Journal of Molecular Biology
How Can Organellar Protein N-terminal Sequences Be Dual Targeting Signals? In silico Analysis and Mutagenesis Approach
Introduction
A eukaryotic cell is divided into functionally distinct, membrane-enclosed compartments, each containing its own set of proteins. Most mitochondrial or chloroplastic proteins are encoded by the nuclear genome, translated on cytosolic ribosomes and directed to the appropriate organelle, by following a specific pathway and guided by signals in their amino acid sequence. In the vast majority of matrix or stroma proteins, this transfer involves a specific N-terminal extension of the protein interacting with specific mitochondrial or plastidial receptors.
The matrix-targeting sequences contain many positively charged, hydrophobic and hydroxylated residues, and often have the potential to form an amphipathic α-helix with a positively charged surface and a hydrophobic one. The hydrophobic residues are supposed to interact with a specific outer membrane receptor, TOM20, and the positive charges are supposed to be important to cross the inner membrane.1 The presequences are involved in separate events: recognition of the mitochondrial receptors, followed by protein import and processing activity.2 Compared to mammalian, yeast or Neurospora crassa, plant mitochondrial presequences are seven to nine residues longer, with an average length of 42 residues (most are 20–60 residues), but have a similar composition, except for a higher Ser content (17% for plants, 10% for N. crassa, 7% in yeast and 3% in mammals).2., 3. The chloroplastic transit peptides (most of them are 30–80 residues, mean 58) are longer and less structured than the plant mitochondrial presequences.3 The plant mitochondrial and plastid N-terminal extensions are roughly similar in amino acid composition, although the plastid ones do not contain many Arg or Leu residues, and are slightly enriched in Ser.3., 4.
Different tools are used for prediction of mitochondrial and plastid targeting sequences. MitoProt,5 iPSORT,6 TargetP,7 and Predotar8 are widely used. The first two are rule-based tools and incorporate existing knowledge about sorting signals. The last two are services based on artificial neural networks. Investigations in Predotar networks suggest that the distribution of positive charges in targeting sequence is critical for discriminating mitochondrial and plastid proteins: mitochondrial presequences are expected to be particularly rich in positive amino acids in the first dozen residues, as positive amino acids are abundant after the first dozen residues in plastid transit peptides. Other determinants are a hydrophobic residue at position 2 in mitochondrial proteins, and a high level of serine in chloroplastic transit peptides.8
A third class of plant organellar proteins has been found recently, corresponding to dual targeted proteins to both mitochondria and chloroplasts.9., 10. As mitochondrial and chloroplastic proteins, they also have an N-terminal extension called an ambiguous presequence, because it is recognized as an import signal to both organelles.4 Only one such dual-targeting sequence has been studied extensively, that of pea glutathione reductase (GR).11., 12., 13. The mitochondrial and chloroplastic signals were overlapping, and effects on import did not always correlate with effects on processing.13 Moreover, positive residues are more important for mitochondrial import than for chloroplastic import.11
In order to further investigate the features of dual targeting sequences, we first compared Arabidopsis thaliana dual, mitochondrial and chloroplastic N-terminal signals. According to these observations, the N-terminal part of three dual targeted proteins was mutated and fused to green fluorescent protein (GFP). Wild-type and mutated constructs were analyzed by in vitro import into isolated mitochondria and chloroplasts. The main advantage of the in vitro import system is that it allows quantification. It also permits the analysis of the different steps, binding to organelles, followed by import and finally processing. By this technique, only the effect of the N-terminal sequence is tested, as the protein is synthetized in vitro before incubation with the organelles. The most important result obtained here is that there are clear differences between organelles for the binding, import and processing of precursor proteins, and that Arg residues and the second amino acid of the sequences are essential organellar determinants of dual targeting sequences.
