Role of membrane contact sites in protein import into mitochondria

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long‐standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence‐carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.     

The assembly pathway of the mitochondrial carrier translocase involves four preprotein translocases

The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.     

Protein import into mitochondria of Neurospora crassa

Biogenesis of mitochondria requires import of several hundreds of different nuclear-encoded preproteins needed for mitochondrial structure and function. Import and sorting of these preproteins is a multistep process facilitated by complex proteinaceous machineries located in the mitochondrial outer and inner membranes. The translocase of the mitochondrial outer membrane, the TOM complex, comprises receptors which specifically recognize mitochondrial preproteins and a protein conducting channel formed by TOM40. The TOM complex is able to insert resident proteins into the outer membrane and to translocate proteins into the intermembrane space. For import of inner membrane or matrix proteins, the TOM complex cooperates with translocases of the inner membrane, the TIM complexes. During the past 30 years, intense research on fungi enabled the identification and mechanistic characterization of a number of different proteins involved in protein translocation. This review focuses on the contributions of the filamentous fungus Neurospora crassa to our current understanding of mitochondrial protein import, with special emphasis on the structure and function of the TOM complex.     

The Mitochondrial Oxidase Assembly Protein1 (Oxa1) Insertase Forms a Membrane Pore in Lipid Bilayers

The inner membrane of mitochondria is especially protein-rich. To direct proteins into the inner membrane, translocases mediate transport and membrane insertion of precursor proteins. Although the majority of mitochondrial proteins are imported from the cytoplasm, core subunits of respiratory chain complexes are inserted into the inner membrane from the matrix. Oxa1, a conserved membrane protein, mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. The molecular mechanism by which Oxa1 mediates insertion of membrane spans, entailing the translocation of hydrophilic domains across the inner membrane, is still unknown. We investigated if Oxa1 could act as a protein-conducting channel for precursor transport. Using a biophysical approach, we show that Oxa1 can form a pore capable of accommodating a translocating protein segment. After purification and reconstitution, Oxa1 acts as a cation-selective channel that specifically responds to mitochondrial export signals. The aqueous pore formed by Oxa1 displays highly dynamic characteristics with a restriction zone diameter between 0.6 and 2 nm, which would suffice for polypeptide translocation across the membrane. Single channel analyses revealed four discrete channels per active unit, suggesting that the Oxa1 complex forms several cooperative hydrophilic pores in the inner membrane. Hence, Oxa1 behaves as a pore-forming translocase that is regulated in a membrane potential and substrate-dependent manner.     

Fates of Sec,Tat, and YidC Translocases in Mitochondria and Other Eukaryotic Compartments

Formation of mitochondria by the conversion of a bacterial endosymbiont was a key moment in the evolution of eukaryotes. It was made possible by outsourcing the endosymbiont’s genetic control to the host nucleus, while developing the import machinery for proteins synthesized on cytosolic ribosomes. The original protein export machines of the nascent organelle remained to be repurposed or were completely abandoned. This review follows the evolutionary fates of three prokaryotic inner membrane translocases Sec, Tat, and YidC. Homologs of all three translocases can still be found in current mitochondria, but with different importance for mitochondrial function. Although the mitochondrial YidC homolog, Oxa1, became an omnipresent independent insertase, the other two remained only sporadically present in mitochondria. Only a single substrate is known for the mitochondrial Tat and no function has yet been assigned for the mitochondrial Sec. Finally, this review compares these ancestral mitochondrial proteins with their paralogs operating in the plastids and the endomembrane system.     

The alpha and the beta: protein translocation across mitochondrial and plastid outer membranes

In the evolution of mitochondria and plastids from endosymbiotic bacteria, most of the proteins that make up these organelles have become encoded by nuclear genes and must therefore be transported across the organellar membranes, following synthesis in the cytosol. The core component of the protein translocation machines in both the mitochondrial and plastid outer membranes appears to be a beta-barrel protein, perhaps a relic from their bacterial ancestry, distinguishing these translocases from the alpha-helical-based protein translocation pores found in all other eukaryotic membranes.     

Protein import into chloroplasts

Most chloroplastic proteins are encoded in the nucleus, synthesized on cytosolic ribosomes and subsequently imported into the organelle. In general, proteins destined for the chloroplast are synthesized as precursor proteins with a cleavable N-terminal presequence that mediates routing to the inside of the chloroplast. These precursor proteins have to be targeted to the correct organellar membrane surface after their release from the ribosome and furthermore they have to be maintained in a conformation suitable for translocation across the two envelope membranes. Recognition and import of most chloroplastic precursor proteins are accomplished by a jointly used translocation apparatus. Different but complementary studies of several groups converged recently in the identification of the outer envelope proteins OEP86, OEP75, OEP70 (a Hsp 70-related protein), OEP34, and of the inner envelope protein IEP110 as components of this translocation machinery. None of these proteins, except for OEP70, shows any homology to components of other protein translocases. The plastid import machinery thus seems to be an original development in evolution. Following translocation into the organelle, chloroplastic proteins are sorted to their suborganellar destination, i.e., the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen. This structural and evolutionary complexity of chloroplasts is reflected by a variety of routing mechanisms by which proteins reach their final location once inside the organelle. This review will focus on recent advances in the identification of components of the chloroplastic protein import machinery, and new insights into the pathways of inter-and intraorganellar sorting.     

