The phosphate carrier has an ability to be sorted to either the TIM22 pathway or the TIM23 pathway for its import into yeast mitochondria

Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria via the TOM40 (translocase of the mitochondrial outer membrane 40) complex, and follow several distinct sorting pathways to reach their destination submitochondrial compartments. Phosphate carrier (PiC) is an inner membrane protein with 6 transmembrane segments (TM1-TM6) and requires, after translocation across the outer membrane, the Tim9-Tim10 complex and the TIM22 complex to be inserted into the inner membrane. Here we analyzed an in vitro import of fusion proteins between various PiC segments and mouse dihydrofolate reductase. The fusion protein without TM1 and TM2 was translocated across the outer membrane but was not inserted into the inner membrane. The fusion proteins without TM1-TM4 were not inserted into the inner membrane but instead translocated across the inner membrane. Functional defects of Tim50 of the TIM23 complex caused either by depletion of the protein or the addition of anti-Tim50 antibodies blocked translocation of the fusion proteins without TM1-TM4 across the inner membrane, suggesting that lack of TM1-TM4 led to switch of its sorting pathway from the TIM22 pathway to the TIM23 pathway. PiC thus appears to have a latent signal for sorting to the TIM23 pathway, which is exposed by reduced interactions with the Tim9-Tim10 complex and maintenance of the import competence.     

Tim23–Tim50 pair coordinates functions of translocators and motor proteins in mitochondrial protein import

Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23–Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23–Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.     

Effect of mutations in Tom40 on stability of the translocase of the outer mitochondrial membrane (TOM) complex, assembly of Tom40, and import of mitochondrial preproteins

Mitochondrial preproteins synthesized in the cytosol are imported through the mitochondrial outer membrane by the translocase of the outer mitochondrial membrane (TOM) complex. Tom40 is the major component of the complex and is essential for cell viability. We generated 21 different mutations in conserved regions of the Neurospora crassa Tom40 protein. The mutant genes were transformed into a tom40 null nucleus maintained in a sheltered heterokaryon, and 17 of the mutant genes gave rise to viable strains. All mutations reduced the efficiency of the altered Tom40 molecules to assemble into the TOM complex. Mitochondria isolated from seven of the mutant strains had defects for importing mitochondrial preproteins. Only one strain had a general import defect for all preproteins examined. Another mutation resulted in defects in the import of a matrix-destined preprotein and an outer membrane beta-barrel protein, but import of the ADP/ATP carrier to the inner membrane was unaffected. Five strains showed deficiencies in the import of beta-barrel proteins. The latter results suggest that the TOM complex distinguishes beta-barrel proteins from other classes of preprotein during import. This supports the idea that the TOM complex plays an active role in the transfer of preproteins to subsequent translocases for insertion into the correct mitochondrial subcompartment.     

The mitochondrial import machinery: preprotein-conducting channels with binding sites for presequences

Mitochondrial preproteins with amino-terminal presequences must cross two membranes to reach the matrix of the organelle. Both outer and inner membranes contain hydrophilic high-conductance channels that are responsible for selective translocation of preproteins. The channels are embedded in dynamic protein complexes, the TOM complex of the outer membrane and the TIM23 complex of the inner membrane. Both channel-forming proteins, Tom40 and Tim23, carry specific binding sites for presequences, but differ in their pore size and response to a membrane potential. Studies with the TOM machinery show that other subunits of the translocase complex also provide specific binding sites for preproteins, modulate the channel activity and are critical for assembly of the channel.     

An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane

The preprotein translocase of the outer mitochondrial membrane (TOM complex) contains one essential subunit, the channel Tom40. The assembly pathway of the precursor of Tom40 involves the TOM complex and the sorting and assembly machinery (SAM complex) with the non-essential subunit Mas37. We have identified Sam50, the second essential protein of the mitochondrial outer membrane. Sam50 contains a beta-barrel domain conserved from bacteria to man and is a subunit of the SAM complex. Yeast mutants of Sam50 are defective in the assembly pathways of Tom40 and the abundant outer membrane protein porin, while the import of matrix proteins is not affected. Thus the protein sorting and assembly machinery of the mitochondrial outer membrane involves an essential, conserved protein.     

Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import.

The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase.     

