Synthetic Partial Extension Peptides of P-450(SCC)and Adrenodoxin Precursors: Effects on the Import of Mitochondrial Enzyme Precursors

Abstract

Various portions of the extension peptides of P-450(SCC) precursor were chemically synthesized. The effects of these peptides on the import of enzyme precursors into mitochondria were examined. Peptides SEP1-15 and SEP1-20, corresponding to the amino terminal portion of the extension peptides, strongly inhibited the import of P-450(SCC) precursor into mitochondria. These peptides were effective at concentrations above 30 microM, and complete inhibition was observed at 100 microM. SEP1-11, which is shorter than SEP1-15 and SEP1-20, showed very weak inhibition. SEP25-39, which corresponds to the carboxy terminal portion of the extension peptide, did not affect the import of the precursor. The import of P-450(11 beta) and adrenodoxin precursors were also inhibited by SEP1-15. Another peptide, AEP1-14, which corresponds to the amino terminal portion of the extension peptide of adrenodoxin precursor, was also synthesized. The peptide inhibited the import of both adrenodoxin and P-450(SCC) precursors into mitochondria. The import of malate dehydrogenase was also inhibited by SEP1-15 and AEP1-14. The rate of the internalization of the precursor into mitochondria was decreased by the peptides. The amount of the precursor bound to the surface of mitochondria and the processing of adrenodoxin precursor were not affected. The respiratory activities of isolated mitochondria were not influenced by SEP1-15 even at 100 microM. We conclude that the inhibitory activities of the synthetic partial extension peptides on the import of enzyme precursors into mitochondria require the presence of about fifteen amino acid residues in the amino terminal portion of the extension peptides, and the inhibition of the import by the peptides was dependent on the blockage of the internalization of the precursors into mitochondria.

Mistargeting of peroxisomal L-alanine:glyoxylate aminotransferase to mitochondria in primary hyperoxaluria patients depends upon activation of a cryptic mitochondrial targeting sequence by a point mutation.

“…Significant levels of AGTLGI and pOTC still associated with mitochondria in the presence of PL but were readily digested by proteinase K (not shown). These results are in agreement with other similar recent studies using synthetic peptides corresponding to known MTSs, which concluded that such peptides inhibit import of a wide range of precursors at a level subsequent to surface binding of precursors (12,13).…”

A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins.

“…excess amount of purified cold pAd was present in the incubation mixture (panel A, closed bar), but not in the presence of mature adrenodoxin (panel A, shadowed bars); (ii) MSF was unable to depolymerize the import-incompetent mutant of pAd (Ou et al, unpublished results) in which Arg at positions 4 and 7 were changed into Ser (panel B); (iii) a synthetic peptide corresponding to the presequence portion of precytochrome P450 (SCC), which is a potent inhibitor of mitochondrial protein import (Furuya et al, 1987), also inhibited the depolymerization activity of MSF (data not shown). These lines of evidence, as well as the fact that MSF was affinity purified with the presequence of yeast COXIV, strongly indicated that MSF specifically recognized the presequences of the mitochondrial precursor proteins.…”

“…The assay mixture (50 ;1) contained 2.5 pl of pAd (Furuya et al, 1987) Sucrose density gradient centrifugation analysis Wheat germ translation mixtures (10 1u) containing pAd and mutated pAd were incubated with or without 200 ng of MSF in buffer A at 30°C for 50 min. The assay mixture (50 ;1) contained 2.5 pl of pAd (Furuya et al, 1987) Sucrose density gradient centrifugation analysis Wheat germ translation mixtures (10 1u) containing pAd and mutated pAd were incubated with or without 200 ng of MSF in buffer A at 30°C for 50 min.…”

Preferential distribution of group‐II‐like phospholipase A2 in mononuclear phagocytic cells in rat spleen and liver

“…It should be noted, however, that the spleen and liver group-I1 PLA2 species are structurally distinct, since the NH2-terminal amino acid sequence of the liver enzyme is different from that of the spleen enzyme at the NH2-terminus [ I l l . Moreover, as mentioned above, the presequence of the spleen PLAzM is characteristic of secretory proteins, whereas the precursor form of the liver mitochondrial PLA2 may have an extension peptide which is essential for its import into mitochondria [34], and is different from the signal peptide of secretory protein. This point remains to be clarified by sequence analysis of rat liver mitochondrial group 11 PLA2 cDNA.…”