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Most mitochondrial proteins are nuclear encoded, synthesized on cytosolic ribosomes, and imported into the mitochondria. We have identified and characterized a 309 amino acid human protein with a molecular weight of 34 kDa that functions as a subunit of the translocase for the import of such proteins. hTom34 (34-kDa Translocase of the Outer Mitochondrial Membrane) is displayed on the surface of mitochondria and is resistant to extraction under alkaline conditions. Antibodies raised against hTom34 specifically inhibit in vitro import of the mitochondrial precursor protein preornithine transcarbamylase into mitochondria isolated from rat liver. Based on trypsin digestion experiments, the receptor has a large (27 kDa) C-terminal domain exposed to the cytosol. This novel component of the protein import machinery possesses a 62 residue motif conserved with the Tom70 family of mitochondrial receptors but otherwise appears to have no counterpart so far characterized in the mitochondria of any other species.

Mitochondrial preornithine transcarbamylase (p-OTC) and premalate dehydrogenase (p-MDH) are the only two matrix-located preproteins so far identified for which the proteolytic processing in vitro requires the formation of genuine processing intermediates, i-OTC and i-MDH, respectively. To establish the processing of other preproteins during import with respect to the two-step processing of p-OTC and p-MDH, the chelators EDTA and 1,10-phenanthroline were used to study the import and processing of rat prechaperonin 60 (p-cpn60) and p-OTC by mitochondria from four cpn60-containing organs. We found no evidence for a secondary processing step in the maturation of p-cpn60, but a clear requirement for two-step processing of p-OTC, even in three organs which do not contain ornithine transcarbamylase. The metal-ion requirement of the p-OTC processing activities in the organelle is consistent with the proposition that the mitochondrial processing protease (MPP) and mitochondrial intermediate peptidase (MIP) activities defined in vitro [Kalousek, F., Hendrick, J.P. & Rosenberg, L. E. (1988) Proc. Natl Acad. Sci. USA 85, 7536-7540] are responsible for precursor processing in vivo. The authenticity of two-step processing in vivo was, furthermore, established by demonstrating that i-OTC accumulates to high levels in Spodoptora frugiperda insect cells supplemented with MnCl2. The inability of the insect cells to process p-OTC fully is not a characteristic of cells grown in culture since cultured rat hepatoma cells process p-OTC to the fully processed m-OTC. Finally, we find that the import and processing of p-cpn60 and p-OTC is inhibited in an identical fashion by presequence-bovine-serum-albumin conjugates. The differences in proteolytic maturation between p-cpn60 and p-OTC are therefore not likely to result from different import pathways as the two precursors compete for common components of the import apparatus.

We have identified a 10-kDa stress-inducible mitochondrial protein. The protein is synthesized at elevated rates in cultured rat hepatoma cells challenged with heat shock or amino acid analogues and, therefore, designated heat shock protein 10 (Hsp10). Hsp10 was purified to homogeneity from rat liver and found to exhibit a native molecular mass of 65 kDa, as opposed to a monomeric molecular mass of 10,813.4 +/- 0.41 Da. The amino acid sequence of rat Hsp10 disclosed extensive sequence similarity with bacterial chaperonin (Cpn) 10. Rat Hsp10 and Escherichia coli Cpn60 were used to reconstitute functional trimeric rat ornithine transcarbamoylase from a chemically denatured state with high efficiency. This process depended completely upon rat Hsp10 and was abolished in the presence of a nonhydrolyzable ATP analogue. We conclude that Hsp10 is a eukaryotic Cpn10 homologue and, therefore, together with Cpn60 essential for mitochondrial protein biogenesis. The Cpn-mediated protein-folding apparatus, thus, exhibits a high degree of conservation between prokaryotes and mitochondria of higher eukaryotes.

Cell-free extracts from phaseolotoxin-producing strains of Pseudomonas syringae pv. phaseolicola grown at 18 degrees C, the optimum temperature for phaseolotoxin production, contain an ornithine carbamoyltransferase activity that is insensitive to phaseolotoxin. Extracts from the same strains grown at 30 degrees C, a temperature at which little or no detectable phaseolotoxin is produced, and from phaseolotoxin-nonproducing strains contain a phaseolotoxin-sensitive ornithine carbamoyltransferase activity. The phaseolotoxin-insensitive ornithine carbamoyltransferase activity is also less senstive to N delta-(phosphonacetyl)-L-ornithine than the phaseolotoxin-sensitive ornithine carbamoyltransferase activity of the corresponding strain.

The uptake of the cytoplasmically synthesized mammalian enzyme, ornithine transcarbamylase, into mitochondria is directed by an N-terminal peptide of 32 amino acids. We have investigated some of the structural requirements for the import of the enzyme from rat liver into isolated mitochondria and into mitochondria of COS cells transfected with cDNA encoding the precursor form of ornithine transcarbamylase. Deletion of 21 amino acids from the N terminus of the leader peptide blocked the import of the precursor; deletion of 5 amino acids at positions 15-19 from the N terminus of the leader peptide had no deleterious effect on the import of the enzyme, nor on the processing and assembly of subunits in mitochondria. The region deleted contained three of eight basic residues in the leader peptide suggesting that specific structural elements containing basic residues, rather than the general basic nature of the leader, may be involved in mitochondrial import.

Urea cycle function was evaluated in liver obtained from six patients with Reye's syndrome and from five control subjects. Reye's syndrome patients demonstrated normal activities for the extramitochondrial portion of the urea cycle, but showed marked abnormalities of the mitochondrial enzymes, i.e., carbamyl phosphate synthetase (CPS) and ornithine transcarbamylase (OTC) (Tables 2,3). CPS activity was reduced to less than 15% of control values in all four patients from whom tissues was obtained during the first 72 hr after the onset of encephalopathy. Two patents from whom tissue was not obtained until after 9 days of symptoms showed no reduction in CPS activity. The OTC activity was also reduced (3-67% of control values) in the four patients from whom tissue was obtained early in the illness. In addition, greater than 60% reduction in Vmax and Km for carbamyl phosphate was noted in all four patients in whom sample size permitted kinetic analysis, including both patients in whom CPS and OTC activity were not markedly reduced. The same kinetic abnormality as well as decreased CPS activity were experimentally produced in normal rate liver incubated in the presence of 1.0 mM 4-pentenoic acid, a short chain fatty acid and known hepatic mitochondrial toxin (Table 4).