L and D presequence peptides derived from the precursor of F1beta subunit of the ATP synthase inhibit mitochondrial protein import by interaction with import machinery.

We investigated the effect of L and D enantiomers of a 25-residue peptide derived from the N-terminal region of the presequence of Nicotiana plumbaginifolia F1beta subunit of the ATP synthase, pF1beta(1, 25), on import into spinach leaf mitochondria. Three in vitro synthesized precursor proteins using different import pathways were used. Import of the precursor proteins of F1beta subunit of the ATP synthase, pre-F1beta, and the alternative oxidase, pre-AOX, required addition of external ATP. whereas the chimeric precursor containing the N-terminal 84 amino acids of the cytochrome b2 precursor protein linked to dihydrofolate reductase, pre-b2(1, 84)-DHFR was not dependent on ATP. Import of pre-F1beta, and pre-AOX was inhibited already at 1 microM and 3 microM concentration of the L and D enantiomers, whereas inhibition of import of pre-b2(1, 84)-DHFR, occurred at concentrations >10 microM of both enantiomers. Binding efficiency of the precursor proteins was not affected by addition of the L and D enantiomers. There was no correlation between inhibition of import of pre-F1beta and pre-AOX and dissipation of membrane potential measured as a decrease of Rhodamine 123 fluorescence quenching. The inhibitory effect of the L and D presequence enantiomers on import of pre-F1beta and pre-AOX was concluded to occur within the outer membrane translocase machinery beyond the initial precursor receptor interaction. Furthermore, the fact that the D enantiomer had the same effect as the natural peptide showed that interaction of the presequence with the import machinery was not dependent on chiral properties of the presequence.

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria.

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic alpha-helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F1 beta subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.

Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria.

Import of the synthetic precursor of the alternative oxidase from soybean was shown to be dependent on a membrane potential and ATP. The membrane potential in soybean mitochondria may be formed either by respiration through the cytochrome pathway, or through the alternative oxidase pathway with NAD(+)-linked substrates. Import of the alternative oxidase precursor in the presence of succinate as respiratory substrate was inhibited by KCN. Import in the presence of malate was insensitive to KCN and SHAM added separately, but was inhibited by KCN and SHAM added together (inhibitors of the cytochrome and alternative oxidases respectively). Import of the alternative oxidase was accompanied by processing of the precursor to a single 32 kDa product in both cotyledon and root mitochondria. This product had a different mobility than the two alternative oxidase bands detected by immunological means (34 and 36 kDa), suggesting that the enzyme had been modified in situ. When the cDNA clone of the alternative oxidase was modified by a single mutation (-2 Arg changed to -2 Gly), the processing of the precursor was inhibited.

Tissue-specific differences of the mitochondrial protein import machinery: in vitro import, processing and degradation of the pre-F1 beta subunit of the ATP synthase in spinach leaf and root mitochondria.

In this study we report the first comparison of the mitochondrial protein import and processing events in two different tissues from the same organism. Both spinach leaf and root mitochondria were able to import and process the in vitro transcribed and translated Neurospora crassa F1 beta subunit of ATP synthase to the mature size product. Temperature optimum for protein import, 20 degrees C, was considerably lower than that found in other systems. In spinach leaf mitochondria, the processing peptidase has been shown to constitute an integral part of the bc1 complex of the respiratory chain. In accordance with these results, the majority of the processing activity in root mitochondria was also localized in the membrane. However, although the same amount of the processing peptidase was present per mg of membrane protein in both leaf and root mitochondria, as determined immunologically, the specific processing activity was several-fold higher in roots. Furthermore, in contrast to the processing enzyme in leaf, a portion of the processing activity could be disassociated from the root membrane with relatively weak salt treatment. The processing event in both the leaf and root membranes was always accompanied by a degradation of the F1 beta precursor. The degradation activity was found to be several-fold higher in roots than in leaves and was also partially dissociated from the membrane after salt treatment. Both the processing and degradation activities were inhibited by orthophenanthroline, a known metalloprotease inhibitor. These results show tissue-specific differences of the processing event catalyzed by the bc1 complex and indicate the presence of two populations of the processing peptidase in root mitochondria.

