Role of N-glycosylation sites and CXC motifs in trafficking of medicago truncatula Nod factor perception protein to plasma membrane

Abstract

The lysin motif receptor-like kinase, NFP (Nod factor perception), is a key protein in the legume Medicago truncatula for the perception of lipochitooligosaccharidic Nod factors, which are secreted bacterial signals essential for establishing the nitrogen-fixing legume-rhizobia symbiosis. Predicted structural and genetic analyses strongly suggest that NFP is at least part of a Nod factor receptor, but few data are available about this protein. Characterization of a variant encoded by the mutant allele nfp-2 revealed the sensitivity of this protein to the endoplasmic reticulum quality control mechanisms, affecting its trafficking to the plasma membrane. Further analysis revealed that the extensive N-glycosylation of the protein is not essential for biological activity. In the NFP extracellular region, two CXC motifs and two other Cys residues were found to be involved in disulfide bridges, and these are necessary for correct folding and localization of the protein. Analysis of the intracellular region revealed its importance for biological activity but suggests that it does not rely on kinase activity. This work shows that NFP trafficking to the plasma membrane is highly sensitive to regulation in the endoplasmic reticulum and has identified structural features of the protein, particularly disulfide bridges involving CXC motifs in the extracellular region that are required for its biological function.

FIGURE 1.

NFP localizes to the PM and NFP-S67F localizes in the ER in N. benthamiana. A, NFP localizes to the PM in N. benthamiana. NFP-RFP was co-expressed in N. benthamiana leaves with a PM marker (PMA4-GFP), and the leaves were analyzed by confocal microscopy at 3 dpi. Superposition of the fluorescence images shows clear co-localization of NFP-RFP with the PM marker at the cell boundary. B, NFP localizes to the PM in M. truncatula roots. Roots of M. truncatula nfp-1 were transformed with the Pro35S:NFP-RFP construct or a control vector. Immunocytolabeling was performed using anti-RFP as primary antibodies and the tyramide signal amplification method (green) on root longitudinal sections corresponding to cortical cells. Proteins are stained with Evans blue (red), and the nucleus is stained with DAPI (blue). C, NFP-S67F is retained in the ER in N. benthamiana. The protein encoded by the nfp-2 allele (NFP-S67F) was fused to RFP and co-expressed with the PM (PMA4-GFP) or ER (HDEL-GFP) markers, and the leaves were analyzed by confocal microscopy at 3 dpi. Co-localization with the ER marker in a reticulated network in the cell and around the nucleus suggests that the protein is retained in the ER. Bars, 20 μm.

FIGURE 2.

N-Glycosylation sites are not conserved between NFP and various LysM-RLKs, and trafficking of NFP to the PM is not blocked by inhibition of N-glycosylation. A, scheme of the extracellular region of NFP showing positions of the Asn (N) and Cys (C) residues (putatively being involved in post-translational modifications) in relation to the LysM domains. SP, signal peptide; TM, transmembrane domain; numbering is from the start of the signal peptide. B, conservation of the NFP Cys and N-glycosylation site residues in the orthologues from L. japonicus (NFR5) and P. sativum (SYM10) and the LYR and LYK proteins from M. truncatula. +, a site found at the same position; +/−, a site found at a close position (see alignment in supplemental Fig. S4). C, tunicamycin effectively inhibits N-glycosylation of NFP. N. benthamiana leaves expressing NFP-YFP at 2 and 3 dpi were treated with 10 μm tunicamycin or solvent only (diluted DMSO) for 20 h prior to analysis by immunoblotting. Tunicamycin treatment led to NFP-YFP detected at the size predicted from the unmodified AA sequence (93 kDa), whereas the protein in the untreated leaves contains about 20 kDa of N-glycans. D, NFP can reach the PM in the absence of N-glycosylation. The same material described in C (leaves at 3 dpi treated with 10 μm tunicamycin for 20 h) was observed by confocal microscopy; two images are shown. The arrow points to labeling of perinuclear ER, whereas most of the NFP protein localizes to the PM. WB, Western blot. Bars, 20 μm.