Section snippets
Organellar N-terminal sequences have a similar overall composition, but in chloroplastic and dual sequences Arg is rare in the first 20 residues and Ala is the most frequent residue at position 2
The N-terminal part (50 or 100 residues) of 35 A. thaliana dual targeted proteins were analyzed and compared to the N-terminal part of A. thaliana proteins known to be only mitochondrial or only chloroplastic, or to the “mature” proteins (that is the above organellar proteins deleted of their first 200 amino acid residues) (Figure 1(a); and Supplementary Data Figure 1) (for the choice and analysis of proteins, see Experimental Procedures; and Supplementary Data Table 1). The three types of
Discussion
Protein import machineries have been studied extensively in the organelles of many organisms, and similarities have been found between the mitochondrial import systems. Orthologues of many components of the translocases of the outer membrane (TOM complex) or the inner membrane (TIM complex) are found in all organisms,9., 27. although the plant mitochondrial import machinery differs in some aspects from that of other organisms. In particular, the plant outer membrane receptors diverge from their
Analysis of A. thaliana dual, mitochondrial and chloroplastic targeting sequences
Three groups of A. thaliana organellar proteins were created, corresponding to clearly established dual targeted, mitochondrial and chloroplastic proteins, and localized in the mitochondrial matrix or the chloroplastic stroma (Supplementary Data Table 1). A total of 35 A. thaliana dual targeted sequences are in the first group, 18 correspond to aaRSs10 and 17 correspond to other sequences.4., 9., 36., 37., 38. The second group corresponds to A. thaliana proteins supposed to be only
Acknowledgements
This work was supported by a grant from the French Ministère délégué à l'Enseignement Supérieur et de la Recherche to C. P. We thank Samira El Farouk for help in mutagenesis and DNA cloning.
References (43)
- et al.
Mitochondrial targeting peptides in plants
Trends Plant Sci.
(1998) - et al.
Interaction of plant mitochondrial and chloroplast signal peptides with the Hsp70 molecular chaperone
Trends Plant Sci.
(2002) - et al.
Dual targeting to mitochondria and chloroplasts
Biochim. Biophys. Acta
(2001) - et al.
Predicting subcellular localization of proteins based on their N-terminal amin acid sequence
J. Mol. Biol.
(2000) - et al.
N-Terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency
J. Mol. Biol.
(2002) - et al.
Processing of the dual targeted precursor protein of glutathione reductase in mitochondria and chloroplasts
J. Mol. Biol.
(2004) - et al.
The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting
FEBS Letters
(2006) - et al.
Characterization of the import pathway of the F(A)d subunit of mitochondrial ATP synthase into isolated plant mitochondria
Arch. Biochem. Biophys.
(1996) - et al.
Signals required for the import and processing of the alternative oxidase into mitochondria
J. Biol. Chem.
(1999) - et al.
Convergent evolution of receptors for protein import into mitochondria
Curr. Biol.
(2006)
A plant outer mitochondrial membrane protein with high amino acid sequence identity to a chloroplast protein import receptor
FEBS Letters
Discrete mutations in the presequence of potato formate dehydrogenase inhibit the in vivo targeting of GFP fusions into mitochondria
Biochem. Biophys. Res. Commun.
One ticket for multiple destinations: dual targeting of proteins to distinct subcellular locations
Curr. Opin. Plant Biol.
Plant organellar protein targeting: a traffic plan still under construction
Trends Cell Biol.
Overlapping destinations for two dual targeted glycyl-tRNA synthetases in Arabidopsis thaliana and Phaseolus vulgaris
J. Biol. Chem.
Versatility of the mitochondrial protein import machinery
Nature Rev. Mol. Cell Biol.
Computational method to predict mitochondrially imported proteins and their targeting sequences
Eur. J. Biochem.
PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization
Trends Biochem. Sci.
Predotar: a tool for rapidly screening proteomes for N-terminal targeting sequences
Proteomics
Protein targeting and import
Dual targeting is the rule for organellar aminoacyl-tRNA synthetases in Arabidopsis thaliana
Proc. Natl Acad. Sci. USA
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2012, Advances in Botanical ResearchCitation Excerpt :The overall amino acid composition of TP is similar to that of MTS. TPs are enriched in positively charged, hydroxylated and hydrophobic residues and contain few acidic residues (Pujol et al., 2007; Zhang & Glaser, 2002; Zybailov et al., 2008). However, TPs are longer than MTSs (5–10 amino acids longer) (Huang et al., 2009; Zhang & Glaser, 2002; Zybailov et al., 2008), they have less propensity to fold into secondary structures (von Heijne & Nishikawa, 1991; Zhang & Glaser, 2002) and they are slightly more hydrophobic than MTSs (Huang et al., 2009).