Spatial orientation of mitochondrial processing peptidase and a preprotein revealed by fluorescence resonance energy transfer

Tomonori G. Nishino, Ken Kitano, Katsuhiko Kojima, Tadashi Ogishima, Akio Ito, Sakae Kitada

Research output: Contribution to journalArticle

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Abstract

Mitochondrial processing peptidase (MPP), which is composed of heterodimeric α-MPP and β-MPP subunits. It specifically recognizes mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4′-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs. FRET efficiencies of 65 and 55% were observed between the EGFP chromophore and CPM-Ser84 and -Lys156 of β-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 Å, respectively. Considering the FRET results and the structural validity based on the crystal structure of the MPP-presequence complex, a plausible model of preEGFP associated with MPP was constructed in silico. The modelled structure indicated that amino acid residues on the C-terminal side of the cleavage site in the preprotein were orientated tail out from the large cavity of MPP and interacted with the glycine-rich loop of α-MPP. Thus, MPP orientates preproteins at the specific cleft between the catalytic domain and the flexible glycine-rich loop which seems to pinch the extended polypeptide.

Original languageEnglish
Pages (from-to)889-895
Number of pages7
JournalJournal of biochemistry
Volume141
Issue number6
DOIs
Publication statusPublished - Jun 1 2007

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Fluorescence Resonance Energy Transfer
Yeast
Yeasts
Glycine
mitochondrial processing peptidase
Crystal structure
Aconitate Hydratase
Chromophores
Reproducibility of Results
Computer Simulation
Catalytic Domain

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Spatial orientation of mitochondrial processing peptidase and a preprotein revealed by fluorescence resonance energy transfer. / Nishino, Tomonori G.; Kitano, Ken; Kojima, Katsuhiko; Ogishima, Tadashi; Ito, Akio; Kitada, Sakae.

In: Journal of biochemistry, Vol. 141, No. 6, 01.06.2007, p. 889-895.

Research output: Contribution to journalArticle

Nishino, Tomonori G. ; Kitano, Ken ; Kojima, Katsuhiko ; Ogishima, Tadashi ; Ito, Akio ; Kitada, Sakae. / Spatial orientation of mitochondrial processing peptidase and a preprotein revealed by fluorescence resonance energy transfer. In: Journal of biochemistry. 2007 ; Vol. 141, No. 6. pp. 889-895.
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