The C-terminal region of TIM17 links the outer and inner mitochondrial membranes in Arabidopsis and is essential for protein import

The translocase of the inner membrane 17 (AtTIM17-2) protein from Arabidopsis has been shown to link the outer and inner mitochondrial membranes. This was demonstrated by several approaches: (i) In vitro organelle import assays indicated the imported AtTIM17-2 protein remained protease accessible in the outer membrane when inserted into the inner membrane. (ii) N-terminal and C-terminal tagging indicated that it was the C-terminal region that was located in the outer membrane. (iii) Antibodies raised to the C-terminal 100 amino acids recognize a 31-kDa protein from purified mitochondria, but cross-reactivity was abolished when mitochondria were protease-treated to remove outer membrane-exposed proteins. Antibodies to AtTIM17-2 inhibited import of proteins via the general import pathway into outer membrane-ruptured mitochondria, but did not inhibit protein import via the carrier import pathway. Together these results indicate that the C-terminal region of AtTIM17-2 is exposed on the outer surface of the outer membrane, and the C-terminal region is essential for protein import into mitochondria.     

Import of carrier proteins into mitochondria

Carrier proteins located in the inner membrane of mitochondria are responsible for the exchange of metabolites between the intermembrane space and the matrix of this organelle. All members of this family are nuclear-encoded and depend on translocation machineries for their import into mitochondria. Recently many new translocation components responsible for the import of carrier proteins were identified. It is now possible to describe a detailed import pathway for this class of proteins. This review highlights the contribution made by translocation components to the process of carrier protein import into mitochondria.     

The protein import motor of mitochondria

Proteins that are destined for the matrix of mitochondria are transported into this organelle by two translocases: the TOM complex, which transports proteins across the outer mitochondrial membrane; and the TIM23 complex, which gets them through the inner mitochondrial membrane. Two models have been proposed to explain how this protein-import machinery works -- a targeted Brownian ratchet, in which random motion is translated into vectorial motion, or a 'power stroke', which is exerted by a component of the import machinery. Here, we review the data for and against each model.     

Mitochondrial preprotein translocases as dynamic molecular machines

Proteomic studies have demonstrated that yeast mitochondria contain roughly 1000 different proteins. Only eight of these proteins are encoded by the mitochondrial genome and are synthesized on mitochondrial ribosomes. The remaining 99% of mitochondrial precursors are encoded within the nuclear genome and after their synthesis on cytosolic ribosomes must be imported into the organelle. Targeting of these proteins to mitochondria and their import into one of the four mitochondrial subcompartments--outer membrane, intermembrane space (IMS), inner membrane and matrix--requires various membrane-embedded protein translocases, as well as numerous chaperones and cochaperones in the aqueous compartments. During the last years, several novel protein components involved in the import and assembly of mitochondrial proteins have been identified. The picture that emerges from these exciting new findings is that of highly dynamic import machineries, rather than of regulated, but static protein complexes. In this review, we will give an overview on the recent progress in our understanding of mitochondrial protein import. We will focus on the presequence translocase of the inner mitochondrial membrane, the TIM23 complex and the presequence translocase-associated motor, the PAM complex. These two molecular machineries mediate the multistep import of preproteins with cleavable N-terminal signal sequences into the matrix or inner membrane of mitochondria.     

Diverse mechanisms and machineries for import of mitochondrial proteins

Mitochondria are ubiquitous organelles that play an essential role in energy conversion and biosynthetic pathways in eukaryotic cells. Most mitochondrial proteins must be imported from the cytosol and sorted into one of the four mitochondrial subcompartments, the outer membrane, the intermembrane space, the inner membrane and the matrix. Studies in recent years revealed a remarkable diversity of mechanisms and machineries that are required for the import of proteins into mitochondria. At least four different pathways for the sorting and assembly of nuclear-encoded mitochondrial proteins have been identified.     

How mitochondria import hydrophilic and hydrophobic proteins

Most mitochondrial proteins are nuclear encoded and have to be transported into the organelle after synthesis on cytosolic ribosomes. Three multimeric protein complexes have been identified that import precursor proteins destined for the mitochondria: the TOM complex in the outer membrane and two TIM complexes in the inner membrane. Recent work has provided a detailed view of the different mechanisms operating during the import of the two major classes of mitochondrial proteins--hydrophilic proteins with cleavable presequences and hydrophobic proteins with multiple internal signals.     

Identification of Tim40 that mediates protein sorting to the mitochondrial intermembrane space

Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria, and sorted to one of the four mitochondrial subcompartments. Here we identified a new inner membrane protein, Tim40, that mediates sorting of small Tim proteins to the intermembrane space. Tim40 is essential for yeast cell growth, and its function in vivo requires six conserved Cys residues but not anchoring of the protein to the inner membrane by its N-terminal hydrophobic segment. Depletion of Tim40 impairs the import of small Tim proteins into mitochondria both in vivo and in vitro. In wild-type mitochondria, Tim40 forms a translocation intermediate with small Tim proteins prior to their assembly in the intermembrane space in vitro. These results suggest the essential role of Tim40 in sorting/assembly of small Tim proteins.     

