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.     

Involvement of contact sites in phosphatidylserine import into liver mitochondria

The synthesis, translocation, and decarboxylation of phosphatidylserine can occur in a cell-free system (Voelker, D. R. (1989) J. Biol. Chem. 264, 8019-8025). We made use of the spatial separation of the site of biosynthesis and the site of decarboxylation of phosphatidylserine to demonstrate that mitochondrial contact sites are intimately involved in the translocation of phosphatidylserine prior to decarboxylation. In that sense, the inhibition of phosphatidylserine decarboxylase leads to an accumulation of this phospholipid in the contact site-enriched fractions without mixing the inner membrane phospholipid pool. On the other hand, newly synthesized phosphatidylethanolamine can be exported very rapidly to the mitochondrial surface in the same way, i.e. via contact sites. These data provide further evidence for the existence of a structural and functional microcompartmentation at the inner mitochondrial membrane surface.     

Presequence binding factor-dependent and -independent import of proteins into mitochondria.

A cytosolic protein factor(s) is involved in the import of precursor proteins into mitochondria. PBF (presequence binding factor) is a protein factor which binds to the precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) but not to the mature OTC, and is required for the mitochondrial import of pOTC. The precursors for aspartate aminotransferase and malate dehydrogenase as well as pOTC synthesized in a reticulocyte lysate were efficiently imported into the mitochondria. However, the precursors synthesized in the lysate depleted for PBF by treatment with pOTC-Sepharose were not imported. Readdition of the purified PBF to the depleted lysate fully restored the import. pOTC synthesized in the untreated lysate sedimented as a complex with a broad peak of around 9 S, whereas pOTC synthesized in the PBF-depleted lysate sedimented at an expected position of monomer (2.5 S). When the purified PBF was readded to the depleted lysate, pOTC sedimented as a complex of about 7 S. In contrast to most mitochondrial proteins, rat 3-oxoacyl-CoA thiolase is synthesized with no cleavable presequence and an NH2-terminal portion of the mature protein functions as a mitochondrial import signal. The thiolase synthesized in the PBF-depleted lysate could be efficiently imported into the mitochondria, and readdition of PBF had little effect on the import. The thiolase synthesized in the untreated, the PBF-depleted, or the PBF-readded lysate sedimented at an expected position of monomer (2.5 S). These observations provide support for the existence of PBF-dependent and -independent pathways of mitochondrial protein import.     

Mitochondrial porin of Neurospora crassa: cDNA cloning, in vitro expression and import into mitochondria.

cDNA encoding porin of Neurospora crassa, the major protein component of the outer mitochondrial membrane, was isolated and the nucleotide sequence was determined. The deduced protein sequence consists of 283 amino acids (29,979 daltons) and shows sequence homology of around 43% to yeast porin; however, no significant homology to bacterial porins was apparent. According to secondary structure predictions, mitochondrial porin consists mainly of membrane-spanning sided beta-sheets. Porin was efficiently synthesized in vitro from the cDNA; this allowed us to study in detail its import into mitochondria. Thereby, three characteristics of import were defined: (i) import depended on the presence of nucleoside triphosphates; (ii) involvement of a proteinaceous receptor-like component on the surface of the mitochondria was demonstrated; (iii) insertion into the outer membrane was resolved into at least two distinct steps: specific binding to high-affinity sites and subsequent assembly to the mature form.     

Functional definition of outer membrane proteins involved in preprotein import into mitochondria

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of beta-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.     

DNA import into mitochondria

In recent decades, it has become evident that the condition for normal functioning of mitochondria in higher eukaryotes is the presence of membrane transport systems of macromolecules (proteins and nucleic acids). Natural competence of the mitochondria in plants, animals, and yeasts to actively uptake DNA may be directly related to horizontal gene transfer into these organelles occurring at much higher rate compared to the nuclear and chloroplast genomes. However, in contrast with import of proteins and tRNAs, little is known about the biological role and molecular mechanism underlying import of DNA into eukaryotic mitochondria. In this review, we discuss current state of investigations in this area, particularly specificity of DNA import into mitochondria and its features in plants, animals, and yeasts; a tentative mechanism of DNA import across the mitochondrial outer and inner membranes; experimental data evidencing several existing, but not yet fully understood mechanisms of DNA transfer into mitochondria. Currently available data regarding transport of informational macromolecules (DNA, RNA, and proteins) into the mitochondria do not rule out that the mechanism of protein and tRNA import as well as tRNA and DNA import into the mitochondria may partially overlap.     

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.     

Unfolding of preproteins upon import into mitochondria.