The transport machinery for the import of preproteins across the outer mitochondrial membrane

In order for proteins to be imported into subcellular compartments, they must first traverse the organellar membranes. In mitochondria, hydrophilic protein channels in both the outer and inner membranes serve such a purpose. Recently, the channel protein of the outer mitochondrial membrane was identified to be Tom40. Tom40 is found in a high molecular weight complex termed the general import pore (GIP) complex where it is tightly associated with the receptor protein Tom22 along with Tom7, Tom6 and Tom5. Tom7 and Tom6 seem to modulate the dynamics of the GIP complex while Tom5 is involved in preprotein transfer from receptors to Tom40. The receptor proteins Tom70 and Tom20 associate with this complex in a weaker manner where they are involved in the initial recognition of preproteins. This review focuses on the identification and characterisation of the transport machinery of the outer mitochondrial membrane and how they are involved in the co-ordination and regulation of events required for the translocation of preproteins into mitochondria.     

A novel import route for an N-anchor mitochondrial outer membrane protein aided by the TIM23 complex

The membrane topology of Om45 in the yeast mitochondrial outer membrane (OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N-terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence-containing proteins, and the membrane potential (ΔΨ). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex.     

Functions of the small proteins in the TOM complex of Neurospora crasssa

The TOM (translocase of the outer mitochondrial membrane) complex of the outer mitochondrial membrane is required for the import of proteins into the organelle. The core TOM complex contains five proteins, including three small components Tom7, Tom6, and Tom5. We have created single and double mutants of all combinations of the three small Tom proteins of Neurospora crassa. Analysis of the mutants revealed that Tom6 plays a major role in TOM complex stability, whereas Tom7 has a lesser role. Mutants lacking both Tom6 and Tom7 have an extremely labile TOM complex and are the only class of mutant to exhibit an altered growth phenotype. Although single mutants lacking N. crassa Tom5 have no apparent TOM complex abnormalities, studies of double mutants lacking Tom5 suggest that it also has a minor role in maintaining TOM complex stability. Our inability to isolate triple mutants supports the idea that the three proteins have overlapping functions. Mitochondria lacking either Tom6 or Tom7 are differentially affected in their ability to import different precursor proteins into the organelle, suggesting that they may play roles in the sorting of proteins to different mitochondrial subcompartments. Newly imported Tom40 was readily assembled into the TOM complex in mitochondria lacking any of the small Tom proteins.     

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.     

Characterization of Neurospora crassa Tom40-deficient mutants and effect of specific mutations on Tom40 assembly

The TOM complex (Translocase of the Outer mitochondrial Membrane) is responsible for the recognition of mitochondrial preproteins synthesized in the cytosol and for their translocation across or into the outer mitochondrial membrane. Tom40 is the major component of the TOM complex and forms the translocation pore. We have created a tom40 mutant of Neurospora crassa and have demonstrated that the gene is essential for the viability of the organism. Mitochondria with reduced levels of Tom40 were deficient for import of mitochondrial preproteins and contained reduced levels of the TOM complex components Tom22 and Tom6, suggesting that the import and/or stability of these proteins is dependent on the presence of Tom40. Mutant Tom40 preproteins were analyzed for their ability to be assembled into the TOM complex. In vitro import assays revealed that conserved regions near the N terminus (residues 51-60) and the C terminus (residues 321-323) of the 349-amino acid protein were required for assembly beyond a 250-kDa intermediate form. Mutant strains expressing Tom40 with residues 51-60 deleted were viable but exhibited growth defects. Slow growing mutants expressing Tom40, where residues 321-323 were changed to Ala residues, were isolated but showed TOM complex defects, whereas strains in which residues 321-323 were deleted could not be isolated. Analysis of the assembly of mutant Tom40 precursors in vitro supported a previous model in which Tom40 precursors progress from the 250-kDa intermediate to a 100-kDa form and then assemble into the 400-kDa TOM complex. Surprisingly, when wild type mitochondria containing Tom40 precursors arrested at the 250-kDa intermediate were treated with sodium carbonate, further assembly of intermediates into the TOM complex occurred, suggesting that disruption of protein-protein interactions may facilitate assembly. Import of wild type Tom40 precursor into mitochondria containing a mutant Tom40 lacking residues 40-48 revealed an alternate assembly pathway and demonstrated that the N-terminal region of pre-existing Tom40 molecules in the TOM complex plays a role in the assembly of incoming Tom40 molecules.     

Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors

The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.     

