Characterization of the preprotein and amino acid transporter gene family in Arabidopsis

Seventeen loci encode proteins of the preprotein and amino acid transporter family in Arabidopsis (Arabidopsis thaliana). Some of these genes have arisen from recent duplications and are not in annotated duplicated regions of the Arabidopsis genome. In comparison to a number of other eukaryotic organisms, this family of proteins has greatly expanded in plants, with 24 loci in rice (Oryza sativa). Most of the Arabidopsis and rice genes are orthologous, indicating expansion of this family before monocot and dicot divergence. In vitro protein uptake assays, in vivo green fluorescent protein tagging, and immunological analyses of selected proteins determined either mitochondrial or plastidic localization for 10 and six proteins, respectively. The protein encoded by At5g24650 is targeted to both mitochondria and chloroplasts and, to our knowledge, is the first membrane protein reported to be targeted to mitochondria and chloroplasts. Three genes encoded translocase of the inner mitochondrial membrane (TIM)17-like proteins, three TIM23-like proteins, and three outer envelope protein16-like proteins in Arabidopsis. The identity of Arabidopsis TIM22-like proteins is most likely a protein encoded by At3g10110/At1g18320, based on phylogenetic analysis, subcellular localization, and complementation of a yeast (Saccharomyces cerevisiae) mutant and coexpression analysis. The lack of a preprotein and amino acid transporter domain in some proteins, localization in mitochondria, plastids, or both, variation in gene structure, and the differences in expression profiles indicate that the function of this family has diverged in plants beyond roles in protein translocation.     

A molecular link between mitochondrial preprotein transporters and respiratory chain complexes

The TIM17:23 complex on the mitochondrial inner membrane is responsible for import of the majority of mitochondrial proteins in plants. In Arabidopsis, Tim17 and Tim23 belong to a large gene family consisting of 16 members termed the Preprotein and Amino acid transporters (PRAT). Recently, two members of this protein family, Tim23-2 and the Complex I subunit B14.7, have been shown to assemble into both Complex I of the respiratory chain and the TIM17:23 complex (Wang et al., 2012), adding to other examples of links between respiratory and protein import complexes. These associations provide a mechanism to coordinate mitochondrial activity and biogenesis.     

Phototropin‐ and photosynthesis‐dependent mitochondrial positioning in Arabidopsis thaliana mesophyll cells

Mitochondria are frequently observed in the vicinity of chloroplasts in photosynthesizing cells, and this association is considered necessary for their metabolic interactions. We previously reported that, in leaf palisade cells of Arabidopsis thaliana, mitochondria exhibit blue‐light‐dependent redistribution together with chloroplasts, which conduct accumulation and avoidance responses under the control of blue‐light receptor phototropins. In this study, precise motility analyses by fluorescent microscopy revealed that the individual mitochondria in palisade cells, labeled with green fluorescent protein, exhibit typical stop‐and‐go movement. When exposed to blue light, the velocity of moving mitochondria increased in 30 min, whereas after 4 h, the frequency of stoppage of mitochondrial movement markedly increased. Using different mutant plants, we concluded that the presence of both phototropin1 and phototropin2 is necessary for the early acceleration of mitochondrial movement. On the contrary, the late enhancement of stoppage of mitochondrial movement occurs only in the presence of phototropin2 and only when intact photosynthesis takes place. A plasma‐membrane ghost assay suggested that the stopped mitochondria are firmly adhered to chloroplasts. These results indicate that the physical interaction between mitochondria and chloroplasts is cooperatively mediated by phototropin2‐ and photosynthesis‐dependent signals. The present study might add novel regulatory mechanism for light‐dependent plant organelle interactions.     

A MICOS–TIM22 Association Promotes Carrier Import into Human Mitochondria

Mitochondrial membrane proteins with internal targeting signals are inserted into the inner membrane by the carrier translocase (TIM22 complex). For this, precursors have to be initially directed from the TOM complex in the outer mitochondrial membrane across the intermembrane space toward the TIM22 complex. How these two translocation processes are topologically coordinated is still unresolved. Using proteomic approaches, we find that the human TIM22 complex associates with the mitochondrial contact site and cristae organizing system (MICOS) complex. This association does not appear to be conserved in yeast, whereby the yeast MICOS complex instead interacts with the presequence translocase. Using a yeast mic10Δ strain and a HEK293T MIC10 knockout cell line, we characterize the role of MICOS for protein import into the mitochondrial inner membrane and matrix. We find that a physiological cristae organization promotes efficient import via the presequence pathway in yeast, while in human mitochondria, the MICOS complex is dispensable for protein import along the presequence pathway. However, in human mitochondria, the MICOS complex is required for the efficient import of carrier proteins into the mitochondrial inner membrane. Our analyses suggest that in human mitochondria, positioning of the carrier translocase at the crista junction, and potentially in vicinity to the TOM complex, is required for efficient transport into the inner membrane.     

