Prechaperonin 60 and preornithine transcarbamylase share components of the import apparatus but have distinct maturation pathways in rat liver mitochondria.
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.
Peralta
D
D
Department of Biochemistry, La Trobe University, Bundoora, Australia.
Lithgow
T
T
Hoogenraad
N J
NJ
Høj
P B
PB
D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015153:Blotting, Western; D018834:Chaperonin 60; D004492:Edetic Acid / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology / Q000648:ultrastructure; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D009928:Organ Specificity; D009954:Ornithine Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010618:Phenanthrolines / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013154:Spleen / Q000201:enzymology / Q000648:ultrastructure; D013737:Testis / Q000201:enzymology / Q000648:ultrastructure
D001426:Bacterial Proteins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D010618:Phenanthrolines; D011498:Protein Precursors; D004492:Edetic Acid; D009954:Ornithine Carbamoyltransferase; C025205:1,10-phenanthroline