Protein transport into mitochondria is conserved between plant and yeast species

Protein targeting into plant mitochondria was investigated by in vitro translocation experiments. The precursor of the mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia was synthesized in vitro, translocated to, processed, and assembled in purified Vicia faba mitochondria. Transport (but not binding) required a membrane potential and external nucleotides and was conserved among plant species. beta subunit precursors from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were imported and correctly processed in plant mitochondria. This translocation used protease-sensitive components of the outer membrane. Conversely, the N. plumbaginifolia beta subunit precursor was efficiently translocated and cleaved in yeast mitochondria. However, a precursor for a chloroplast protein was not targeted to plant or yeast mitochondria. We conclude that the machinery for protein import into mitochondria is specific and conserved in plant and yeast organisms. These results are discussed in the context of a poly- or monophyletic origin of mitochondria.     

Cryopreservation of Chlorophyll Synthesis and Apoprotein Stabilization in Barley Etioplasts

Methods for the cryopreservation of protein import and integration in pea chloroplasts and of protein import or protein synthesis in tobacco mitochondria were modified to yield enzymatically active cryopreserved etioplasts from barley (Hordeum vulgare L.). The cryoprotectants ethylene glycol and dimethy sulfoxide were about 64 and 77% effective, respectively, for the cryopreservation of etioplast intactness. Phototransformation of protochlorophyllide a, esterification of chlorophyllide a or zinc-pheophorbide a, and stabilization of the de novo synthesized plastid-encoded chlorophyll-apoproteins P700, CP47, CP43, D2, and D1 were successfully preserved in liquid nitrogen. Cryopreservation of freshly prepared intact etioplasts completely retained enzymatic activities for accumulation of chlorophyll a or resulted in a slightly decreased yield of zinc-pheophytin a.     

Highly efficient DNA synthesis in isolated mitochondria from rat liver.

We have developed a highly efficient DNA-synthesizing system with isolated intact rat liver mitochondria. The ATP requirements for this in organello DNA synthesis are provided by endogenous synthesis in the presence of exogenous ADP and an oxidizable substrate. In this system, mitochondrial DNA synthesis strikingly proceeds at a constant rate for about 5 h at 37 degrees C. Gel electrophoresis, hybridization and restriction enzyme analyses show that intact mitochondria synthesize nucleic acids with a size of 16.5 kb, that correspond to mitochondrial DNA, and that both DNA strands are replicated. This in organello DNA synthesis requires the supply of dNTPs and decreases at high ADP concentration in the incubation medium.     

Isolation and characterization of mitochondria from human B lymphoblastoid cell lines.

Mitochondria were isolated from detergent-treated Epstein-Barr virus-transformed human lymphocytes to examine their potential use in the study of the functional expression of genetic disorders of the respiratory chain. The increase of cytochrome c oxidase activity in the mitochondrial fraction indicated a 6-fold purification of intact mitochondria. Polarographic and spectrophotometric studies revealed that the isolated mitochondria were functionally well preserved. Furthermore, the isolated mitochondria supported an active in organello protein synthesis, which was dependent on the presence of a respiratory substrate generating ATP and was essentially abolished by chloramphenicol or by a specific respiratory chain inhibitor, such as antimycin. Thus, B lymphoblastoid cell lines constitute a valuable source of mitochondria to investigate mitochondrial functions in patients affected by respiratory chain disorders.     

Protein import into mitochondria in a homologous yeast in vitro system

To study the import of proteins into mitochondria we developed a homologous in vitro system in which mitochondria and cell-free translation extract are both derived from the yeast Saccharomyces cerevisiae. This system allows the synthesis of precursor proteins in the presence of isolated mitochondria and offers a means of analyzing yeast mutants defective in mitochondrial protein import. The in vitro import of an artificial precursor protein into yeast mitochondria in the presence of its substrate analog was analyzed subsequent to synthesis in either a yeast or rabbit reticulocyte cell-free translation reaction. Results suggest that a component(s) present in the yeast cytosolic extract may interact with the precursor protein.     

Protein synthesis in mitochondria purified from roots, leaves and flowers of sugar beet

Highly purified, intact and functional mitochondria were isolated from roots and leaves of a number of fertile and male-sterile lines of sugar beet ( Beta vulgaris L.). Intact and functional mitochondria were successfully isolated from the flowers of fertile plants, but not from the flowers of male-sterile plants. Several alternative methods for the homogenization of male-sterile flowers were tried. Their failure suggests that the mitochondria from male-sterile flowers are more sensitive to mechanical damage than mitochondria from fertile, or other organs of male-sterile, plants.
In organello protein synthesis was optimized with respect to the total concentration of amino acids, the concentration of [³⁵S]-methionine, pH and respiratory substrate. Inhibitor experiments showed that the mitochondrial preparations contained mitochondrial translational activity only. With the exception of one band, no processing or proteolytic breakdown in either root or leaf mitochondrial protein synthesis products could be detected in pulse-chase experiments. Submitochondrial fractionation experiments showed the presence of two soluble polypeptides, whereas all other polypeptides were membrane bound.
The polypeptide patterns of root, leaf and flower mitochondria were very similar with the exception of 4 polypeptides involved in glycine oxidation. These 4 polypeptides were present in large amounts in leaf mitochondria and just detectable in flower mitochondria. The patterns of polypeptides syntesized in mitochondria isolated from roots, leaves and flowers also showed a number of organ-specific differences. Six qualitative and 6 quantitative differences were found between mitochondria isolated from these three organs. No unique polypeptides were found to be synthesized either by flower mitochondria or by mitochondria from roots and leaves of male-sterile plants compared to their male-fertile counterparts.     

