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.     

Import of proteins into mitochondria

A mounting body of evidence suggests that cytoplasmically synthesized proteins destined to be imported into the mitochondrial interior must at least partly unfold to penetrate across the mitochondrial membranes. During post-translational import, this unfolding process appears to be a major rate-limiting step. It can be blocked by ligands that stabilize the protein's native conformation and appears to be accompanied by the cleavage of ATP outside the mitochondrial inner membrane.     

The cytosolic factor required for import of precursors of mitochondrial proteins into mitochondria

The mechanism of import of proteins into mitochondria was studied by using the peptide of the presequence of ornithine aminotransferase (the extrapeptide), which was chemically synthesized and is composed of 34 amino acids. When the extrapeptide was incubated with isolated mitochondria in the presence of a rabbit reticulocyte lysate at 25 degrees C, it was imported into the mitochondrial matrix, and the import depended on the inner membrane potential, but not added ATP. The import of several precursors of mitochondrial proteins was competitively inhibited by the presence of excess extrapeptide in the reaction system, indicating that the extrapeptide and mitochondrial proteins were imported by the same machinery. Import of the extrapeptide was significantly stimulated by addition of a rabbit reticulocyte lysate, and a component of the lysate (the cytosolic factor) stimulating import of the extrapeptide was purified about 20,000 times by successive column chromatography on DEAE-cellulose and aminopentyl-Sepharose 4B. The binding of the extrapeptide to liposomes composed of egg lecithin and partially purified receptor of the precursor of mitochondrial protein (Ono, H., and Tuboi, S., (1985) Biochem. Int. 10, 351-357) required the cytosolic factor when the concentration of the peptide was less than 1.5 X 10(-8) M, suggesting that the physiological binding of the precursors of mitochondrial proteins to the receptor is dependent on the cytosolic factor. The extrapeptide and the cytosolic factor were shown to form a complex. From these results, the mechanism of binding of the extrapeptide to the receptor of the mitochondrial outer membrane is suggested to be as follows: the peptide (the precursor of mitochondrial protein) and the cytosolic factor form a complex, and then the complex is recognized by and bound to the receptor.     

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.     

Import of proteins into mitochondria and chloroplasts

Although mitochondria and chloroplasts synthesize some of their own proteins, they must import most of them from the cytosol. Import is mediated by molecular chaperones in the cytosol, receptors and channels in the organelle membranes and ATP-driven 'import motors' inside the organelles. Many of these components are now known, allowing informed guesses on how they might work.     

Import of proteins into mitochondria: a multi-step process

Translocation of precursor proteins from the cytosol into mitochondria is a multi-step process. The generation of translocation intermediates, i.e. the reversible accumulation of precursors at distinct stages of their import pathway into mitochondria ('translocation arrest'), has allowed the experimental characterization of distinct functional steps of protein import. These steps include: ATP-dependent unfolding of precursors; specific recognition of precursors by distinct receptors on the mitochondrial surface; interaction of precursors; specific recognition of precursors by distinct receptors on the mitochondrial surface; interaction of precursors with a general insertion protein ('GIP') in the outer mitochondrial membrane; membrane-potential-dependent translocation into the inner membrane at contact sites between both membranes; proteolytic processing of precursors; and intramitochondrial sorting of precursors via the matrix space ('conservative sorting'). The functional characteristics unveiled by studying mitochondrial protein import appear to be of general interest for investigations on intracellular protein sorting.     

Import of precursor proteins into mitochondria: site of polypeptide unfolding

The matrix-targeting signal of mitochondrial preornithine carbamyl transferase has been fused to either murine dihydrofolate reductase (pODHFR) or bacterial chloramphenicol acetyltransferase (pOCAT). Loosening of the tightly folded "native" structure of the two proteins following their synthesis in a rabbit reticulocyte lysate was assayed by the acquisition of protease sensitivity (pODHFR and pOCAT) or by the loss of enzyme activity (pOCAT). By these criteria, the bulk population of both precursor proteins was tightly folded following release from the ribosome, even in the presence of ATP and excess reticulocyte lysate. Neither protein unfolded as a consequence of binding to the surfaces of anionic liposomes or intact mitochondria. However, a non-native form of full-length pOCAT, exhibiting a loss of enzymatic activity and an enhanced protease sensitivity, was detected in association with a submitochondrial fraction that banded between the inner and outer mitochondrial membrane fractions on sucrose density gradients. Delivery of the precursor molecule to this position required ATP and a proteinaceous component on the surface of the organelle.     

The structure of precursor proteins during import into mitochondria

Precursor proteins must be at least partially unfolded during import into mitochondria, but their actual conformation during translocation is not known. Are proteins fully unfolded and threaded through the import machinery amino acid by amino acid, or do they retain some partial structure? The folding pathway of most proteins in vitro contains a partially folded intermediate known as the molten globule state, and it has been suggested that proteins are in the molten globule state during translocation across membranes. Here we show that precursors are normally fully unfolded during import into mitochondria. However, precursors containing residual structure can be imported, if less efficiently.     

