to guanidinium chloride unfolded substrate proteins regardless of the presence or absence of presequences. This suggests that Tom20 functions as a receptor not only for presequences but also for mature portions exposed in unfolded preproteins. Aggregation suppression assays on citrate synthase showed that the cytosolic domain of Tom20 has a chaperone-like activity to prevent this protein from aggregating. This activity was inhibited by a presequence peptide, suggesting that the binding site of Tom20 for presequence is identical or close to the active site for the chaperone-like activity. The cytosolic domain of Tom22 also showed a similar activity for citrate synthase, whereas Tom70 did not. These results suggest that the cytosolic domains of Tom20 and Tom22 function to maintain their substrate preproteins unfolded and prevent them from aggregating on the mitochondrial surface.     

Identification and functional analysis of human Tom22 for protein import into mitochondria

Mitochondria have a receptor complex in the outer membrane which recognizes and translocates mitochondrial proteins synthesized in the cytosol. We report here the identification and functional analysis of human Tom22 (hTom22). hTom22 has an N-terminal negatively charged region exposed to the cytosol, a putative transmembrane region, and a C-terminal intermembrane space region with little negative charge. Tom22 forms a complex with Tom20, and its cytosolic domain functions as an import receptor as in fungi. An import inhibition assay, using pre-ornithine transcarbamylase (pOTC) derivatives and a series of hTom22 deletion mutants, showed that the C-terminal segment of the cytosolic domain is important for presequence binding, whereas the N-terminal domain is important for binding to the mature portion of pOTC. No evidence for pOTC interaction with the Tom22 intermembrane space domain was obtained. Binding studies revealed that the presequence is critical for pOTC binding to Tom20, whereas both the presequence and mature portion are important for binding to Tom22. A cell-free immunoprecipitation assay indicated that an internal segment of the Tom22 cytosolic domain is important for interaction with Tom20.     

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.     

Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex

Preproteins with N-terminal presequences are imported into mitochondria at translocation contact sites that include the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Little is known about the functional cooperation of these translocases. We have characterized translocation contact sites by a productive TOM-TIM-preprotein supercomplex to address the role of three translocase subunits that expose domains to the intermembrane space (IMS). The IMS domain of the receptor Tom22 is required for stabilization of the translocation contact site supercomplex. Surprisingly, the N-terminal segment of the channel Tim23, which tethers the TIM23 complex to the outer membrane, is dispensable for both protein import and generation of the TOM-TIM supercomplex. Tim50, with its large IMS domain, is crucial for generation but not for stabilization of the supercomplex. Thus, Tim50 functions as a dynamic factor and the IMS domain of Tom22 represents a stabilizing element in formation of a productive translocation contact site supercomplex.     

Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23-Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22-Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23-Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.     

NMR analyses on the interactions of the yeast Tim50 C-terminal region with the presequence and Tim50 core domain

The mitochondrial targeting signal in the presequence of mitochondrial precursor proteins is recognized by Tom20 and subsequently by Tim50 in mitochondria. Yeast Tim50 contains two presequence binding sites in the conserved core domain and in the fungi-specific C-terminal presequence binding domain (PBD). We report the NMR analyses on interactions of a shorter variant of PBD (sPBD), a shorter variant of PBD, with presequences. The presequence is recognized by sPBD in a similar manner to Tom20. sPBD can also bind to the core domain of Tim50 through the presequence binding region, which could promote transfer of the presequence from sPBD to the core domain in Tim50.     

Assembly of Tom6 and Tom7 into the TOM core complex of Neurospora crassa

Translocation of preproteins across the mitochondrial outer membrane is mediated by the translocase of the outer mitochondrial membrane (TOM) complex. We report the molecular identification of Tom6 and Tom7, two small subunits of the TOM core complex in the fungus Neurospora crassa. Cross-linking experiments showed that both proteins were found to be in direct contact with the major component of the pore, Tom40. In addition, Tom6 was observed to interact with Tom22 in a manner that depends on the presence of preproteins in transit. Precursors of both proteins are able to insert into the outer membrane in vitro and are assembled into authentic TOM complexes. The insertion pathway of these proteins shares a common binding site with the general import pathway as the assembly of both Tom6 and Tom7 was competed by a matrix-destined precursor protein. This assembly was dependent on the integrity of receptor components of the TOM machinery and is highly specific as in vitro-synthesized yeast Tom6 was not assembled into N. crassa TOM complex. The targeting and assembly information within the Tom6 sequence was found to be located in the transmembrane segment and a flanking segment toward the N-terminal, cytosolic side. A hybrid protein composed of the C-terminal domain of yeast Tom6 and the cytosolic domain of N. crassa Tom6 was targeted to the mitochondria but was not taken up into TOM complexes. Thus, both segments are required for assembly into the TOM complex. A model for the topogenesis of the small Tom subunits is discussed.     

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 biophysical analysis of the tetratricopeptide repeat-rich mitochondrial import receptor, Tom70, reveals an elongated monomer that is inherently flexible, unstable, and unfolds via a multistate pathway

Proteins destined for all submitochondrial compartments are translocated across the outer mitochondrial membrane by the TOM (translocase of the outer membrane) complex, which consists of a number of specialized receptor subunits that bind mitochondrial precursor proteins for delivery into the translocation channel. One receptor, Tom70, binds large, hydrophobic mitochondrial precursors. The current model of Tom70-mediated import involves multiple dimers of the receptor recognizing a single molecule of substrate. Here we show via a battery of biophysical and spectroscopic techniques that the cytosolic domain of Tom70 is an elongated monomer. Thermal and urea-induced denaturation revealed that the receptor, which unfolds via a multistate pathway, is a relatively unstable molecule undergoing major conformational change at physiological temperatures. The data suggest that the malleability of the monomeric Tom70 receptor is an important factor in mitochondrial import.     

