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Identification and functional assay of the interaction motifs in the partner protein O s NAR 2.1 of the two‐component system for high‐affinity nitrate transport
Author(s) -
Liu Xiaoqin,
Huang Daimin,
Tao Jinyuan,
Miller Anthony J.,
Fan Xiaorong,
Xu Guohua
Publication year - 2014
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/nph.12986
Subject(s) - amino acid , biochemistry , point mutation , chemistry , protein–protein interaction , bimolecular fluorescence complementation , transporter , blot , arginine , yeast , biology , mutation , gene
Summary A partner protein, NAR 2, is essential for high‐affinity nitrate transport of the NRT 2 protein in plants. However, the NAR 2 motifs that interact with NRT 2s for their plasma membrane ( PM ) localization and nitrate transporter activity have not been functionally characterized. In this study, O s NAR 2.1 mutations with different carbon (C)‐terminal deletions and nine different point mutations in the conserved regions of NAR 2 homologs in plants were generated to explore the essential motifs involved in the interaction with O s NRT 2.3a. Screening using the membrane yeast two‐hybrid system and X enopus oocytes for nitrogen‐15 ( 15 N) uptake demonstrated that either R 100 G or D 109 N point mutations impaired the O s NAR 2.1 interaction with O s NRT 2.3a. Western blotting and visualization using green fluorescent protein fused to either the N‐ or C‐terminus of O s NAR 2.1 indicated that Os NAR 2.1 is expressed in both the PM and cytoplasm. The split‐yellow fluorescent protein ( YFP )/ B i FC analyses indicated that Os NRT 2.3a was targeted to the PM in the presence of Os NAR 2.1, while either R100 G or D109 N mutation resulted in the loss of Os NRT 2.3a‐ YFP signal in the PM . Based on these results, arginine 100 and aspartic acid 109 of the Os NAR 2.1 protein are key amino acids in the interaction with Os NRT 2.3a, and their interaction occurs in the PM but not cytoplasm.