Mechanisms Of Mitochondrial Import Research Articles

Kinetic identification of a mitochondrial zinc uptake transport process in prostate cells

Prostate cells accumulate high cellular and mitochondrial concentrations of zinc, generally 3–10-fold higher than other mammalian cells. However, the mechanism of mitochondrial import and accumulation of zinc from cytosolic sources of zinc has not been established for these cells or for any mammalian cells. Since the cytosolic concentration of free Zn 2+ ions is negligible (estimates vary from 10 −9 to 10 −15 M), we postulated that loosely bound zinc–ligand complexes (Zn-Ligands) serve as zinc donor sources for mitochondrial import. Zinc chelated with citrate (Zn-Cit) is a major form of zinc in prostate and represents an important potential cytosolic source of transportable zinc into mitochondria. The mitochondrial uptake transport of zinc was studied with isolated mitochondrial preparations obtained from rat ventral prostate. The uptake rates of zinc from Zn-Ligands (citrate, aspartate, histidine, cysteine) and from ZnCl 2 (free Zn 2+) were essentially the same. No zinc uptake occurred from either Zn-EDTA, or Zn-EGTA. Zinc uptake exhibited Michaelis–Menten kinetics and characteristics of a functional energy-independent facilitative transporter associated with the mitochondrial inner membrane. The uptake and accumulation of zinc from various Zn-Ligand preparations with log K f (formation constant) values less than 11 was the same as for ZnCl 2; and was dependent upon the total zinc concentration independent of the free Zn 2+ ion concentration. Zn-Ligands with log K f values grater than 11 were not zinc donors. Therefore the putative zinc transporter exhibits an effective log K f∼11 and involves a direct exchange of zinc from Zn-Ligand to transporter. The uptake of zinc by liver mitochondria exhibited transport kinetics similar to prostate mitochondria. The results demonstrate the existence of a mitochondrial zinc uptake transporter that exists for the import of zinc from cytosolic Zn-Ligands. This provides the mechanism for mitochondrial zinc accumulation from the cytosol which contains a negligible concentration of free Zn 2+. The uniquely high accumulation of mitochondrial zinc in prostate cells appears to be due to their high cytosolic level of zinc-transportable ligands, particularly Zn-Cit.

Nucleotide sequence of a full-length cDNA coding for the mitochondrial precursor protein of the β-subunit of F1-ATPase fromNeurospora crassa

Subunits of mitochondrial H-ATPases are investigated under a variety of different aspects: (i) mechanisms of energy coupling (1, 2); (ii) evolution of ATPases (3) and (iii) mechanisms of mitochondrial import and assembly of the nuclear coded subunits (4—7). For the latter reason, we have cloned and expressed a full-length cDNA coding for the nuclear coded /8-subunit of the Fj-ATPase from Neurospora crassa. The nucleotide sequence and the deduced amino acid sequence are shown in Figure 1. The protein is synthesized in the cytosol as a precursor of 55,470 Da which is cleaved inside mitochondria to the mature protein. The mature protein shares 70 — 80% of sequence similarity to known mitochondrial /3 subunits, for example yeast (8), bovine (9) or human (10).

Complementation of a Schizosaccharomyces pombe mutant lacking the beta subunit of the mitochondrial ATPase by the ATP2 gene of Saccharomyces cerevisiae.

A chimeric plasmid carrying the structural gene (ATP2) for the mitochondrial ATPase beta subunit of Saccharomyces cerevisiae has been used to complement a mutant of Schizosaccharomyces pombe lacking the beta subunit (Boutry, M., and Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477). Transformation with ATP2 restored the growth rate of S. pombe mutant on glycerol as well as the mitochondrial ATPase and 32Pi-ATP exchange activities to approximately 20% of the parental strain. Mitochondria prepared from the transformant contained a normal amount of a hybrid F1-ATPase consisting of the S. cerevisiae beta subunit assembled with the remaining subunits of the S. pombe ATPase complex. The presence of the S. cerevisiae beta subunit in the S. pombe ATPase complex conferred a sensitivity to the energy transfer inhibitors citreoviridin and oligomycin which was like that of the intact S. cerevisiae enzyme. The S. cerevisiae beta subunit assembled into the hybrid ATPase complex was the same size as the mature subunit in S. cerevisiae. These data indicate that the mechanism of mitochondrial import and the assembly of the cytoplasmically synthesized subunits is similar or identical in these evolutionary divergent yeasts. In addition, this study provides a new approach for the construction of hybrid mitochondrial ATPase complexes which can be used to examine the function of selected subunits in energy transduction.

Mitochondrial importation of lipids and liponucleotides from microsomes independent of and facilitated by purified cytosol proteins.

The mitochondrial importation of microsomal lipids and liponucleotides in the presence and in the absence of partially purified cytosol protein(s) isolated from guinea pig liver was studied by the aid of isomeric (5-, 12-, and 16-(N-oxyl-4',4'-dimethyloxazolidine)stearoyl) spin-labelled radioactive phosphatide acid, phosphatidylcholine, neutral lipids, and CDP-diglycerides. Using a conventional procedure for the protein purification, cytosol protein(s) was purified approximately 1000-fold in respect to its ability to catalyze the translocation of isomeric spin-labelled lipids and liponucleotides from the microsomal to mitochondrial membranes. The highest activity of this protein was exhibited with biosynthesized spin-labelled lipids and liponucleotides bound to the microsomal membranes as substrates and the lowest, with the synthetic liponucleotides and derived lipids bound to the microsomal membranes. The partially purified protein was active in catalyzing the mitochondrial import of phospholipids from microsomes after heat treatment up to 90 degrees C. In addition to the cytosol protein catalyzing mechanism of mitochondrial import of lipids and liponucleotides from microsomal membranes, another cytosol protein independent mechanism of the mitochondrial importation of the same lipids and liponucleotides was also demonstrated in an agreement with our previous reports on the existence of cytosol protein independent intermembranous translocation of phospholipids. These experimental findings are discussed in terms of possible physiological significance and reaction mechanisms involved in the mitochondrial import of lipids and liponucleotides from the microsomal membranes of guinea pig liver.