Henrik Biverståhl

Dual Targeting to Mitochondria and Chloroplasts: Characterization of Thr–tRNA Synthetase Targeting Peptide

There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins of Arabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides (dTPs). The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-tRNA synthetase (ThrRS-dTP) studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFP. These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS-dTP(2-60) to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP(2-60) inhibited import of pF1beta into mitochondria and of pSSU into chloroplasts at microM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP(2-60) indicated that the peptide has the propensity for forming alpha-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.

Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing.

Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.