Processing of mitochondrial presequences

Mitochondrial proteins are synthesized as precursor proteins on either cytosolic or mitochondrial ribosomes. The synthesized precursors from both translation origins possess targeting signals that guide the protein to its final destination in one of the four subcompartments of the organelle. The majority of nuclear-encoded mitochondrial precursors and also mitochondrial-encoded preproteins have an N-terminal presequence that serves as a targeting sequence. Specific presequence peptidases that are found in the matrix, inner membrane and intermembrane space of mitochondria proteolytically remove the signal sequence upon import or sorting. Besides the classical presequence peptidases MPP, IMP and Oct1, several novel proteases have recently been described to possess precursor processing activity, and analysis of their functional relevance revealed a tight connection between precursor processing, mitochondrial dynamics and protein quality control. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.     

Electron tomographic and ultrastructural analysis of the Cryptosporidium parvum relict mitochondrion, its associated membranes, and organelles

Sporozoites of the apicomplexan Cryptosporidium parvum possess a small, membranous organelle sandwiched between the nucleus and crystalloid body. Based upon immunolabelling data, this organelle was identified as a relict mitochondrion. Transmission electron microscopy and tomographic reconstruction reveal the complex arrangement of membranes in the vicinity of this organelle, as well as its internal organization. The mitochondrion is enveloped by multiple segments of rough endoplasmic reticulum that extend from the outer nuclear envelope. In tomographic reconstructions of the mitochondrion, there is either a single, highly-folded inner membrane or multiple internal subcompartments (which might merge outside the reconstructed volume). The infoldings of the inner membrane lack the tubular "crista junctions" found in typical metazoan, fungal, and protist mitochondria. The absence of this highly conserved structural feature is congruent with the loss, through reductive evolution, of the normal oxidative phosphorylation machinery in C. parvum. It is proposed that the retention of a relict mitochondrion in C. parvum is a strategy for compartmentalizing away from the cytosol toxic ferrous iron and sulfide, which are needed for iron sulfur cluster biosynthesis, an essential function of mitochondria in all eukaryotes.     

Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.

Most chloroplast and mitochondrial proteins are synthesized with N-terminal presequences that direct their import into the appropriate organelle. In this report we have analyzed the specificity of standard in vitro assays for import into isolated pea chloroplasts and mitochondria. We find that chloroplast protein import is highly specific because mitochondrial proteins are not imported to any detectable levels. Surprisingly, however, pea mitochondria import a range of chloroplast protein precursors with the same efficiency as chloroplasts, including those of plastocyanin, the 33-kDa photosystem II protein, Hcf136, and coproporphyrinogen III oxidase. These import reactions are dependent on the Deltaphi across the inner mitochondrial membrane, and furthermore, marker enzyme assays and Western blotting studies exclude any import by contaminating chloroplasts in the preparation. The pea mitochondria specifically recognize information in the chloroplast-targeting presequences, because they also import a fusion comprising the presequence of coproporphyrinogen III oxidase linked to green fluorescent protein. However, the same construct is targeted exclusively into chloroplasts in vivo indicating that the in vitro mitochondrial import reactions are unphysiological, possibly because essential specificity factors are absent in these assays. Finally, we show that disruption of potential amphipathic helices in one presequence does not block import into pea mitochondria, indicating that other features are recognized.     

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 amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.     

Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins

Mitochondria import nuclear-encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.     

Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria

The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex in different translocation modes. Membrane-integrated part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments.     

MINOS1 is a conserved component of mitofilin complexes and required for mitochondrial function and cristae organization

The inner membrane of mitochondria is especially protein rich and displays a unique morphology characterized by large invaginations, the mitochondrial cristae, and the inner boundary membrane, which is in proximity to the outer membrane. Mitochondrial inner membrane proteins appear to be not evenly distributed in the inner membrane, but instead organize into functionally distinct subcompartments. It is unknown how the organization of the inner membrane is achieved. We identified MINOS1/MIO10 (C1orf151/YCL057C-A), a conserved mitochondrial inner membrane protein. mio10-mutant yeast cells are affected in growth on nonfermentable carbon sources and exhibit altered mitochondrial morphology. At the ultrastructural level, mutant mitochondria display loss of inner membrane organization. Proteomic analyses reveal MINOS1/Mio10 as a novel constituent of Mitofilin/Fcj1 complexes in human and yeast mitochondria. Thus our analyses reveal new insight into the composition of the mitochondrial inner membrane organizing machinery.     

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.     