A synthetic presequence reversibly inhibits protein import into yeast mitochondria

We show that a synthetic peptide corresponding to the N-terminal 22 residues of the cytochrome c oxidase subunit IV presequence blocked import of pre-subunit IV into yeast mitochondria. The 22-residue peptide pL4-(1-22) did not alter the electrical potential across the mitochondrial inner membrane (the delta psi). Inhibition of import was reversible and could be overcome by the addition of increased amounts of precursor. Two other peptides, pL4-(1-16) and pL4-(1-23), which correspond to, respectively, the N-terminal 16 and 23 residues of the same presequence, also blocked import of pre-subunit IV. However, pL4-(1-16) was a much weaker inhibitor of import, while the inhibitory effect of pL4-(1-23) was due to its ability to completely collapse the delta psi. pL4-(1-22) seems to be a general inhibitor of mitochondrial import, in that it also blocked uptake of several other proteins. These included the precursors of the yeast proteins cytochrome c oxidase subunit Va, the F1-ATPase beta subunit, mitochondrial malate dehydrogenase, and the ATP/ADP carrier. In addition, uptake of two non-yeast precursor proteins (human ornithine transcarbamylase and a cytochrome oxidase subunit IV-dihydrofolate reductase fusion), was also blocked by the peptide. Subsequent studies revealed that pL4-(1-22) did not block the initial recognition or binding of proteins to mitochondria. Rather, our results suggest that the peptide acts at a subsequent translocation step which is common to the import pathways of many different precursor proteins.     

Zinc-dependent intermembrane space proteins stimulate import of carrier proteins into plant mitochondria

Mitochondrial inner membrane carrier proteins are imported into mitochondria from yeast, fungi and mammals by specific machinery, some components of which are distinct from those utilized by other proteins. Import of two different carriers into plant mitochondria showed that one contains a cleavable presequence which was processed during import, while the other imported in a valinomycin-sensitive manner without processing. Mild osmotic shock of mitochondria released intermembrane space (IMS) components and impaired carrier protein import. Adding back the released IMS proteins as a concentrate in the presence of micromolar ZnCl2 stimulated carrier import into IMS-depleted mitochondria, but did not stimulate import of a non-carrier control precursor protein, the alternative oxidase. Anion-exchange separation of IMS components before addition to IMS-depleted mitochondria revealed a correlation between several 9-10 kDa proteins and stimulation of carrier import. MS/MS sequencing of these proteins identified them as plant homologues of the yeast zinc-finger carrier import components Tim9 and Tim10. Stimulation of import was dependent on either Zn2+ or Cd2+ and inhibited by both N-ethylmalamide (NEM) and a divalent cation chelator, consistent with a functional requirement for a zinc finger protein. This represents direct functional evidence for a distinct carrier import pathway in plant mitochondria, and provides a tool for determining the potential function of other IMS proteins associated with protein import.     

Import and assembly of proteins into mitochondria of mammalian cells

Most of our knowledge regarding the process of protein import into mitochondria has come from research employing fungal systems. This review outlines recent advances in our understanding of this process in mammalian cells. In particular, we focus on the characterisation of cytosolic molecular chaperones that are involved in binding to mitochondrial-targeted preproteins, as well as the identification of both conserved and novel subunits of the import machineries of the outer and inner mitochondrial membranes. We also discuss diseases associated with defects in import and assembly of mitochondrial proteins and what is currently known about the regulation of import in mammals.     

Import of precursor proteins into mitochondria from soybean tissues during development

Characterisation of the amount of protein import of the alternative oxidase (AOX) and the F(A)d precursor proteins (previously shown to use different import pathways) into mitochondria from developing soybean tissues indicated that they displayed different patterns. Import of the AOX declined in both cotyledon and root mitochondria with increasing age, whereas the import of the F(A)d into cotyledon mitochondria remained high throughout the same period. Using primary leaf mitochondria, it was evident that import of AOX remained high while it declined in cotyledon and root mitochondria. The amount of import of the AOX into mitochondria from different tissues closely matched the amount of the Tom 20 receptor.     

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.     

Import of RNA into mitochondria

RNAs that function in mitochondria, in contrast to the majority of mitochondrial proteins, are generally encoded by the mitochondrial genome. However, evidence has been presented for transport of nucleus-encoded tRNAs into mitochondria in diverse organisms. While mitochondrial protein import has been characterized in great detail, virtually nothing is known about the pathway of RNA import into mitochondria. Only very recently have in vivo systems for RNA import been established, and these are now providing some insight into this intriguing process.     

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.     

A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites

Bovine pancreatic trypsin inhibitor (which contains three intramolecular disulfide bridges) was chemically coupled to the COOH terminus of a purified artificial mitochondrial precursor protein. When the resulting chimeric precursor was presented to energized isolated yeast mitochondria, its trypsin inhibitor moiety prevented the protein from completely entering the organelle; the protein remained stuck across both mitochondrial membranes, with its NH2 terminus in the matrix and its trypsin inhibitor moiety still exposed on the mitochondrial surface. The incompletely imported protein appeared to "jam" mitochondrial protein import sites since it blocked import of three authentic mitochondrial precursor proteins; it did not collapse the potential across the mitochondrial inner membrane. Quantification of the inhibition indicated that each isolated mitochondrial particle contains between 10(2) and 10(3) protein import sites.