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

被引:36
作者
Berglund, Anna-Karin [1 ]
Spanning, Erika [1 ]
Biverstahl, Henrik [1 ]
Maddalo, Gianluca [1 ]
Tellgren-Roth, Christian [2 ]
Maler, Lena [1 ]
Glaser, Elzbieta [1 ]
机构
[1] Stockholm Univ, Arrhenius Labs Nat Sci, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden
[2] Uppsala Univ, Rudbecklab, Dept Genet & Pathol, SE-75185 Uppsala, Sweden
基金
瑞典研究理事会;
关键词
Dual targeting; mitochondria; chloroplast; targeting peptide; aminoacyl-tRNA synthetase; PROTEIN SECONDARY STRUCTURE; PEA GLUTATHIONE-REDUCTASE; STROMAL PROCESSING PEPTIDASE; CYTOCHROME BC(1) COMPLEX; IMPORT RECEPTOR TOM20; N-TERMINAL DOMAIN; PRECURSOR PROTEINS; CHLAMYDOMONAS-REINHARDTII; PLANT MITOCHONDRIAL; STRUCTURAL BASIS;
D O I
10.1093/mp/ssp048
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 pF(1)beta into mitochondria and of pSSU into chloroplasts at mu M 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.
引用
收藏
页码:1298 / 1309
页数:12
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