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.
Sigyarto
C
C
Department of Biochemistry and Biophysics, Stockholm University, Sweden.
Hugosson
M
M
Moberg
P
P
Andreu
D
D
Glaser
E
E
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D018724:Spinacia oleracea / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D000099188:Alternative Oxidase
D000596:Amino Acids; D004792:Enzyme Precursors; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D021382:Protein Sorting Signals; D021122:Protein Subunits; D006180:Proton-Translocating ATPases; D000099188:Alternative Oxidase