Expanded impact of protein N-myristoylation in plants

Abstract

N-MYR controls the function of the plant protein complex SnRK1, described as one of the most important plant regulatory protein in stress and energy signalling. In plant cells, N-MYR is involved in a significantly higher number of metabolic pathways than in yeast or human. Some N-myristoylated protein families are solely encountered in plant cells. This lipid modification could be involved in the control of the redox imbalances originating from different stresses in plants. This prevalence of N-MYR in such proteins is unique to the plant kingdom. We hypothesize that this expansion of the mechanism in plants improves the control of the damages induced by environmental changes.

N-myristoylation (N-MYR) is an irreversible protein lipidation, now recognized as a major modification since it is believed to involve nearly 2% of all plant proteins.^1,2 The modification corresponds to the irreversible link of a lipid, myristate (C:14) to the N-terminal glycine of some proteins. The targeted proteins show specific aminoacids within the first 9 residues of the polypeptide sequence. The reaction is catalyzed by the N-myristoyltransferase (NMT). In A. thaliana two related genes encode two isoforms, AtNMT1 and AtNMT2.³ Functionality, the most well-known role for this modification is to target the modified protein to a membrane where it plays crucial roles in signal transduction pathways.

In recent studies,⁴ we have demonstrated the importance of the N-MYR in plant viability. Characterization of knockout mutant lines (nmt1-1) indicates that AtNMT1 is the main catalyst in charge of N-MYR and that it is strictly required for plant growth. AtNMT1 impairment induces extremely severe defects during embryonic development at the shoot apical meristem (SAM), causing growth arrest at early state of development (DAI 4). The main challenge was to identify the molecular aspects associated to the severe phenotype observed. Different approaches led to link the defect with the protein complex SnRK1, featuring two N-myristoylated subunits β. In this context, we have demonstrated that N-MYR of the SnRK1 kinase was involved in the differentiation of the shoot apical meristem, through trafficking from the nucleus and/or the cytoplasm to the membrane. The role of this heterotrimeric kinase was not guessed so far, neither was the involvement of N-MYR.

SnRK1 (SNF1-related protein kinase) is a plant heterotrimeric Ser/Thr kinase complex, highly conserved in living organisms, S. cerevisiae and humans being the best studied cases. This complex is made up of three different subunits: α, β and γ. Each subunit type is usually encoded by more than one gene, allowing several possible combinations, complicating the understanding of the role of the complex. In A. thaliana there are two α (α1, α2), three β (β1, β2 and β3) and two γ (γ and βγ) subunits.⁵ Subunit α corresponds to the catalytic domain. Both β and γ subunits have regulatory functions in the complex, but their molecular roles are not well understood. In yeast and human complexes, where the role of β subunit is better understood than in plants, N-MYR is essential for the function of protein complexes and for subcellular location. N-MYR of several β subunits is conserved throughout evolution in Eukaryotes. In Arabidopsis subunit β1 and β2, but not β3 are N-myristoylated.^3,6 In A. thaliana, the catalytic subunits of the complex SnRK1 (AKINα1 or AKINα2) have recently been described as a central integrator of the transcription networks, in plant stress and energy signalling.⁷ SnRK1 appears to be involved in the control of the molecular mechanism by which plants sense and adapt to several environmental conditions. N-MYR could correspond to the mechanism by which such a function is regulated.

It is now well-known that plants have a significantly higher content of N-myristoylated proteins than yeast or human.^3,8 By using a combination of predictive software^9,10 (TermiNator) with in vitro assays,¹¹ both designed in our laboratory, 437 A. thaliana proteins have been identified as N-myristoylated. This corresponds to the so-called N-myristoylome. This modification seems to be involved in important plant metabolic pathways, as we have described for SnRK1. Moreover, some proteins of the N-myristoylome belong to protein families only found in the plant kingdom or even belonging to proteins families in which no yeast or human ortholog is N-myristoylated. This gives plant N-MYR a unique role within all living organisms. This is the case of the small G-protein Ara6 also known as RabF1.³

Within the Arabidopsis N-myristoylome several proteins are involved in plant rescue metabolism against the damage produced by the external unstable conditions. It is recognized that N-MYR in plant cells have a critical role in controlling membrane signalling pathways that lead to specific plant immunity.¹² In fact, we have shown that mutant lines expressing low levels of AtNMT1 exhibit yellow and necrotic leaves, indicative of plant defence response.⁴ However, N-MYR seems to be also involved in redox homeostasis regulation. Like all aerobic organisms, plants maintain most cytoplasmic thiols in their reduced form. Redox homeostasis is mainly preserved by the glutathione pool present in cells. Molecular events have emerged throughout plant evolution that allowed plant cells to better adapt to this control. Unlike many eukaryotic organisms like humans, plants synthesize tocopherols and higher concentrations of ascorbate to get better control of the redox imbalance. Thioredoxins (TRX) or glutaredoxins (GRX) have been recently described as oxido-reductive proteins involved in redox regulation,^13,14 and the number of genes encoding both proteins types in plants is much higher than the others eukaryotic organisms.¹⁵ In addition, members of TRXs or GRXs are solely N-myristoylated in plant (Traverso et al., this laboratory, unpublished data). Other proteins related to the redox control have also been predicted or showed as N-myristoylated in our laboratory (glutathion-dependent dehydroascorbate reductases, glutathione peroxidases etc.,). All of them, together with TRXs and GRXs, share both (i) their involvement in the redox homeostasis regulation mainly due to their oxidoreductive activity and (ii) the fact that no homologue has been described as N-MYR in other eukaryotic cells, i.e., N-MYR of these proteins is specific of plant (Fig. 1).

Figure 1.

A phylogenic tree in TRXs h from A. thaliana. The TRXs modified co-translationally by lipids are underlined. These modifications are only encountered in subgroup II and III of the h-type TRXs. This pattern is found in all plant studied but not in non-photosynthetic organisms. Proteins exclusively modified in the plant kingdom have been observed in other oxide-reductive protein families (data not shown).

Adaptation to different environmental conditions is a trait shared by all living organism. Plants imperatively need to avoid the most deleterious conditions in the context of their lack of mobility and the existence of a photosynthetic metabolism. Plant cells require therefore more sophisticated systems to get acclimatized to environmental changes that could lead otherwise to biotic or abiotic stresses. According to our data, we hypothesize that N-MYR could concur to an evolutionary mechanism highly expanded in plant genomes and allowing to improve the control of the environmental change effects.

Addendum to: Pierre M, Traverso JA, Boisson B, Domenichini S, Bouchez D, Giglione C, Meinnel T. N-myristoylation regulates the SnRK1 pathway in Arabidopsis. Plant Cell. 2007;19:2804–2821. doi: 10.1105/tpc.107.051870.