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Journal of Biochemistry Advance Access published online on April 10, 2007
Journal of Biochemistry, 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

Tomonori G. Nishino, Ken Kitano{ddagger}, Katsuhiko Kojima, Tadashi Ogishima, Akio Ito and Sakae Kitada**

Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan; and {ddagger}Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan.

**To whom correspondence should be addressed: Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan. 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 analyzed 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)-labeled 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 Å 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 modeled 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


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