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

ABSTRACT: 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.

SUBMITTER: Pavlov PF

PROVIDER: S-EPMC1220334 | biostudies-other | 1999 Jul

REPOSITORIES: biostudies-other

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Project description:Dengue virus infections, which have been reported in nearly 140 countries, pose a significant threat to human health. The genome of dengue virus encodes three structural and seven nonstructural (NS) proteins along with two untranslated regions, one each on both ends. Among them, dengue protease (NS3) plays a pivotal role in polyprotein processing and virus multiplication. NS3 is also known to regulate several host proteins to induce and maintain pathogenesis. Certain viral proteins are known to interact with mitochondrial membrane proteins and interfere with their functions, but the association of a virus-coded protein with the mitochondrial matrix is not known. In this report, by using in silico analysis, we show that NS3pro alone is capable of mitochondrial import; however, this is dependent on its innate mitochondrial transport signal (MTS). Transient-transfection and protein import studies confirm the import of NS3pro to the mitochondrial matrix. Similarly, NS3pro-helicase (amino acids 1 to 464 of NS3) also targets the mitochondria. Intriguingly, reduced levels of matrix-localized GrpE protein homolog 1 (GrpEL1), a cochaperone of mitochondrial Hsp70 (mtHsp70), were noticed in NS3pro-expressing, NS3pro-helicase-expressing, and virus-infected cells. Upon the use of purified components, GrpEL1 undergoes cleavage, and the cleavage sites have been mapped to KR81A and QR92S. Importantly, GrpEL1 levels are seriously compromised in severe dengue virus-infected clinical samples. Our studies provide novel insights into the import of NS3 into host mitochondria and identify a hitherto unknown factor, GrpEL1, as a cleavage target, thereby providing new avenues for dengue virus research and the design of potential therapeutics.IMPORTANCE Approximately 40% of the world's population is at risk of dengue virus infection. There is currently no specific drug or potential vaccine for these infections. Lack of complete understanding of the pathogenesis of the virus is one of the hurdles that must be overcome in developing antivirals for this virus infection. In the present study, we observed that the dengue virus-coded protease imports to the mitochondrial matrix, and our report is the first ever of a virus-coded protein, either animal or human, importing to the mitochondrial matrix. Our analysis indicates that the observed mitochondrial import is due to an inherited mitochondrial transport signal. We also show that matrix-localized GrpE protein homolog 1 (GrpEL1), a cochaperone of mitochondrial Hsp70 (mtHsp70), is also the substrate of dengue virus protease, as observed in vitro and ex vivo in virus-infected cells and dengue virus-infected clinical samples. Hence, our studies reveal an essential aspect of the pathogenesis of dengue virus infections, which may aid in developing antidengue therapeutics.

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

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