Presence of a member of the mitochondrial carrier family in hydrogenosomes: conservation of membrane-targeting pathways between hydrogenosomes and mitochondria

A number of microaerophilic eukaryotes lack mitochondria but possess another organelle involved in energy metabolism, the hydrogenosome. Limited phylogenetic analyses of nuclear genes support a common origin for these two organelles. We have identified a protein of the mitochondrial carrier family in the hydrogenosome of Trichomonas vaginalis and have shown that this protein, Hmp31, is phylogenetically related to the mitochondrial ADP-ATP carrier (AAC). We demonstrate that the hydrogenosomal AAC can be targeted to the inner membrane of mitochondria isolated from Saccharomyces cerevisiae through the Tim9-Tim10 import pathway used for the assembly of mitochondrial carrier proteins. Conversely, yeast mitochondrial AAC can be targeted into the membranes of hydrogenosomes. The hydrogenosomal AAC contains a cleavable, N-terminal presequence; however, this sequence is not necessary for targeting the protein to the organelle. These data indicate that the membrane-targeting signal(s) for hydrogenosomal AAC is internal, similar to that found for mitochondrial carrier proteins. Our findings indicate that the membrane carriers and membrane protein-targeting machinery of hydrogenosomes and mitochondria have a common evolutionary origin. Together, they provide strong evidence that a single endosymbiont evolved into a progenitor organelle in early eukaryotic cells that ultimately give rise to these two distinct organelles and support the hydrogen hypothesis for the origin of the eukaryotic cell.     

Protein import into chloroplasts: new aspects of a well-known topic

Protein import into plant chloroplasts is a fascinating topic that is being investigated by many research groups. Since the majority of chloroplast proteins are synthesised as precursor proteins in the cytosol, they have to be posttranslationally imported into the organelle. For this purpose, most preproteins are synthesised with an N-terminal presequence, which is both necessary and sufficient for organelle recognition and translocation initiation. The import of preproteins is facilitated by two translocation machineries in the outer and inner envelope of chloroplasts, the Toc and Tic complexes, respectively. Translocation of precursor proteins across the envelope membrane has to be highly regulated to react to the metabolic requirements of the organelle. The aim of this review is to summarise the events that take place at the translocation machineries that are known so far. In addition, we focus in particular on alternative import pathways and the aspect of regulation of protein transport at the outer and inner envelope membrane.     

The human mitochondrial import receptor, hTom20p, prevents a cryptic matrix targeting sequence from gaining access to the protein translocation machinery

Yeast Mas70p and NADH cytochrome b5 reductase are bitopic integral proteins of the mitochondrial outer membrane and are inserted into the lipid-bilayer in an Nin-Ccyto orientation via an NH2-terminal signal- anchor sequence. The signal anchor of both proteins is comprised of a short, positively charged domain followed by the predicted transmembrane segment. The positively charged domain is capable of functioning independently as a matrix-targeting signal in yeast mitochondria in vitro but does not support import into mammalian mitochondria (rat or human). Rather, this domain represents a cryptic signal that can direct import into mammalian mitochondria only if proximal components of the outer membrane import machinery are removed. This can be accomplished either by treating the surface of the intact mitochondria with trypsin or by generating mitoplasts. The import receptor Tom20p (Mas20p/MOM19) is responsible for excluding the cryptic matrix-targeting signal from mammalian mitochondria since replacement of yeast Tom20p with the human receptor confers this property to the yeast organelle while at the same time maintaining import of other proteins. In addition to contributing to positive recognition of precursor proteins, therefore, the results suggest that hTom20p may also have the ability to screen potential matrix-targeting sequences and exclude certain proteins that would otherwise be recognized and imported by distal components of the outer and inner membrane protein- translocation machinery. These findings also indicate, however, that cryptic signals, if they exist within otherwise native precursor proteins, may remain topogenically silent until the precursor successfully clears hTom20p, at which time the activity of the cryptic signal is manifested and can contribute to subsequent translocation and sorting of the polypeptide.     

Protein import into mitochondria: origins and functions today (Review)

Mitochondria are organelles derived from α-proteobacteria over the course of one to two billion years. Mitochondria from the major eukaryotic lineages display some variation in functions and coding capacity but sequence analysis demonstrates them to be derived from a single common ancestral endosymbiont. The loss of assorted functions, the transfer of genes to the nucleus, and the acquisition of various ‘eukaryotic’ proteins have resulted in an organelle that contains approximately 1000 different proteins, with most of these proteins imported into the organelle across one or two membranes. A single translocase in the outer membrane and two translocases in the inner membrane mediate protein import. Comparative sequence analysis and functional complementation experiments suggest some components of the import pathways to be directly derived from the eubacterial endosymbiont's own proteins, and some to have arisen ‘de novo’ at the earliest stages of ‘mitochondrification’ of the endosymbiont. A third class of components appears lineage-specific, suggesting they were incorporated into the process of protein import long after mitochondria was established as an organelle and after the divergence of the various eukaryotic lineages. Protein sorting pathways inherited from the endosymbiont have been co-opted and play roles in intraorganelle protein sorting after import. The import apparatus of animals and fungi show significant similarity to one another, but vary considerably to the plant apparatus. Increasing complexity in the eukaryotic lineage, i.e., from single celled to multi-cellular life forms, has been accompanied by an expansion in genes encoding each component, resulting in small gene families encoding many components. The functional differences in these gene families remain to be elucidated, but point to a mosaic import apparatus that can be regulated by a variety of signals.