How Can Organellar Protein N-terminal Sequences Be Dual Targeting Signals? In silico Analysis and Mutagenesis Approach

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Abstract

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.

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.

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