Figure 5: ND2-TM-6-8 is the smallest identified region sufficient to co-localize with GluN1.
From: An evolutionary switch in ND2 enables Src kinase regulation of NMDA receptors
(a) Cartoon model of ND2, with TM regions numbered 1–11. The model was used to rationally design a sequence of GFP-tagged ND2 fragments; ND2-TM-6-11 (residues 151–347), ND2-TM-10-11 (residues 250–347), ND2-TM-6-8+cytoplasmic loop (residues 151–240), ND2-TM-6-8 (residues 151–223), ND2-TM-6-7 (residues 151–200), ND2-TM-7-8 (residues 175–223) and ND2-TM-8+cytoplasmic loop (residues 151–240). Inset—homology model of ND2, generated using ND2 homologues from E. coli (PDB IDs 3RKO, NuoN subunit) and T. thermophilus (PDB code: 4HE8). (b) Cumulative frequency distribution of thresholded PCC values for GluN1 with GFP-ND2, (mean PCC=0.66±0.02; n=41), GFP-ND2-TM-6-11, (0.63±0.03; n=31), GFP-ND2-TM-10-11 (0.33±0.02; n=77) and GFP-ND2-TM-6-8+cytoplasmic loop (0.75±0.02; n=30). (c) Cumulative frequency distribution of thresholded PCC values for GluN1 with GFP-ND2-TM-6-8+cytoplasmic loop, (mean PCC=0.75±0.02; n=30), GFP-ND2-TM-6-8, (0.71±0.02; n=24), GFP-ND2-TM-6-7, (0.05±0.03; n=28), GFP-ND2-TM-7-8, (0.26±0.03; n=29) and GFP-ND2-TM-8+cytoplasmic loop (0.25±0.03; n=27). (d) Schematic depicting GluN1ΔCTD-Vc and ND2-TM-6-8-Vn fragments interaction. (e) × 100 images of HEK293 cells transfected with GluN1ΔCTD-Vc and either ND2-TM-6-8-Vn or ND2-TM-6-7-Vn constructs. Scale bar, 5 μm. (f) Histogram depicting normalized Venus intensity signal observed in GluN1-positive HEK293 cells transfected with GluN1ΔCTD-Vc alone, or GluN1ΔCTD-Vc with either ND2-TM-6-8-Vn or ND2-TM-6-7-Vn. Data were normalized to average Venus fluorescence intensity determined for GluN1-positive HEK293 cells transfected with GluN1ΔCTD-Vc alone 
. No significant difference was observed in Venus intensity between GluN1ΔCTD-Vc (1.00±0.17, n=17) and GluN1ΔCTD-Vc+ND2 ND2-TM-6-7-Vn populations (1.33±0.17, n=15), but there was a significant difference between GluN1ΔCTD-Vc alone and GluN1ΔCTD-Vc+ND2-TM-6-8-Vn (2.19±0.12, n=30). Statistically significant differences between populations are indicated by the symbols ‘***’ and ‘****’ (P<0.001 and P<0.0001, respectively) and were evaluated by Kruskal–Wallis non-parametric analysis of variance with Dunn’s multiple post hoc comparison tests. n=# of cells. Results are presented as mean±s.e.m. See also Supplementary Figs 9 and 10.
