A human pathology-related mutation prevents import of an aminoacyl-tRNA synthetase into mitochondria

Mutations in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase, a key enzyme for mitochondrial translation, are correlated with leukoencephalopathy. A Ser?? to Gly?? mutation is located in the predicted targeting signal of the protein. We demonstrate in the present study, by in vivo and in vitro approaches, that this pathology-related mutation impairs the import process across mitochondrial membranes.     

A purified precursor polypeptide requires a cytosolic protein fraction for import into mitochondria.

The beta-subunit of mitochondrial ATPase is coded by a nuclear gene, synthesized outside the mitochondria as a larger precursor and imported into mitochondria. The beta-subunit precursor was purified from yeast, both as a homogeneous, unlabeled polypeptide and in radiochemically pure form. Both precursor preparations were cleaved to the mature beta-subunit by partially purified processing protease from the mitochondrial matrix. However, import of the radiochemically pure precursor into isolated yeast mitochondria required a cytosolic fraction from yeast or reticulocytes. The cytosolic factor was non-dialyzable and trypsin-sensitive; its apparent mol. wt. was approximately 40 000 as judged by gel filtration. Import of some proteins into mitochondria thus requires proteins of the 'soluble' cytoplasm.     

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.     

The presequence of Arabidopsis serine hydroxymethyltransferase SHM2 selectively prevents import into mesophyll mitochondria

Serine hydroxymethyltransferases (SHMs) are important enzymes of cellular one-carbon metabolism and are essential for the photorespiratory glycine-into-serine conversion in leaf mesophyll mitochondria. In Arabidopsis (Arabidopsis thaliana), SHM1 has been identified as the photorespiratory isozyme, but little is known about the very similar SHM2. Although the mitochondrial location of SHM2 can be predicted, some data suggest that this particular isozyme could be inactive or not targeted into mitochondria. We report that SHM2 is a functional mitochondrial SHM. In leaves, the presequence of SHM2 selectively hinders targeting of the enzyme into mesophyll mitochondria. For this reason, the enzyme is confined to the vascular tissue of wild-type Arabidopsis, likely the protoxylem and/or adjacent cells, where it occurs together with SHM1. The resulting exclusion of SHM2 from the photorespiratory environment of mesophyll mitochondria explains why this enzyme cannot substitute for SHM1 in photorespiratory metabolism. Unlike the individual shm1 and shm2 null mutants, which require CO(2)-enriched air to inhibit photorespiration (shm1) or do not show any visible impairment (shm2), double-null mutants cannot survive in CO(2)-enriched air. It seems that SHM1 and SHM2 operate in a redundant manner in one-carbon metabolism of nonphotorespiring cells with a high demand of one-carbon units; for example, during lignification of vascular cells. We hypothesize that yet unknown kinetic properties of SHM2 might render this enzyme unsuitable for the high-folate conditions of photorespiring mesophyll mitochondria.     

A receptor for protein import into potato mitochondria

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.     

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.     

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.     

Distinct steps in the import of ADP/ATP carrier into mitochondria

Transport of the precursor to the ADP/ATP carrier from the cytosol into the mitochondrial inner membrane was resolved into several consecutive steps. The precursor protein was trapped at distinct stages of the import pathway and subsequently chased to the mature form. In a first reaction, the precursor interacts with a protease-sensitive component on the mitochondrial surface. It then reaches intermediate sites in the outer membrane which are saturable and where it is protected against proteases. This translocation intermediate can be extracted at alkaline pH. We suggest that it is anchored to the membrane by a so far unknown proteinaceous component. The membrane potential delta psi-dependent entrance of the ADP/ATP carrier into the inner membrane takes place at contact sites between outer and inner membranes. Completion of translocation into the inner membrane can occur in the absence of delta psi. A cytosolic component which is present in reticulocyte lysate and which interacts with isolated mitochondria is required for the specific binding of the precursor to mitochondria.     

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.     

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.     

Reconstitution of phosphatidylserine import into rat liver mitochondria

The synthesis translocation and decarboxylation of phosphatidylserine occurs in a cell-free system. The principal membrane components necessary are microsomes (source of phosphatidylserine synthase) and mitochondria (source of phosphatidylserine decarboxylase). The interorganelle translocation of phosphatidylserine can be measured by quantitating the decarboxylation of phosphatidyl[1'-14C]serine initially present in prelabeled microsomal membranes using a 14CO2 trapping assay. The decarboxylation of microsomal phosphatidylserine by intact mitochondria is 1) dependent upon substrate (microsomal membrane) concentration, 2) different from decarboxylation of liposomal phosphatidylserine, 3) resistant to proteases, 4) independent of soluble factors, and 5) unaffected by the addition of partially purified phospholipid exchange proteins but accelerated by purified nonspecific phospholipid exchange protein. The rate-limiting step in the reconstituted translocation-decarboxylation system is not the decarboxylation reaction but the initial translocation event between the microsomal membrane and the outer mitochondrial membrane. These data are interpreted to demonstrate that phosphatidylserine import into the mitochondria can occur via collision complexes formed between the endoplasmic reticulum or vesicles derived therefrom and the outer mitochondrial membrane.     

