Rapid degradation of the presequence of the f1beta precursor of the ATP synthase inside mitochondria.

We have investigated the fate of the presequence of an overexpressed protein derived from the precursor of the F(1)beta subunit of ATP synthase after import and processing in mitochondria. Our studies revealed a rapid degradation of the presequence inside mitochondria catalysed by matrix-located protease(s). In contrast, the mature portion of the precursor was not degraded. This is the first experimental evidence of the rapid degradation of a mitochondrial presequence in organello after in vitro import and processing.

The precursor of the F1beta subunit of the ATP synthase is covalently modified upon binding to plant mitochondrial.

We present evidence for a unique covalent modification of a nuclear-encoded precursor protein targeted to plant mitochondria. We investigated the early events of in vitro import for the mitochondrial precursor of the ATP synthase F1beta subunit from Nicotiana plumbaginifolia (pF1beta) into plant mitochondria. When pF1beta of 59 kDa was incubated with mitochondria isolated from different higher-plant species, a band of 61 kDa was generated. The 61 kDa protein was a covalently modified form of the 59 kDa pF1beta. The modification was dependent on the 25 amino acid long N-terminal region of the presequence of pF1beta. The modification was catalysed by an enzyme located in the outer mitochondrial membrane which was specific for higher plants and could not be washed off from the membrane by urea, KCl or EDTA. The modification was ATP- and Ca(2+)-dependent, but it was not affected by inhibitors of protein kinases. No inhibition of the modification was observed with phosphatase, methylation or acylation inhibitors. The modification occurs prior to translocation through the mitochondrial outer membrane. Inhibition of the modification process does not affect the import of the precursor protein, hence precursor modification was not a prerequisite for import. Both the modified and the unmodified pF1beta proteins were strongly associated with the mitochondrial outer membrane.

Chemical cleavage of the overexpressed mitochondrial F1beta precursor with CNBr: a new strategy to construct an import-competent preprotein.

We have isolated a soluble import-competent 15 kDa N-terminal fragment of the overexpressed Nicotiana plumbaginifolia F1beta precursor of the ATP synthase (N15pF1beta). The isolation was achieved after chemical cleavage, with CNBr, of the insoluble precursor collected in inclusion bodies, followed by purification of the fragment using ion-exchange chromatography. The purity of the final product was estimated to be more than 99%. N15pF1beta contained a presequence of 54 amino acid residues (except for the N-terminal methionine residue) and 82 N-terminal residues of the mature protein. N15pF1beta was shown to be imported into isolated potato tuber mitochondria and to be processed by the isolated mitochondrial processing peptidase (MPP) integrated into the cytochrome bc1 complex of the respiratory chain. Addition of N15pF1beta at micromolar concentrations resulted in the inhibition of import of F1beta precursor and alternative oxidase precursor, synthesized in vitro, into isolated mitochondria as well as the processing of these precursors catalysed by the isolated MPP-bc1 complex. N15pF1beta conjugated via a biotin link to avidin blocked import sites even after the reisolation of mitochondria and inhibited the import of the mitochondrial precursors, indicating that it can be used as a substrate for the generation of a stable translocation intermediate. Our results present a novel procedure for the production of an N-terminal fragment of the F1beta precursor that contains all information necessary for mitochondrial targeting and processing and that can be used for structural and functional studies of the mitochondrial protein import system. This procedure has a general value because it can be used for the production of chemical quantities of any mitochondrial import substrate and presequence peptide.

Inhibition of protein import into mitochondria by amphiphilic cations: potential targets and mechanism of action.

In this paper we describe for the first time the inhibitory effect of three amphiphilic cations, trifluoperazine, propranolol and dibucaine on mitochondrial protein import. The amphiphilic cations did not affect binding of mitochondrial precursor proteins to mitochondria. Import into mitoplasts was affected in a similar manner to intact mitochondria, indicating that the protein import machinery of the inner membrane of mitochondria was responsible for the observed effect. At concentrations which completely inhibited protein import, the amphiphilic cations did not affect the membrane potential (DeltaPsi) across the inner membrane. The inhibitory potency of amphiphilic cations reflects their lipid/water partition coefficient and relatively high concentrations of the drugs were required for complete inhibition, hence we propose that the mechanism of protein import inhibition by amphiphilic cations is due to membrane perturbing effects. We discuss the implications of our findings in view of the possible connection between various inner mitochondrial membrane channels and the protein import pore.

Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import.

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F1 beta subunit of the ATP synthase (pF1 beta) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu2+ were inhibitory. Inhibition by Cu2+ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondrial than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu2+ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF1 beta with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu2+ were inhibitory. NEM, MPB, DTNB and Cu2+ inhibited import of the NEM-modified pF1 beta into intact mitochondria. Import of pF1 beta through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu2+ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.

[Protein composition of venoms from several species of tropical ants and their effect on mitochondrial H+-ATPase]. / Issledovanie belkovogo sostava iadov nekotorykh vidov tropicheskikh murav'ev i ikh vliianiia na H+-ATPazu mitokhondrii.

Protein compositions of venoms of South-American stinging ants, Ectatomma tuberculatum, Paraponera clavata (subfamily Ponerinae), and "tangarana" were analyzed. The venom of E. tuberculatum displayed the most complex protein composition (more than 15 polypeptides). The water-soluble fraction of the venoms of P. clavata and "tangarana" contained acidic proteins (pI < 3.5 to 5.2), whereas the venom of E. tuberculatum contained predominantly basic proteins (pI 8 to > 9.5). N-Terminal residues and N-terminal sequences of a number of polypeptides were determined. High-molecular-mass polypeptides of the P. clavata venom slightly stimulated the ATPase activity of mitochondrial F1-ATPase. Low-molecular-mass nonprotein components of this venom significantly inhibited the ATPase activity of submitochondrial particles and F1-ATPase from bovine heart mitochondria. The venoms of E. tuberculatum and "tangarana" produced no effect on the ATPase activity.

[Study of the structural organization of OSCP--subunits of H+-ATPase of porcine heart mitochondria]. / Issledovanie strukturnoi organizatsii OSCP--sub''edinitsy H+-ATP-azy mitokhondrii serdtsa svin'i.

Unmodified and citraconilated OSCP of the pig heart mitochondrial H(+)-ATPase were hydrolysed by proteinase from Staphylococcus aureus V8 and trypsin, respectively. To purify the individual peptides, various types of HPLC and covalent chromatography on SH-Sepharose were used. By the automatic Edman method complete or partial amino acid sequences of the peptides obtained were determined, thus allowing for the reconstruction of the primary structure of pig OSCP. A linear antigenic determinant recognizable by A1 monoclonal antibody against bovine OSCP, was localized. Studies showed Gly43 residue (bovine OSCP) to be replaced by Ala43 (pig OSCP), which is responsible for a decrease of the affinity of the monoclonal antibody A1 to pig OSCP. Comparative analysis of primary structures of bovine and pig OSCP was carried out.