Characterization of a human import component of the mitochondrial outer membrane,TOMM70A

Functional mitochondria require up to 1000 proteins to function properly, with 99% synthesized as precursors in the cytoplasm and transported into the mitochondria with the aid of cytosolic chaperones and mitochondrial translocators (import components). Proteins to be imported are chaperoned to the mitochondria by the cytosolic heat shock protein (cHSP70) and are immediately pursued by Translocators of the Outer Membrane (TOMs), followed by transient interactions of the unfolded proteins with Translocators of the Inner Membrane (TIMs). In the present study, we describe a human gene, TOMM70A, orthologous to the yeast Tom70 import component. TOMM70A is ubiquitously expressed in human tissues, maps on chromosome 3q13.1-q13.2 and consists of 12 coding exons spanning over 37 kb. TOMM70A localizes in the mitochondria of COS-7 cells, and in organello import assays confirmed its presence in the Outer Mitochondrial membrane (OM) of rat liver mitochondria. TOMM70A could play a significant role in the import of nuclear-encoded mitochondrial proteins with internal targeting sites such as ADP/ATP carriers and the uncoupling proteins.     

The Involvement of Protein Phosphorylation/Dephosphorylation in the Redox Control of Translation in Cereal Mitochondria

A comparative analysis of a dependence of protein synthesis in mitochondria of cultivated (Zea mays) and wild (Elymus sibiricus) cereals on redox conditions showed that the addition of oxidized glutathione enhanced and the addition of reduced glutathione suppressed translation in organello. Inhibitors of protein kinases and protein phosphatases modified substantially the effects of redox agents on protein synthesis in mitochondria. It is supposed that protein phosphorylation in mitochondria may be a mechanism mediating the interrelation between the redox state of the respiratory chain and the activity of mitochondrial translation.     

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.     

The First Steps of Protein Import into Mitochondria

Protein import into mitochondria is initiated by the recognition and binding of precursor proteins by import components in the cytosol, on the mitochondrial surface, and in the mitochondrial outer membrane. Following their synthesis on cytoplasmic ribosomes, some precursor proteins interact with molecular chaperones in the cytosol which function in maintaining the precursor protein in an import-competent state and may also aid in the delivery of the precursor to the mitochondria. A multisubunit protein import receptor then recognises and binds precursor proteins before feeding them into the outer membrane import site. Some proteins are sorted from the import site into the outer membrane, but most precursor proteins travel through the outer membrane import site into the mitochondria, where the later steps of protein import take place.     

Absence of expression from RNA internalised into electroporated mammalian mitochondria

Transfection of mammalian mitochondria has proved to be notoriously difficult. Whilst there have been sporadic reports of import of foreign nucleic acids into isolated organelles, these imported nucleic acids have never been demonstrated to be functional. Inability to manipulate mitochondrial gene expression has hampered our understanding of RNA processing, maturation and translation in mitochondria. In an attempt to establish a model system for mt-RNA expression, we have electroporated rat liver mitochondria and mitoplasts in the presence of various RNA constructs built around the mitochondrial reporter gene mt-luciferase. Following electroporation, a fraction of the RNA was shown to be stably maintained, mitochondria remained coupled for oxidative phosphorylation and intramitochondrial protein synthesis was unaffected. In no case, however, was this RNA translated.     

Coupling of protein synthesis and mitochondrial import in a homologous yeast in vitro system.

We made use of a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae to investigate the coupling of protein synthesis and import. Mitochondrial precursor proteins were synthesized in a yeast lysate either in the presence or absence of isolated yeast mitochondria. We were, therefore, able to analyze protein import into mitochondria either in a strictly posttranslational reaction (when isolated mitochondria were added only after protein synthesis has been arrested by the addition of cycloheximide) or in a reaction in which synthesis and import were permitted to occur simultaneously. We found that the import of a precursor protein consisting of the amino-terminal mitochondrial targeting sequence of cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase is very inefficient in a strictly posttranslational reaction, whereas efficient import is observed if precursor synthesis and import are coupled. The same result was obtained when we analyzed the import of bulk endogenous yeast mitochondrial proteins in this system. Finally, we found that the insertion of the yeast outer membrane protein porin is also several times more efficient when synthesis and insertion are coupled.     

Protein import into yeast mitochondria: the inner membrane import site protein ISP45 is the MPI1 gene product.

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross-linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p-beta-galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right-side-out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.     

Sorting of precursor proteins between isolated spinach leaf mitochondria and chloroplasts

The precursors of the F₁-ATPase -subunits fromNicotiana plumbaginifolia andNeurospora crassa were imported into isolated spinach (Spinacia oleracea L.) leaf mitochondria. Both F₁ precursors were imported and processed to mature size products. No import of the mitochondrial precursor proteins into isolated intact spinach chloroplasts was seen. Moreover, the precursor of the 33 kDa protein of photosynthetic water-splitting enzyme was not imported into the leaf mitochondria. This study provides the first experimental report ofin vitro import of precursor proteins into plant mitochondria isolated from photosynthetic tissue and enables studies of protein sorting between mitochondria and chloroplasts in a system which is homologous with respect to organelles. The results suggest a high organellar specificity in the plant cell for the cytoplasmically synthesized precursor proteins.