Short term 22Na+ and 42K+ uptake in intact, mid-vegetative plants

Spergularia marina (L.) Griseb. is. a rapidly growing, annual, coastal halophyte. Because of its small size, it is suitable for isotope studies of ion transport well beyond the seedling stage. The purpose of this report is to establish the similarities and differences between ²²Na⁺ and ⁴²K⁺ uptake in S. marina and in more commonly used mesophytic crop species. Vegetative plants were used 18 days after transfer to solution culture. Plants were grown either on Na⁺-free medium or on 0.2 × sea water. ²²Na⁺ uptake was linear with time for several hours. The rate was relatively insensitive to external concentration between 1 and 180 mol Na⁺ m?³, particularly in Na⁺-free plants. Transport to the shoot accounted for 40 to 70% of the total uptake, dependent on salinity but largely independent of time. ⁴²K⁺ uptake decreased with increasing salinity in Na⁺-free plants and increased in 0.2 × sea water plants. Both uptake and transport to the shoot were non-linear with time, upward concavity suggesting recovery from a manipulative and/or osmotic injury. Steady state root contents were compared with predicted contents based on cortical cell electrical potentials using the Nernst equation. Reasonable agreement was found in all cases except Na⁺ content of 0.2 × sea water plants, in which active efflux was indicated. Uptake studies conducted in the presence of chemical modifiers (dicyclohexylcarbodiimide, dinitrophenol and fusicoccin) showed responses of ⁴²K⁺ uptake as expected from studies on agronomic species, and implied the presence of a similar active uptake here despite the appearance of equilibrium. Active Na⁺ uptake was suggested at low Na⁺ levels. We conclude that S. marina is a promising experimental system combining the rapid nutrient acquisition strategy of agionomically important annuals with a high degree of salt tolerance.     

K+(Rb+) and Ca2+ fluxes in young winter wheat plants exposed to sub-zero temperatures

Ice crystal formation temperature was determined in the region of the crown in one group of 7-day-old intact unhardened high-salt plants of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) with TA (Thermal Analysis) and DTA (Differential Thermal Analysis) methods. After exposure of another group of plants, grown for the first 7 days in the same way as the first group, to various sub-zero temperatures (-1 to 5°C), influx in roots of Rb⁺(⁸⁶Rb⁺) and Ca²⁺(⁴⁵Ca²⁺) and contents of K⁺ and Ca²⁺ were determined at intervals during 7 days of recovery. Ice crystal formation in the crown tissue was probably extracellular and took place at about -4°C. There was a large loss of K⁺ from the roots after treatment at sub-zero temperatures. This loss increased as the temperature of the sub-zero treatment decreased. During recovery, roots of plants exposed to -1, -2 and -3°C gradually reabsorbed K⁺. Reabsorption of K⁺ in roots of plants exposed to -4°C was greatly impaired. Rb⁺ influx decreased and Ca²⁺ influx increased after sub-zero temperature treatments of the plants. Active Rb⁺ influx mechanisms and active extrusion of Ca²⁺ were impaired or irreversibly damaged by the exposure. While Rb⁺ influx mechanisms were apparently repaired during recovery in plants exposed to temperatures down to -3°C, Ca²⁺ extrusion mechanisms were not. The temperature for ice crystal formation in the region of the crown tissue coincides with the temperature at which the plants lost the ability to reabsorb K⁺ and to repair Rb⁺ influx mechanisms during the recovery period. Plants were lethally damaged at temperatures below ?4°C.     

In vivo import of plastocyanin and a fusion protein into developmentally different plastids of transgenic plants

Transgenic tomato plants that constitutively express a foreign plastocyanin gene were used to study protein transport in different tissues. Normally expression of endogenous plastocyanin genes in plants is restricted to photosynthetic tissues only, whereas this foreign plastocyanin protein is found to be present in all tissues examined. The protein is transported into the local plastids in these tissues and it is processed to the mature size. We conclude that plastids of developmentally different tissues are capable of importing precursor proteins that are normally not found in these tissues. Most likely such plastids, though functionally and morphologically differentiated, have similar or identical protein import mechanisms when compared to the chloroplasts in green tissue.     

70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria

We have developed an in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS). When mRNA for a nuclear-encoded yeast mitochondrial matrix protein, Put2, was translated in a wheat germ cell-free system, import into posttranslationally added yeast mitochondria was negligible. However, when a yeast PRS was added, significant import was observed. The import stimulating activity of the yeast PRS was shown to consist of at least two distinct factors. One of these is the recently purified 70-kD heat shock-related protein Ssalp/Ssa2p, two proteins that are 98% homologous. The other factor is an N-ethylmaleimide- sensitive protein(s). Both factors act synergistically.     

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.     

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 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.     

Convergent evolution of receptors for protein import into mitochondria

BACKGROUND: Mitochondria evolved from intracellular bacterial symbionts. Establishing mitochondria as organelles required a molecular machine to import proteins across the mitochondrial outer membrane. This machinery, the TOM complex, is composed of at least seven component parts, and its creation and evolution represented a sizeable challenge. Although there is good evidence that a core TOM complex, composed of three subunits, was established in the protomitochondria, we suggest that the receptor component of the TOM complex arose later in the evolution of this machine. RESULTS: We have solved by nuclear magnetic resonance the structure of the presequence binding receptor from the TOM complex of the plant Arabidopsis thaliana. The protein fold suggests that this protein, AtTom20, belongs to the tetratricopeptide repeat (TPR) superfamily, but it is unusual in that it contains insertions lengthening the helices of each TPR motif. Peptide titrations map the presequence binding site to a groove of the concave surface of the receptor. In vitro functional assays and peptide titrations suggest that the plant Tom20 is functionally equivalent to fungal and animal Tom20s. CONCLUSIONS: Comparison of the sequence and structure of Tom20 from plants and animals suggests that these two presequence binding receptors evolved from two distinct ancestral genes following the split of the animal and plant lineages. The need to bind equivalent mitochondrial targeting sequences and to make similar interactions within an equivalent protein translocation machine has driven the convergent evolution of two distinct proteins to a common structure and function.