Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway

The NITROGEN LIMITATION ADAPTATION (NLA) protein is a RING-type E3 ubiquitin ligase that plays an essential role in the regulation of nitrogen and phosphate homeostasis. NLA is localized to two different subcellular sites (the plasma membrane and the nucleus), and contains four distinct domains: (i) a RING domain that mediates degradation of phosphate transporters at the plasma membrane; (ii) an SPX domain that facilitates NLA's interaction with the phosphate transporters, and also exists in other proteins that regulate the nuclear transcription factors that control the phosphate starvation response pathway; (iii) a linker domain that lies between the RING and SPX domains; and (iv) a C-terminal domain, which, like the linker region, is of unknown function. Here we carried out a mutational analysis of NLA, which indicated that all the domains are not only essential for proper functioning of the protein, but also mediate its localization to the plasma membrane and (or) nucleus, as well as to different subdomains within the nucleus. Overall, the results provide new insights to the distinct protein motifs within NLA and the role(s) that this protein serves at different subcellular sites with respect to the regulation of nitrogen-dependent phosphate homeostasis as well as other possible physiological functions. Keywords: nitrate-dependent phosphate homeostasis, NITROGEN LIMITATION ADAPTATION, subcellular protein localization, RING domain, SPX domain. La proteine NLA (acronyme de << NITROGEN LIMITATION ADAPTATION >>) est une ubiquitine ligase E3 a domaine RING qui joue un role essentiel dans la regulation de l'homeostasie de l'azote et du phosphate. La NLA se localise dans deux sites subcellulaires differents (la membrane plasmique et le noyau), et elle comprend quatre domaines distincts: (i) un domaine RING qui intervient dans la degradation des transporteurs de phosphate a la membrane plasmique; (ii) un domaine SPX, qui facilite l'interaction de la NLA avec les transporteurs de phosphate, et qui existe aussi a l'interieur d'autres proteines qui regulent les facteurs de transcription nucleaires controlant la voie de reponse a la privation en phosphate; (iii) un domaine linker qui se trouve entre les domaines RING et SPX et (iv) un domaine C-terminal qui, comme la region linker, est de fonction inconnue. Les auteurs ont realise une analyse mutationnelle de la NLA qui indiquait que les quatre domaines sont non seulement essentiels au fonctionnement adequat de la proteine, mais qu'ils interviennent aussi dans sa localisation a la membrane plasmique et/ou dans le noyau, de meme que dans differents sous-domaines a l'interieur du noyau. Dans l'ensemble, les resultats apportent un eclairage nouveau sur les motifs proteiques distincts a l'interieur de la NLA et sur les roles que joue cette proteine dans differents sites subcellulaires quant a la regulation de l'homeostasie du phosphate dependante de l'azote, de meme que sur d'autres fonctions physiologiques possibles. [Traduit par la Redaction] Mots-cles: homeostasie de phosphate dependante du nitrate, NLA (<< NITROGEN LIMITATION ADAPTATION >>), localisation subcellulaire de la proteine, domaine RING, domaine SPX.
Introduction As a major component of most cellular macromolecules, including proteins, nucleic acids, and chlorophyll, nitrogen is essential for plant growth and development (Hawkesford et al. 2012). Consequently, plants subjected [...]