NMR structures of a mitochondrial transit peptide from the green alga Chlamydomonas reinhardtii

Abstract

The 26-amino-acid pre-sequence of the ATP synthase subunit that directs the protein from the cytosol to mitochondria in the unicellular green alga Chlamydomonas reinhardtii has been synthesised and analysed using NMR spectroscopy/circular dichroism and compared to a chloroplast transit peptide from the same organism. The results demonstrate that the peptide, though mainly unstructured in water, undergoes a strong conformational change in a 36% water/64% 2,2,2-trifluoroethanol mixture. In this solvent condition, an ~-helix was characterised by NMR from residue 2 to 26. Structure calculations under NMR restraints lead to a population of models of which 60% are kinked at position 9-10. Structural analysis indicates two hydrophobic sectors on the models with a discontinuity at the 9-10 kink level. The structures suggest a different interaction mode with the mitochondrial membrane compared to the chloroplast transit peptide.

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase

“…The internal segment between the N‐ and C‐terminal domains is enriched in proline indicating that it is a more flexible part of the presequence. The C‐terminal part of the pF 1 β presequence has been previously shown to interact with both the spinach MPP/ bc 1 complex and rat MPP (Sjöling et al ., 1994; Sjöling et al ., 1996). Therefore, this study focused on the C‐terminal part of the presequence.…”

“…In addition, a proline residue between the distal and proximal arginines (Hammen et al ., 1996; Niidome et al ., 1994; Ogishima et al ., 1995; Thornton et al ., 1993) and individual residues at positions +1, +2 and +3 from the cleavage site were shown to be of importance (Klaus et al ., 1996; Lain et al ., 1998; Ogishima et al ., 1995). It has also been suggested that MPP recognizes higher‐order structural elements (Hammen et al ., 1994; Luciano and Géli, 1996; Sjöling and Glaser, 1998; Sjöling et al ., 1994; Sjöling et al ., 1996; Waltner and Weiner, 1995).…”

NMR investigations of the dual targeting peptide of Thr‐tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana

“…The content of Pro and Ser residues is higher in cTPs, while the prevalence of basic amino acid residues is higher in mTPs. mTPs have been shown to form amphiphilic α‐helices in membrane mimetic media , and it has been demonstrated that they bind with their hydrophobic side into a hydrophobic groove in the Tom20 receptor . An N‐terminal α‐helix has been observed in the targeting peptides both in alternative oxidase (AOX) and in F 1 β in SDS micelles, which also appears to be important for targeting.…”

“…The mTPs are on average 42 amino acid residues long and contain more arginines especially in the N‐terminal portion of the mTPs, while the cTPs are on average 58 amino acid residues long and contain more serines and prolines . Structurally, most mTPs are predicted to form an amphiphilic α‐helix, which has been shown to play an important role in mitochondrial import , while cTPs appear to be unstructured in most studies . Nevertheless, some cTPs have been reported to form helical structures in a membrane mimetic environment .…”

Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts

“…1,2 Another important aspect for mitochondrial proteins is the propensity for their presequences to form an amphiphilic α-helix. 13,14 The hydrophobic side of the amphiphilic α-helix has been shown to bind to a hydrophobic groove in the import receptor Tom20. 15,16 The amphiphilic α-helix of a plant presequence have also been shown to bind to the plant import receptor Tom20.…”