Prechaperonin 60 and preornithine transcarbamylase share componunts of the import apparatus but have distinct maturation pathways in rat liver mitochondria

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Abstract
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 processinw 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‐PTC 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 insest cells to process p‐OTC fully is not a characteristic of cells grown in culture since cultured rat hepatoma cells process p‐OTC 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‐qlbumin conjugates. The differenses 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.

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