Mdm10 is an ancient eukaryotic porin co-occurring with the ERMES complex

Mitochondrial β-barrel proteins fulfill central functions in the outer membrane like metabolite exchange catalyzed by the voltage-dependent anion channel (VDAC) and protein biogenesis by the central components of the preprotein translocase of the outer membrane (Tom40) or of the sorting and assembly machinery (Sam50). The mitochondrial division and morphology protein Mdm10 is another essential outer membrane protein with proposed β-barrel fold, which has so far only been found in Fungi. Mdm10 is part of the endoplasmic reticulum mitochondria encounter structure (ERMES), which tethers the ER to mitochondria and associates with the SAM complex. In here, we provide evidence that Mdm10 phylogenetically belongs to the VDAC/Tom40 superfamily. Contrary to Tom40 and VDAC, Mdm10 exposes long loops towards both sides of the membrane. Analyses of single loop deletion mutants of Mdm10 in the yeast Saccharomyces cerevisiae reveal that the loops are dispensable for Mdm10 function. Sequences similar to fungal Mdm10 can be found in species from Excavates to Fungi, but neither in Metazoa nor in plants. Strikingly, the presence of Mdm10 coincides with the appearance of the other ERMES components. Mdm10's presence in both unikonts and bikonts indicates an introduction at an early time point in eukaryotic evolution.     

Two Modular Forms of the Mitochondrial Sorting and Assembly Machinery Are Involved in Biogenesis of α-Helical Outer Membrane Proteins

The mitochondrial outer membrane contains two translocase machineries for precursor proteins—the translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The TOM complex functions as the main mitochondrial entry gate for nuclear-encoded proteins, whereas the SAM complex was identified according to its function in the biogenesis of β-barrel proteins of the outer membrane. The SAM complex is required for the assembly of precursors of the TOM complex, including not only the β-barrel protein Tom40 but also a subset of α-helical subunits. While the interaction of β-barrel proteins with the SAM complex has been studied in detail, little is known about the interaction between the SAM complex and α-helical precursor proteins. We report that the SAM is not static but that the SAM core complex can associate with different partner proteins to form two large SAM complexes with different functions in the biogenesis of α-helical Tom proteins. We found that a subcomplex of TOM, Tom5-Tom40, associates with the SAM core complex to form a new large SAM complex. This SAM-Tom5/Tom40 complex binds the α-helical precursor of Tom6 after the precursor has been inserted into the outer membrane in an Mim1 (mitochondrial import protein 1)-dependent manner. The second large SAM complex, SAM-Mdm10 (mitochondrial distribution and morphology protein), binds the α-helical precursor of Tom22 and promotes its membrane integration. We suggest that the modular composition of the SAM complex provides a flexible platform to integrate the sorting pathways of different precursor proteins and to promote their assembly into oligomeric complexes.     

The Tom40 assembly process probed using the attachment of different intramitochondrial sorting signals

The TOM40 complex is a protein translocator in the mitochondrial outer membrane and consists of several different subunits. Among them, Tom40 is a central subunit that constitutes a protein-conducting channel by forming a β-barrel structure. To probe the nature of the assembly process of Tom40 in the outer membrane, we attached various mitochondrial presequences to Tom40 that possess sorting information for the intermembrane space (IMS), inner membrane, and matrix and would compete with the inherent Tom40 assembly process. We analyzed the mitochondrial import of those fusion proteins in vitro. Tom40 crossed the outer membrane and/or inner membrane even in the presence of various sorting signals. N-terminal anchorage of the attached presequence to the inner membrane did not prevent Tom40 from associating with the TOB/SAM complex, although it impaired its efficient release from the TOB complex in vitro but not in vivo. The IMS or matrix-targeting presequence attached to Tom40 was effective in substituting for the requirement for small Tim proteins in the IMS for the translocation of Tom40 across the outer membrane. These results provide insight into the mechanism responsible for the precise delivery of β-barrel proteins to the outer mitochondrial membrane.     

Biogenesis of the protein import channel Tom40 of the mitochondrial outer membrane: intermembrane space components are involved in an early stage of the assembly pathway

Tom40 forms the central channel of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The precursor of Tom40 is encoded in the nucleus, synthesized in the cytosol, and imported into mitochondria via a multi-step assembly pathway that involves the mature TOM complex and the sorting and assembly machinery of the outer membrane (SAM complex). We report that opening of the mitochondrial intermembrane space by swelling blocks the assembly pathway of the beta-barrel protein Tom40. Mitochondria with defects in small Tim proteins of the intermembrane space are impaired in the Tom40 assembly pathway. Swelling as well as defects in the small Tim proteins inhibit an early stage of the Tom40 import pathway that is needed for formation of a Tom40-SAM intermediate. We propose that the biogenesis pathway of beta-barrel proteins of the outer mitochondrial membrane not only requires TOM and SAM components, but also involves components of the intermembrane space.     