Unfolding of preproteins and translocation across the mitochondrial membranes requires their interaction with mt-Hsp70 and Tim44 at the inner face of the inner membrane and ATP as an energy source. We measured the temperature dependence of the rates of unfolding and import into the matrix of two folded passenger domains, the tightly folded heme-binding domain (HBD) of cytochrome b2 and the loosely folded mouse dihydrofolate reductase (DHFR). Despite the stability of the HBD, its rates of thermal breathing were fast and the preprotein was imported rapidly at all temperatures. In contrast, rates of unfolding and import of DHFR were strongly temperature dependent and import was significantly slower than unfolding. In addition, import rates of DHFR were strongly dependent on the length of the presequence. We propose that the mitochondrial import motor does not exert a constant pulling force. Rather, mt-Hsp70 appears to release a translocating polypeptide chain such that the precursor can then slide back and refold on the surface of the mitochondria. Refolding competes with translocation, and passengers may undergo several rounds of unfolding and refolding prior to their import.     

Protein import into plant mitochondria

Plant Molecular Biology -     

High-affinity binding sites for prostaglandin E on human lymphocytes.

Binding sites on human lymphocytes for prostaglandins were examined by incubating cells with [³H]prostaglandin (PG) A₁, E₁, E₂, F_1α, and F_2α. Specific reversible binding for [³H]PGE₁ and E₂ was found with a K_d of ~2 × 10^?9M and a B max of ~200 binding sites per cell, assuming uniform distribution. We detected no specific binding of [³H]PGA₁, F_1α, or F_2α to lymphocytes. Also, the addition of 10- to 1000-fold greater amounts of unlabeled PGA, F_1α, or F_2α did not inhibit the binding of [³H]PGE. The time course of [³H]PGE binding appeared to be bimodal with one component complete within 5 min at 37 °C and another component of binding increasing over a 40-min incubation. We feel that the rapid component of binding may represent cell surface receptors for PGE while the slower component may represent a specific uptake mechanism for PGE into the cell. Glass adherent cells had fewer binding sites than nonadherent cells. Preincubation of the cells overnight resulted in a loss of binding sites.     

Directed import of macromolecules into mitochondria

Mitochondria are multifunctional eukaryotic organelles that provide cells with energy via oxidative phosphorylation. They participate in the formation of Fe-S clusters, oxidation of fatty acids, and synthesis of certain amino acids and play an important role in apoptosis. Mitochondria have their own genome and are able to transcribe and translate it. However, most macromolecules functioning in mitochondria, such as proteins and some small RNAs, are imported from the cytoplasm. Protein import into mitochondria is a universal process, and its mechanism is very similar in all eukaryotic cells. Today this mechanism is known in detail. At the same time, the RNA import was discovered only in several eukaryotic groups. Nevertheless, it is proposed that this process is typical for most species. A set of imported RNA molecules varies in different organisms. Although the knowledge about the mechanisms of RNA import is less extensive than that of protein import, it becomes clear that these mechanisms greatly differ between different species. The review summarizes information about the import of such macromolecules into mitochondria.     

Directed import of macromolecules into mitochondria

Mitochondria are multifunctional organelles of eukaryotic cells that provide the energy for the cells by oxidative phosphorylation, play an important role in the apoptosis and take part in Fe-S clusters formation, fatty acids oxidation and synthesis of some aminoacids. They contain their own genome and are able to transcribe and to translate it. However, the vast majority of the macromolecules which function inside the mitochondria are imported into these organelles from the cytoplasm. The imported macromolecules include proteins and several types of small RNAs. Protein import is a universal process and its mechanism is conserved among all species. This mechanism is now known in detail. RNA import was shown to occur in several groups of eukaryotes, while the pool of imported RNA molecules varies in different organisms. Although the knowledge about the mechanisms of RNA import is less extensive than for the proteins, it becomes clear that these mechanisms are not universal among all the species possessing this pathway. In this review, we summarize the data about the import of macromolecules mentioned above into mitochondria.     

Mechanisms of protein import into mitochondria

Apart from a handful of proteins encoded by the mitochondrial genome, most proteins residing in this organelle are nuclear-encoded and synthesised in the cytosol. Thus, delivery of proteins to their final destination depends on a network of specialised import components that form at least four main translocation complexes. The import machinery ensures that proteins earmarked for the mitochondrion are recognised and delivered to the organelle, transported across membranes, sorted to the correct compartment and assisted in overcoming energetic barriers.     

Characterization of dicyclohexylcarbodiimide binding sites in beef-heart mitochondria.

Binding studies with [¹⁴C]-dicyclohexylcarbodiimide showed the presence of binding sites in the beef-heart mitochondrial membrane at a concentration of 1.8 nmol/mg protein (1.4 sites per cytochrome a+a₃). Saturation of these sites correlated with the inhibition of the ATPase activity. The maximum binding capacity could be related with the amount of F₁-ATPase in mitochondria from various tissues.     

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.