Tim9, a new component of the TIM22.54 translocase in mitochondria.

We have identified Tim9, a new component of the TIM22.54 import machinery, which mediates transport of proteins into the inner membrane of mitochondria. Tim9, an essential protein of Saccharomyces cerevisiae, shares sequence similarity with Tim10 and Tim12. Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct hetero-oligomeric assemblies with Tim10 and Tim12. One complex contains Tim9 and Tim10. The other complex contains Tim9, Tim10 and Tim12 and is tightly associated with Tim22 in the inner membrane. The TIM9.10 complex is more abundant than the TIM9.10.12 complex and mediates partial translocation of mitochondrial carriers proteins across the outer membrane. The TIM9.10.12 complex assists further translocation into the inner membrane in association with TIM22.54.     

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).     

The Channel-Forming Sym1 Protein Is Transported by the TIM23 Complex in a Presequence-Independent Manner

The majority of multispanning inner mitochondrial membrane proteins utilize internal targeting signals, which direct them to the carrier translocase (TIM22 complex), for their import. MPV17 and its Saccharomyces cerevisiae orthologue Sym1 are multispanning inner membrane proteins of unknown function with an amino-terminal presequence that suggests they may be targeted to the mitochondria. Mutations affecting MPV17 are associated with mitochondrial DNA depletion syndrome (MDDS). Reconstitution of purified Sym1 into planar lipid bilayers and electrophysiological measurements have demonstrated that Sym1 forms a membrane pore. To address the biogenesis of Sym1, which oligomerizes in the inner mitochondrial membrane, we studied its import and assembly pathway. Sym1 forms a transport intermediate at the translocase of the outer membrane (TOM) complex. Surprisingly, Sym1 was not transported into mitochondria by an amino-terminal signal, and in contrast to what has been observed in carrier proteins, Sym1 transport and assembly into the inner membrane were independent of small translocase of mitochondrial inner membrane (TIM) and TIM22 complexes. Instead, Sym1 required the presequence of translocase for its biogenesis. Our analyses have revealed a novel transport mechanism for a polytopic membrane protein in which internal signals direct the precursor into the inner membrane via the TIM23 complex, indicating a presequence-independent function of this translocase.     

Protein import into and across the mitochondrial inner membrane: role of the TIM23 and TIM22 translocons

Import of nuclear-encoded mitochondrial proteins requires the action of at least two different import machines, called translocons, in the mitochondrial inner membrane (IM). The TIM23 complex mediates the translocation of proteins into the mitochondria matrix, whereas the TIM22 complex is required for the insertion of polytopic proteins into the IM. While the two translocons are distinct and composed of separate subunits, the essential reactions in each complex are carried out by homologous proteins. In addition, the core components of both the TIM23 and TIM22 translocons have been shown to form aqueous pores in the mitochondrial IM. In this review, we summarize what is known about import of proteins across the mitochondrial IM.     

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.     

Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported proteins into the yeast mitochondrial inner membrane

Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.     

The translocator maintenance protein Tam41 is required for mitochondrial cardiolipin biosynthesis

The mitochondrial inner membrane contains different translocator systems for the import of presequence-carrying proteins and carrier proteins. The translocator assembly and maintenance protein 41 (Tam41/mitochondrial matrix protein 37) was identified as a new member of the mitochondrial protein translocator systems by its role in maintaining the integrity and activity of the presequence translocase of the inner membrane (TIM23 complex). Here we demonstrate that the assembly of proteins imported by the carrier translocase, TIM22 complex, is even more strongly affected by the lack of Tam41. Moreover, respiratory chain supercomplexes and the inner membrane potential are impaired by lack of Tam41. The phenotype of Tam41-deficient mitochondria thus resembles that of mitochondria lacking cardiolipin. Indeed, we found that Tam41 is required for the biosynthesis of the dimeric phospholipid cardiolipin. The pleiotropic effects of the translocator maintenance protein on preprotein import and respiratory chain can be attributed to its role in biosynthesis of mitochondrial cardiolipin.     