Protein translocation pathways of the mitochondrion

The biogenesis of mitochondria depends on the coordinated import of precursor proteins from the cytosol coupled with the export of mitochondrially coded proteins from the matrix to the inner membrane. The mitochondria contain an elaborate network of protein translocases in the outer and inner membrane along with a battery of chaperones and processing enzymes in the matrix and intermembrane space to mediate protein translocation. A mitochondrial protein, often with an amino-terminal targeting sequence, is escorted through the cytosol by chaperones to the TOM complex (translocase of the outer membrane). After crossing the outer membrane, the import pathway diverges; however, one of two TIM complexes (translocase of inner membrane) is generally utilized. This review is focused on the later stages of protein import after the outer membrane has been crossed. An accompanying paper by Lithgow reviews the early stages of protein translocation.     

Evolution of mitochondrial TAT translocases illustrates the loss of bacterial protein transport machines in mitochondria

Background

Bacteria and mitochondria contain translocases that function to transport proteins across or insert proteins into their inner and outer membranes. Extant mitochondria retain some bacterial-derived translocases but have lost others. While BamA and YidC were integrated into general mitochondrial protein transport pathways (as Sam50 and Oxa1), the inner membrane TAT translocase, which uniquely transports folded proteins across the membrane, was retained sporadically across the eukaryote tree.

Results

We have identified mitochondrial TAT machinery in diverse eukaryotic lineages and define three different types of eukaryote-encoded TatABC-derived machineries (TatAC, TatBC and TatC-only). Here, we investigate TatAC and TatC-only machineries, which have not been studied previously. We show that mitochondria-encoded TatAC of the jakobid Andalucia godoyi represent the minimal functional pathway capable of substituting for the Escherichia coli TatABC complex and can transport at least one substrate. However, selected TatC-only machineries, from multiple eukaryotic lineages, were not capable of supporting the translocation of this substrate across the bacterial membrane. Despite the multiple losses of the TatC gene from the mitochondrial genome, the gene was never transferred to the cell nucleus. Although the major constraint preventing nuclear transfer of mitochondrial TatC is likely its high hydrophobicity, we show that in chloroplasts, such transfer of TatC was made possible due to modifications of the first transmembrane domain.

Conclusions

At its origin, mitochondria inherited three inner membrane translocases Sec, TAT and Oxa1 (YidC) from its bacterial ancestor. Our work shows for the first time that mitochondrial TAT has likely retained its unique function of transporting folded proteins at least in those few eukaryotes with TatA and TatC subunits encoded in the mitochondrial genome. However, mitochondria, in contrast to chloroplasts, abandoned the machinery multiple times in evolution. The overall lower hydrophobicity of the Oxa1 protein was likely the main reason why this translocase was nearly universally retained in mitochondrial biogenesis pathways.

     

Cooperation of translocase complexes in mitochondrial protein import

Most mitochondrial proteins are synthesized in the cytosol and imported into one of the four mitochondrial compartments: outer membrane, intermembrane space, inner membrane, and matrix. Each compartment contains protein complexes that interact with precursor proteins and promote their transport. These translocase complexes do not act as independent units but cooperate with each other and further membrane complexes in a dynamic manner. We propose that a regulated coupling of translocases is important for the coordination of preprotein translocation and efficient sorting to intramitochondrial compartments.     

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.     

Protein import into mitochondria

This review is focused on the import of processable precursor proteins into the mitochondrial matrix; the import of carrier proteins into the inner mitochondrial membrane is also briefly discussed. Post- and cotranslational theories of the import, specific features of the presequence structures, and effects of some cytosolic factors on the import of precursor proteins are reviewed. The data on the structure of the protein translocases of the outer (TOM complex) and the inner (TIM complex) membranes of mitochondria and the current models of the precursor protein import by these translocases are also summarized.     

Evolution and diversification of mitochondrial protein import systems

More than 95% of mitochondrial proteins are encoded in the nucleus, synthesised in the cytosol and imported into the organelle. The evolution of mitochondrial protein import systems was therefore a prerequisite for the conversion of the α-proteobacterial mitochondrial ancestor into an organelle. Here, I review that the origin of the mitochondrial outer membrane import receptors can best be understood by convergent evolution. Subsequently, I discuss an evolutionary scenario that was proposed to explain the diversification of the inner membrane carrier protein translocases between yeast and mammals. Finally, I illustrate a scenario that can explain how the two specialised inner membrane protein translocase complexes found in most eukaryotes were reduced to a single multifunctional one in trypanosomes.     