FIGURE 3.

Trafficking of NFP-S67F to the PM is blocked in M. truncatula, and NFP-S67F protein interacts with the ER-located BIP chaperones in N. benthamiana. A, NFP-S67F lacks Golgi matured N-glycans in M. truncatula. Protein extracts from nfp-1 roots expressing NFP-RFP or the indicated mutated variants were treated with PNGase F and analyzed by immunoblotting. The lower size band after treatment indicates a sensitivity of NFP N-glycans to PNGase F and reflects an absence of maturation of NFP N-glycans in the Golgi apparatus and hence retention of the protein in the ER. The higher band size reflects transport through the Golgi to the PM. B, NFP-S67F interacts with the BIP chaperones in N. benthamiana. NFP-RFP or NFP-S67F-RFP expressed in N. benthamiana leaves were solubilized and purified using anti-RFP antibodies. Immunoblotting reveals that NFP-S67F, but not NFP, interacts strongly with BIP chaperones. NT, nontransformed leaves. C, BIP chaperones are not overexpressed in roots of nfp-2 mutant plants. An immunoblot of protein extracts from roots of WT, nfp-1, and nfp-2 plants reveals similar quantities of BIP chaperones, suggesting that the accumulation of NFP-S67F protein in the ER does not lead to an “unfolded protein response.” The ponceau red staining shows the protein loading. WB, Western blot.

FIGURE 4.

NFP contains three disulfide bridges and mutants in the CXC motifs are retained in the ER. A, NFP bears three S-S bridges. A truncated version of NFP, without its intracellular region, fused to RFP (NFPΔIR-RFP) was expressed in N. benthamiana leaves. The microsomal fraction derived from these leaves was first treated or not with DTT, and then free Cys residues were labeled with maleimide-PEG2-biotin. NFP was then solubilized and purified using anti-RFP antibodies. Proteins were separated by SDS-PAGE and analyzed by immunoblotting; fusion proteins were detected using anti-RFP antibodies, and labeled Cys residues were detected using anti-biotin antibodies. The result suggests that the six Cys residues of NFP extracellular region are involved in S-S bridges, and only the single Cys residue of the RFP is labeled in the absence of DTT. B, NFP mutated in the Cys pairs localizes to the ER in M. truncatula. Protein extracts from nfp-1 roots expressing the indicated NFP-RFP mutated variants were treated as described in Fig. 3A. The sensitivity of the proteins to PNGase F suggests lack of N-glycan modification and hence retention in the ER. C, NFP mutated in the CXC motifs interacts with BIP chaperones. NFP and the quadruple Cys to Ala mutant proteins were expressed and purified from N. benthamiana leaves, and complexes with BIP were analyzed by SDS-PAGE and immunoblotting. The mutant NFP protein co-purifies BIP chaperones, suggesting that it is retained in the ER by binding to BIP chaperones. NT, nontransformed leaves; WB, Western blot.

FIGURE 5.

Molecular modeling of the NFP extracellular region. A, molecular modeling showing the suggested positions of the S-S bridges. LysM domains are colored blue (LysM1), red (LysM2), and green (LysM3) ribbons, whereas the preceding loops are represented in pale blue, pale red, and pale green, respectively. Cys residues involved in S-S bridges are shown as yellow sticks. The S-S bridge positions deduced from mutation and complementation analysis (Table 1) were used for modeling. B, superposed molecular modeling of NFP and NFP S67F. An NFP model is shown before (violet ribbons) and after mutation (pink ribbons); local structural changes are highlighted at the LysM1 level. The arrow points to the position of residue 67. C and D, solvent-accessible surfaces colored according to the lipophilic potential, from brown (hydrophobic) to blue (polar), calculated for the NFP model before (C) and after the S67F mutation (D). The mutation zone is circled.