The Mitochondrial ADP/ATP Carrier Associates with the Inner Membrane Presequence Translocase in a Stoichiometric Manner

The majority of mitochondrial proteins are synthesized with amino-terminal signal sequences. The presequence translocase of the inner membrane (TIM23 complex) mediates the import of these preproteins. The essential TIM23 core complex closely cooperates with partner protein complexes like the presequence translocase-associated import motor and the respiratory chain. The inner mitochondrial membrane also contains a large number of metabolite carriers, but their association with preprotein translocases has been controversial. We performed a comprehensive analysis of the TIM23 interactome based on stable isotope labeling with amino acids in cell culture. Subsequent biochemical studies on identified partner proteins showed that the mitochondrial ADP/ATP carrier associates with the membrane-embedded core of the TIM23 complex in a stoichiometric manner, revealing an unexpected connection of mitochondrial protein biogenesis to metabolite transport. Our data indicate that direct TIM23-AAC coupling may support preprotein import into mitochondria when respiratory activity is low.     

The Mitochondrial Import Machinery for Preproteins

Most mitochondrial proteins are transported from the cytosol into the or-ganelle. Due to the division of mitochondria into an outer and inner membrane, an inter-membrane space and a matrix, an elaborated system for recognition and transport of preproteins has evolved. The translocase of the outer mitochondrial membrane (TOM) and the translocases of the inner mitochondrial membrane (TIM) mediate these processes. Receptor proteins on the cytosolic face of mitochondria recognize the cargo proteins and transfer them to the general import pore (GIP) of the outer membrane. Following the passage of preproteins through the outer membrane they are transported with the aid of the TIM23 complex into either the matrix, inner membrane, or intermembrane space. Some preprotein families utilize the TIM22 complex for their insertion into the inner membrane. The identification of protein components, which are involved in these transport processes, as well as significant insights into the molecular function of some of them, has been achieved in recent years. Moreover, we are now approaching a new era in which elaborated techniques have already allowed and will enable us to gather information about the TOM and TIM complexes on an ultrastructural level.     

Ups1p and Ups2p antagonistically regulate cardiolipin metabolism in mitochondria

Cardiolipin, a unique phospholipid composed of four fatty acid chains, is located mainly in the mitochondrial inner membrane (IM). Cardiolipin is required for the integrity of several protein complexes in the IM, including the TIM23 translocase, a dynamic complex which mediates protein import into the mitochondria through interactions with the import motor presequence translocase–associated motor (PAM). In this study, we report that two homologous intermembrane space proteins, Ups1p and Ups2p, control cardiolipin metabolism and affect the assembly state of TIM23 and its association with PAM in an opposing manner. In ups1Δ mitochondria, cardiolipin levels were decreased, and the TIM23 translocase showed altered conformation and decreased association with PAM, leading to defects in mitochondrial protein import. Strikingly, loss of Ups2p restored normal cardiolipin levels and rescued TIM23 defects in ups1Δ mitochondria. Furthermore, we observed synthetic growth defects in ups mutants in combination with loss of Pam17p, which controls the integrity of PAM. Our findings provide a novel molecular mechanism for the regulation of cardiolipin metabolism.     

The TIM22 complex mediates the import of sideroflexins and is required for efficient mitochondrial one-carbon metabolism

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in AGK cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis. We mapped the proteomic changes in Sengers patient fibroblasts and AGK^KO cell lines to understand the effects of AGK dysfunction on mitochondria. This uncovered down-regulation of a number of proteins at the inner mitochondrial membrane, including many SLC25 carrier family proteins, which are predicted substrates of the complex. We also observed down-regulation of SFXN proteins, which contain five transmembrane domains, and show that they represent a novel class of TIM22 complex substrate. Perturbed biogenesis of SFXN proteins in cells lacking AGK reduces the proliferative capabilities of these cells in the absence of exogenous serine, suggesting that dysregulation of one-carbon metabolism is a molecular feature in the biology of Sengers syndrome.     

The mitochondrial import machinery for preproteins.

Most mitochondrial proteins are transported from the cytosol into the organelle. Due to the division of mitochondria into an outer and inner membrane, an intermembrane space and a matrix, an elaborated system for recognition and transport of preproteins has evolved. The translocase of the outer mitochondrial membrane (TOM) and the translocases of the inner mitochondrial membrane (TIM) mediate these processes. Receptor proteins on the cytosolic face of mitochondria recognize the cargo proteins and transfer them to the general import pore (GIP) of the outer membrane. Following the passage of preproteins through the outer membrane they are transported with the aid of the TIM23 complex into either the matrix, inner membrane, or intermembrane space. Some preprotein families utilize the TIM22 complex for their insertion into the inner membrane. The identification of protein components, which are involved in these transport processes, as well as significant insights into the molecular function of some of them, has been achieved in recent years. Moreover, we are now approaching a new era in which elaborated techniques have already allowed and will enable us to gather information about the TOM and TIM complexes on an ultrastructural level.     