A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria

Mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria with the help of protein translocases. For the majority of precursor proteins, the role of the translocase of the outer membrane (TOM) and mechanisms of their transport across the outer mitochondrial membrane are well recognized. However, little is known about the mode of membrane translocation for proteins that are targeted to the intermembrane space via the redox-driven mitochondrial intermembrane space import and assembly (MIA) pathway. On the basis of the results obtained from an in organello competition import assay, we hypothesized that MIA-dependent precursor proteins use an alternative pathway to cross the outer mitochondrial membrane. Here we demonstrate that this alternative pathway involves the protein channel formed by Tom40. We sought a translocation intermediate by expressing tagged versions of MIA-dependent proteins in vivo. We identified a transient interaction between our model substrates and Tom40. Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22. Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.     

The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane

The biogenesis of mitochondrial outer membrane proteins involves the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The two known subunits of the SAM complex, Mas37 and Sam50, are required for assembly of the abundant outer membrane proteins porin and Tom40. We have identified an unexpected subunit of the SAM complex, Mdm10, which is involved in maintenance of mitochondrial morphology. Mitochondria lacking Mdm10 are selectively impaired in the final steps of the assembly pathway of Tom40, including the association of Tom40 with the receptor Tom22 and small Tom proteins, while the biogenesis of porin is not affected. Yeast mutants of TOM40, MAS37, and SAM50 also show aberrant mitochondrial morphology. We conclude that Mdm10 plays a specific role in the biogenesis of the TOM complex, indicating a connection between the mitochondrial protein assembly apparatus and the machinery for maintenance of mitochondrial morphology.     

The Tim54p–Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane

We have identified a new protein, Tim54p, located in the yeast mitochondrial inner membrane. Tim54p is an essential import component, required for the insertion of at least two polytopic proteins into the inner membrane, but not for the translocation of precursors into the matrix. Several observations suggest that Tim54p and Tim22p are part of a protein complex in the inner membrane distinct from the previously characterized Tim23p-Tim17p complex. First, multiple copies of the TIM22 gene, but not TIM23 or TIM17, suppress the growth defect of a tim54-1 temperature-sensitive mutant. Second, Tim22p can be coprecipitated with Tim54p from detergent-solubilized mitochondria, but Tim54p and Tim22p do not interact with either Tim23p or Tim17p. Finally, the tim54-1 mutation destabilizes the Tim22 protein, but not Tim23p or Tim17p. Our results support the idea that the mitochondrial inner membrane carries two independent import complexes: one required for the translocation of proteins across the inner membrane (Tim23p–Tim17p), and the other required for the insertion of proteins into the inner membrane (Tim54p–Tim22p).     

A Highlights from MBoC Selection: Assembly of the Mitochondrial Protein Import Channel: Role of Tom5 in Two-Stage Interaction of Tom40 with the SAM Complex

The preprotein translocase of the outer mitochondrial membrane (TOM) consists of a central β-barrel channel, Tom40, and six proteins with α-helical transmembrane segments. The precursor of Tom40 is imported from the cytosol by a pre-existing TOM complex and inserted into the outer membrane by the sorting and assembly machinery (SAM). Tom40 then assembles with α-helical Tom proteins to the mature TOM complex. The outer membrane protein Mim1 promotes membrane insertion of several α-helical Tom proteins but also affects the biogenesis of Tom40 by an unknown mechanism. We have identified a novel intermediate in the assembly pathway of Tom40, revealing a two-stage interaction of the precursor with the SAM complex. The second SAM stage represents assembly of Tom5 with the precursor of Tom40. Mim1-deficient mitochondria accumulate Tom40 at the first SAM stage like Tom5-deficient mitochondria. Tom5 promotes formation of the second SAM stage and thus suppresses the Tom40 assembly defect of mim1Δ mitochondria. We conclude that the assembly of newly imported Tom40 is directly initiated at the SAM complex by its association with Tom5. The involvement of Mim1 in Tom40 biogenesis can be largely attributed to its role in import of Tom5.