The Tim8-Tim13 complex of Neurospora crassa functions in the assembly of proteins into both mitochondrial membranes

The Tim8 and Tim13 proteins in yeast are known to exist in the mitochondrial intermembrane space and to form a hetero-oligomeric complex involved in the import of the mitochondrial inner membrane protein Tim23, the central component of the TIM23 translocase. Here, we have isolated tim8 and tim13 mutants in Neurospora crassa and have shown that mitochondria lacking the Tim8-Tim13 complex were deficient in the import of the outer membrane beta-barrel proteins Tom40 and porin. Cross-linking studies showed that the Tom40 precursor contacts the Tim8-Tim13 complex. The complex is involved at an early point in the Tom40 assembly pathway because cross-links can only be detected during the initial stages of Tom40 import. In mitochondria lacking the Tim8-Tim13 complex, the Tom40 precursor appears in a previously characterized early intermediate of Tom40 assembly more slowly than in wild type mitochondria. Thus, our data suggest a model in which one of the first steps in Tom40 assembly may be interaction with the Tim8-Tim13 complex. As in yeast, the N. crassa Tim23 precursor was imported inefficiently into mitochondria lacking the Tim8-Tim13 complex when the membrane potential was reduced. Tim23 import intermediates could also be cross-linked to the complex, suggesting a dual role for the Tim8-Tim13 intermembrane space complex in the import of proteins found in both the outer and inner mitochondrial membranes.     

The role of the Tim8p-Tim13p complex in a conserved import pathway for mitochondrial polytopic inner membrane proteins

Tim23p is imported via the TIM (translocase of inner membrane)22 pathway for mitochondrial inner membrane proteins. In contrast to precursors with an NH2-terminal targeting presequence that are imported in a linear NH2-terminal manner, we show that Tim23p crosses the outer membrane as a loop before inserting into the inner membrane. The Tim8p-Tim13p complex facilitates translocation across the intermembrane space by binding to the membrane spanning domains as shown by Tim23p peptide scans with the purified Tim8p-Tim13p complex and crosslinking studies with Tim23p fusion constructs. The interaction between Tim23p and the Tim8p-Tim13p complex is not dependent on zinc, and the purified Tim8p-Tim13p complex does not coordinate zinc in the conserved twin CX3C motif. Instead, the cysteine residues seemingly form intramolecular disulfide linkages. Given that proteins of the mitochondrial carrier family also pass through the TOM (translocase of outer membrane) complex as a loop, our study suggests that this translocation mechanism may be conserved. Thus, polytopic inner membrane proteins, which lack an NH2-terminal targeting sequence, pass through the TOM complex as a loop followed by binding of the small Tim proteins to the hydrophobic membrane spanning domains.     

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.     

Tim23 links the inner and outer mitochondrial membranes

Tim23, a key component of the mitochondrial preprotein translocase, is anchored in the inner membrane by its C-terminal domain and exposes an intermediate domain in the intermembrane space that functions as a presequence receptor. We show that the N-terminal domain of Tim23 is exposed on the surface of the outer membrane. The two-membrane-spanning topology of Tim23 is a novel characteristic in membrane biology. By the simultaneous integration into two membranes, Tim23 forms contacts between the outer and inner mitochondrial membranes. Tethering the inner membrane translocase to the outer membrane facilitates the transfer of precursor proteins from the TOM complex to the TIM23 complex and increases the efficiency of protein import.     

Recognition of mitochondrial targeting sequences by the import receptors Tom20 and Tom22

The Tom20 and Tom22 receptor subunits of the TOM (translocase of the outer mitochondrial membrane) complex recognize N-terminal presequences of proteins that are to be imported into the mitochondrion. In plants, Tom20 is C-terminally anchored in the mitochondrial membrane, whereas Tom20 is N-terminally anchored in animals and fungi. Furthermore, the cytosolic domain of Tom22 in plants is smaller than its animal/fungal counterpart and contains fewer acidic residues. Here, NMR spectroscopy was used to explore presequence interactions with the cytosolic regions of receptors from the plant Arabidopsis thaliana and the fungus Saccharomyces cerevisiae (i.e., AtTom20, AtTom22, and ScTom22). It was found that AtTom20 possesses a discontinuous bidentate hydrophobic binding site for presequences. The presequences on plant mitochondrial proteins comprise two or more hydrophobic binding regions to match this bidentate site. NMR data suggested that while these presequences bind to ScTom22, they do not bind to AtTom22. AtTom22, however, binds to AtTom20 at the same binding site as presequences, suggesting that this domain competes with the presequences of imported proteins, thereby enabling their progression along the import pathway.     

The cytosolic domain of human Tom22 modulates human Bax mitochondrial translocation and conformation in yeast

The role of the mitochondrial protein receptor Tom22p in the interaction of pro-apoptotic protein Bax with yeast mitochondria was investigated. Co-immunoprecipitation assays showed that human Bax interacted with different TOM subunits, including Tom22p. Expression of the cytosolic receptor domain of human Tom22 increased Bax mitochondrial localization, but decreased the proportion of active Bax. BN-PAGE showed that the cytosolic domain of Tom22 interfered with the oligomerization of Bax. These data suggest that the interaction with the cytosolic domain of Tom22 helps Bax to acquire a conformation able to interact with the outer mitochondrial membrane.