Identification,expression, and import of components 17 and 23 of the inner mitochondrial membrane translocase from Arabidopsis

Characterization of components 17 and 23 of the inner mitochondrial membrane translocase (TIM17:23) from Arabidopsis indicated that there were three genes present for TIM17 and TIM23 and two for TIM44. AtTIM17 differed from the yeast (Saccharomyces cerevisiae) and mammalian homologs in that two genes encoded proteins that were longer and one gene encoded a shorter protein. All Arabidopsis TIM23 predicted proteins appeared to lack the first 34 amino acids compared with yeast TIM23. All AtTIM17 and AtTIM23 genes were expressed but displayed different tissue and developmental profiles. Complementation of deletion mutants in yeast indicated that for AtTIM17, the extension at the C terminus not present in yeast had to be removed to achieve complementation, whereas for TIM23, a preprotein and amino acid transporter domain had to be present for complementation. Import assays with AtTIM17 and AtTIM23 indicated that they both contained internal signals for integration into the inner mitochondrial membrane in a membrane potential-dependent manner. The C terminus of imported AtTIM17-2 was susceptible to degradation by externally added protease with intact mitochondria. Removal of the 85 C-terminal amino acids resulted in import and full protection of the truncated protein. This suggests that the novel extension at the C terminus of AtTIM17-2 links the outer and inner membrane in a manner analogous to yeast TIM23.     

Biogenesis of beta-barrel membrane proteins of mitochondria

beta-Barrel membrane proteins have several important functions in outer membranes of Gram-negative bacteria and in the organelles of endosymbiotic origin, mitochondria and chloroplasts. The biogenesis of beta-barrel membrane proteins was, until recently, an unresolved process. A breakthrough was achieved when a specific pathway for the insertion of beta-barrel outer-membrane proteins was identified in both mitochondria and Gram-negative bacteria. The key component of this pathway is Tob55 (also known as Sam50) in mitochondria and Omp85 in bacteria, both beta-barrel membrane proteins themselves. Tob55 is part of the hetero-oligomeric TOB (topogenesis of mitochondrial outer-membrane beta-barrel proteins) or SAM (sorting and assembly of mitochondria) complex, which is present in the mitochondrial outer membrane. Tob55 belongs to an evolutionarily conserved protein family, the members of which are present in almost all eukaryotes and in Gram-negative bacteria and chloroplasts. Thus, is it emphasized that the insertion pathway of mitochondrial beta-barrel membrane proteins was conserved during evolution of mitochondria from endosymbiotic bacterial ancestors.     

Identification of Tam41 maintaining integrity of the TIM23 protein translocator complex in mitochondria

Newly synthesized mitochondrial proteins are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes. We report the identification of yeast Tam41, a new member of mitochondrial protein translocator systems. Tam41 is a peripheral inner mitochondrial membrane protein facing the matrix. Disruption of the TAM41 gene led to temperature-sensitive growth of yeast cells and resulted in defects in protein import via the TIM23 translocator complex at elevated temperature both in vivo and in vitro. Although Tam41 is not a constituent of the TIM23 complex, depletion of Tam41 led to a decreased molecular size of the TIM23 complex and partial aggregation of Pam18 and -16. Import of Pam16 into mitochondria without Tam41 was retarded, and the imported Pam16 formed aggregates in vitro. These results suggest that Tam41 facilitates mitochondrial protein import by maintaining the functional integrity of the TIM23 protein translocator complex from the matrix side of the inner membrane.     

Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane

The mitochondrial inner membrane contains two separate translocons: one required for the translocation of matrix-targeted proteins (the Tim23p-Tim17p complex) and one for the insertion of polytopic proteins into the mitochondrial inner membrane (the Tim54p-Tim22p complex). To identify new members of the Tim54p-Tim22p complex, we screened for high-copy suppressors of the temperature-sensitive tim54-1 mutant. We identified a new gene, TIM18, that encodes an integral protein of the inner membrane. The following genetic and biochemical observations suggest that the Tim18 protein is part of the Tim54p-Tim22p complex in the inner membrane: multiple copies of TIM18 suppress the tim54-1 growth defect; the tim18::HIS3 disruption is synthetically lethal with tim54-1; Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and Tim18p, along with Tim54p and Tim22p, is detected in an approximately 300-kDa complex after blue native electrophoresis. We propose that Tim18p is a new component of the Tim54p-Tim22p machinery that facilitates insertion of polytopic proteins into the mitochondrial inner membrane.