Antibacterial peptides and mitochondrial presequences affect mitochondrial coupling, respiration and protein import.

Cecropins A and P1, antibacterial peptides from insects and from pig and some related peptides released respiratory control, inhibited protein import and at higher concentrations also inhibited respiration. However, PR-39, an antibacterial peptide from pig intestine, was found to be almost inert towards mitochondria. The concentrations at which the three mitochondrial functions were effected varied for different peptides. Melittin, magainin and Cecropin-A-(1,13)-Melittin(1,13)-NH2, a hybrid between cecropin A and melittin, were most potent, while the two cecropins acted at higher concentrations. The biosynthesis of cecropin A is known and the intermediates are synthesized. We have used four peptides from this pathway to investigate their effects on coupling, respiration and protein import into mitochondria. Mature cecropin A followed by the preproprotein were most aggressive whereas the intermediates were less active or inert. The efficiency of different derivatives of cecropin A as uncouplers correlates well with their capacity to release membrane potential measured as fluorescence quenching of Rhodamine 123. Inhibition of respiration was found to be dependent on membrane potential and was most pronounced with mature cecropin A, less so with its three precursors. We also found that three peptides derived from mitochondrial presequences showed antibacterial activity. It is concluded that, there are similarities in the functions of antibacterial peptides and mitochondrial presequences, uncoupling activity in mitochondria cannot be correlated with the antibacterial activity (contrary to a previous suggestion), the processing of preprocecropin A may have evolved in such a way that there is a minimum of membrane damage from the intermediates in the pathway, and peptides produced for delivery outside of an animal have evolved to be more aggressive against mitochondria than peptides for delivery inside of the animal.

[Design of heterologous mitochondria: import of cattle cytochrome P-450SCC precursors in plant mitochondria]. / Konstruirovanie geterologicheskikh mitokhondrii: import predshestvennika tsitokhroma P-450SCC byka v rastitel'nye mitokhondrii.

It has been shown that pre-P-450scc of bovine adrenal cortex mitochondria synthesized in a rabbit reticulocyte lysate cell-free system, is translocated into isolated soybean cotyledon mitochondria, thereby taking the mature form size. This finding is suggestive of the occurrence of a specific receptor and maturase for pre-P-450scc in plant mitochondria. Thus, plant mitochondria can be used as recipients for the mammalian cholesterol hydroxylase system in an attempt to study the mechanism of its formation and preservation.

Import of the mammalian cytochrome P450 (scc) precursor into plant mitochondria.

According to previous reports (Matocha M. F. and Waterman M. R. (1984) J. Biol. Chem. 259, 8672-8678 and (1986) Arch. Biochem. Biophys. 250, 456-460) import of the cytochrome P450 (scc) precursor into mitochondria is tissue-specific. The present paper shows that in vitro synthesized bovine cytochrome P450 (scc) precursor can be imported into isolated soybean cotyledon mitochondria and processed therein to the mature size product. This shows that heterologous import of the cytochrome P450 (scc) precursor is possible and that import into mitochondria of this precursor is not restricted to steroidogenic tissues.

Absorption, gastrointestinal transit, and tablet erosion of felodipine extended-release (ER) tablets.

The gastrointestinal transit and tablet erosion of felodipine extended release (ER) tablets 10 mg were studied by gamma scintigraphy in eight healthy young males after administration under fasting and nonfasting conditions. Plasma concentrations of felodipine were also measured. Gastric emptying after administration together with food (mean, 3.2 hr) was slower in all subjects compared to emptying under fasting conditions (mean, 0.6 hr). The mean small intestinal transit times for the two study conditions did not differ significantly (5.1 and 4.7 hr, respectively). Tablets did not leave the colon in any subject within 14 hr after administration. Felodipine was shown to be absorbed in the colon, although the major part of the dose was absorbed in the small intestine. The absorption rate of felodipine was related to erosion of the hydrophilic matrix tablet. Tablet erosion and hence drug absorption were slower in the more distal parts of the gastrointestinal tract. Administration together with food did not significantly affect tablet erosion.