How can organellar protein N-terminal sequences be dual targeting signals? In silico analysis and mutagenesis approach.
Organellar nuclear-encoded proteins can be mitochondrial, chloroplastic or localized in both mitochondria and chloroplasts. Most of the determinants for organellar targeting are localized in the N-terminal part of the proteins, which were therefore analyzed in Arabidopsis thaliana. The mitochondrial, chloroplastic and dual N-terminal sequences have an overall similar composition. However, Arg is rare in the first 20 residues of chloroplastic and dual sequences, and Ala is more frequent at position 2 of these two types of sequence as compared to mitochondrial sequences. According to these observations, mutations were performed in three dual targeted proteins and analyzed by in vitro import into isolated mitochondria and chloroplasts. First, experiments performed with wild-type proteins suggest that the binding of precursor proteins to mitochondria is highly efficient, whereas the import and processing steps are more efficient in chloroplasts. Moreover, different processing sites are recognized by the mitochondrial and chloroplastic processing peptidases. Second, the mutagenesis approach shows the positive role of Arg residues for enhancing mitochondrial import or processing, as expected by the in silico analysis. By contrast, mutations at position 2 have dramatic and unpredicted effects, either enhancing or completely abolishing import. This suggests that the nature of the second amino acid residue of the N-terminal sequence is essential for the import of dual targeted sequences.
Pujol
Claire
C
Institut de Biologie Moléculaire des Plantes, Laboratoire Propre du CNRS (UPR 2357) Conventionné avec l'Université Louis Pasteur (Strasbourg 1), 12 rue du Général Zimmer, 67084 Strasbourg, France.
Maréchal-Drouard
Laurence
L
Duchêne
Anne-Marie
AM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D016415:Sequence Alignment
D000596:Amino Acids; D029681:Arabidopsis Proteins; D024101:Mitochondrial Proteins; D021382:Protein Sorting Signals; D000604:Amino Acyl-tRNA Synthetases