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Journal of Biochemistry Advance Access originally published online on April 10, 2007
Journal of Biochemistry 2007 141(6):889-895; doi:10.1093/jb/mvm095
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© 2007 The Japanese Biochemical Society.

Spatial Orientation of Mitochondrial Processing Peptidase and a Preprotein Revealed by Fluorescence Resonance Energy Transfer{dagger}

Tomonori G. Nishino1, Ken Kitano2, Katsuhiko Kojima1, Tadashi Ogishima1, Akio Ito1 and Sakae Kitada1,*

1Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan; and 2Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan

*To whom correspondence should be addressed. Tel: +81-92-642-4182, Fax: +81-92-642-2607, E-mail: s.kitscc{at}mbox.nc.kyushu-u.ac.jp

Received February 6, 2007; Accepted April 2, 2007


  Abstract

Mitochondrial processing peptidase (MPP), which is composed of heterodimeric {alpha}-MPP and ß-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 ß-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 {alpha}-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.

Key Words: FRET, GFP, mitochondria, processing, protease

Abbreviations: ACON, aconitase; COX IV, cytochrome c oxidase subunit IV; CPM, 7-dietylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin; EGFP, enhanced green fluorescent protein; FRET, fluorescence resonance energy transfer; MDH, malate dehydrogenase; MPP, mitochondrial processing peptidase; preEGFP, presequence-fused EGFP; TIM, translocase on the inner mitochondrial membrane; TOM, translocase on the outer mitochondrial membrane

{dagger}Following Schechter and Berger (1), the enzyme binding sites are denoted S1, ... S2, ..., Si and Formula , Formula , ..., Formula away from the scissile peptide bond toward the N- and C-termini, respectively. The amino acid residues in the substrates are referred to as P1, P2, ..., Pi and Formula , Formula , ..., Formula in accordance with the binding site.


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