Evolution of mitochondrial protein biogenesis

Mitochondria and the nucleus are key features that distinguish eukaryotic cells from prokaryotic cells. Mitochondria originated from a bacterium that was endosymbiotically taken up by another cell more than a billion years ago. Subsequently, most mitochondrial genes were transferred and integrated into the host cell's genome, making the evolution of pathways for specific import of mitochondrial proteins necessary. The mitochondrial protein translocation machineries are composed of numerous subunits. Interestingly, many of these subunits are at least in part derived from bacterial proteins, although only few of them functioned in bacterial protein translocation. We propose that the primitive α-proteobacterium, which was once taken up by the eukaryote ancestor cell, contained a number of components that were utilized for the generation of mitochondrial import machineries. Many bacterial components of seemingly unrelated pathways were integrated to form the modern cooperative mitochondria-specific protein translocation system.     

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.     

Zim17, a novel zinc finger protein essential for protein import into mitochondria

Translocation of precursor proteins across the mitochondrial membranes requires the coordinated action of multisubunit translocases in the outer and inner membrane, and the driving force for translocation across the inner membrane is provided by the matrix-located heat shock protein 70 (mtHsp70). The central components of the protein import machinery are essential. Here we describe Zim17, an essential protein with a zinc finger motif involved in protein import into mitochondria. Comparative genomics suggested a correction to the open reading frame of YNL310c, the gene encoding Zim17 in Saccharomyces cerevisiae. The revised open reading frame codes for a classic mitochondrial targeting signal, which is processed from Zim17 in the mitochondrial matrix. Loss of Zim17 selectively diminishes import of proteins into the matrix of mitochondria, but this loss of Zim17 is partially suppressed by overexpression of the J-protein Pam18/Tim14. We propose that Zim17 functions as an example of a "fractured" J-protein, where a protein like Zim17 contributes a zinc finger domain to Type III J-proteins, in toto providing for substrate loading onto Hsp70.     

A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates.

Translocation of nuclear encoded preproteins into the mitochondrial matrix requires the coordinated action of two translocases: one (Tom) located in the outer mitochondrial membrane and the other (Tim) located in the inner membrane. These translocases reversibly cooperate during protein import. We have previously constructed a chimeric precursor (pPGPrA) consisting of an authentic mitochondrial precursor at the N terminus (Delta(1)-pyrroline-5-carboxylate dehydrogenase, pPut) linked, through glutathione S-transferase, to protein A. When pPGPrA is expressed in yeast, it becomes irreversibly arrested during translocation across the outer and inner mitochondrial membranes. Consequently, the two membranes of mitochondria become progressively "zippered" together, forming long stretches in which they are in close contact (Schülke, N., Sepuri, N. B. V., and Pain, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7314-7319). We now demonstrate that trapped PGPrA intermediates hold the import channels stably together and inhibit mitochondrial protein import and cell growth. Using IgG-Sepharose affinity chromatography of solubilized zippered membranes, we have isolated a multisubunit complex that contains all Tom and Tim components known to be essential for import of matrix-targeted proteins, namely Tom40, Tom22, Tim17, Tim23, Tim44, and matrix-localized Hsp70. Further characterization of this complex may shed light on structural features of the complete mitochondrial import machinery.     

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.     

Ancestral and derived protein import pathways in the mitochondrion of Reclinomonas americana

The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or "translocase in the inner mitochondrial membrane complex." Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution.     

Import of hybrid forms of CYP11A1 into yeast mitochondria

Heterologous expression in yeast of mCYP11A1 fusions with different topogenic signals of yeast mitochondrial proteins for artificial channeling to different translocases of the inner membrane was used to gain insight in the mechanism of its topogenesis in mitochondria. To ensure insertion of the CYP11A1 domain into the inner mitochondrial membrane during the process of translocation, topogenic sequences containing transmembrane segments of Bcs1p(1-83), DLD(1-72), and full-sized AAC protein were used when constructing modified forms of CYP11A1, and the Su9(1-112) addressing signal was included to stimulate membrane insertion of CYP11A1 after its translocation to the matrix. Alternatively, to promote slippage of the hybrid molecules into the matrix, the hybrid of mCYP11A1 with the precursor of steroidogenic mitochondria matrix protein adrenodoxin (preAd) was designed. The extra sequences used for intramitochondrial sorting of CYP11A1 apparently ensured predicted topology of hybrid molecules in yeast mitochondria. All of the addressing sequences, containing transmembrane domains, provided effective insertion of the hybrid proteins AAC-mCYP11A1, Bcs1p(1-83)-mCYP11A1, DLD(1-72)-mCYP11A1 and Su9(1-116)-mCYP11A1 into the inner membrane. preAd-mCYP11A1 hybrid molecules were shown to be translocated across the inner membrane and tightly associated with the membrane on its matrix side but not membrane inserted. Measuring specific activities of hybrid proteins in the mitochondrial fractions upon addition of Ad and AdR showed that the hybrids predetermined for cotranslocational insertion of CYP11A1 into the inner membrane were more active in the reaction of cholesterol side-chain cleavage than those destined for insertion on the matrix side of the IM, the Ad-mCYP11A1 hybrid demonstrating only residual enzyme activity. The data obtained reinforce the proposal that complete transfer of the polypeptide chain into the matrix is not a necessary stage in its topogenesis, but rather persistent interaction of the polypeptide chain with the membrane during the process of translocation is of importance for heme binding, folding and membrane insertion.