ND2 is a Src unique domain-interacting protein. (A) Schematic diagram illustrating the domain structure of ND2, clones isolated from the yeast two-hybrid screen, and recombinant GST-tagged fusion proteins. (B) Blot of ND2–GST fusion proteins probed with biotinylated Src unique domain followed by SA-HRP. (C) Blot of ND2.1–GST probed with biotinylated domains of Src and Fyn followed by SA-HRP. (D) Immunoblots of coimmunoprecipitates from brain homogenate probed with anti-ND2, anti-Src, or anti-Fyn. Nonspecific IgG was used as a negative control for immunoprecipitation. In these experiments, the cell lysates were prepared by using nondenaturing conditions; under denaturing conditions, we found no coimmunoprecipitation of Src by anti-ND2 or of ND2 by anti-Src (not shown). (E) Immunoblot of coimmunoprecipitates from cultured Src^+/+ and Src^-/- fibroblasts probed with anti-ND2. Nonspecific IgG was used as a negative control for immunoprecipitation, and immunoblotting of ND2 protein from both cell lines was used as a positive control.

ND2 interacts with Src at the postsynaptic density. (A) Immunoblots of coimmunoprecipitates from PSD preparations probed with anti-ND2 or anti-Src as indicated. Nonspecific IgG (either rabbit or mouse) was used as a negative control for both antibodies. (B) Recombinant ND2.1–GST fusion protein, but not ND2.2–GST, ND2.3–GST, or GST alone, pulls Src from PSD preparations.

Blocking expression of ND2 prevents Src-dependent regulation of NMDA receptor activity. (A) Immunoblots of total soluble protein obtained from cultured rat hippocampal neurons treated with 50 μg/ml chloramphenicol (CAP) for 48 h probed with anti-ND2, anti-NR1, and anti-Src as indicated. (B) Immunoblot of coimmunoprecipitates obtained from cultured hippocampal neurons, either untreated or treated with CAP for 48 h, probed with anti-NR1 or anti-Src. (C) Summary histograms (Left) of ATP level or mitochondrial membrane potential (▵ψ_M), as assessed by TMRM fluorescence dequenching (Right), in cultured hippocampal neurons either untreated or treated with CAP for 48 h. (D) The up-regulation of NMDAR activity by the Src activator peptide EPQ(pY)EEIPIA, labeled (pY)EEI, is prevented in CAP-treated neurons. (E) The reduction of NMDAR activity by Src40–58 peptide is also prevented in neurons treated with CAP for 48 h. Composite traces are shown in black, the NMDAR component is shown in dark gray, and the AMPA receptor (AMPAR) component is shown in light gray. (Scale bars are 50 ms/10 pA.) (F) Summary histogram of electrophysiology data. NMDA component data were calculated as Q_20′/Q_2′, and AMPA component data were calculated as A_20′/A_2′. CAP treatment for 48 h prevents modulation of NMDAR function by the Src activator and Src40–58 peptides, whereas neither of these reagents affected the AMPAR component of mEPSCs. Asterisks indicate a significant difference, Student's t test, P < 0.05.

ND2 is present at the PSD. (A) Immunoblots of PSD proteins probed with anti-ND2, anti-cytochrome c oxidase I (Cyto1), anti-ND4, anti-PSD95, anti-NR1, anti-Src, and anti-synaptophysin. The PSD preparation contained PSD95, NR1, and Src, but lacked the presynaptic marker synaptophysin. (B) Immunoblots of mitochondrial proteins probed with anti-ND2, anti-Cyto1, and anti-ND4. Neither NR1 nor NR2A/B was detected in the mitochondrial fraction (not shown). (C) Immunoblots of PSD proteins showing the specificity of the N-terminal ND2 antibody by preadsorption with the antigenic peptide used to derive the antibody. (D) Immunoblots of PSD and mitochondrial proteins probed with two independent rabbit polyclonal antibodies directed against two disparate regions of ND2. The N-terminal ND2 antibody was used for all subsequent experiments shown. (E) Postembedding immunogold electron microscopy images of rat hippocampus CA1 synapses. ND2 immunoreactivity in PSDs is visualized by secondary antibody conjugated to 10-nm gold particles. pre, presynaptic. (Scale bar is 200 nm.)

ND2 interacts with Src at the NMDAR complex. (A) Immunoblots of coimmunoprecipitates from PSD preparations probed with anti-ND2 or with anti-NMDA receptor subunit 1 (NR1) as indicated. Nonspecific IgG (either rabbit or mouse) was used as a negative control for both antibodies. (B) Immunoblot of coimmunoprecipitates from PSD preparations using anti-GluR2, anti-GABA_ARα, anti-GABA_ARβ2/3, and anti-Kv3.1 antibodies to immunoprecipitate and probed with anti-ND2. (C) Dot blot of ND2–GST fusion proteins probed with biotinylated Src40–58 or scrambled Src40–58 peptides followed by SA–HRP. (D) Blot of ND2.1–GST probed with biotinylated Src unique domain in the presence of either Src40–58 or scrambled Src40–58 followed by SA-HRP. (E) Immunoblots of coimmunoprecipitates obtained from PSD proteins in the presence of either Src40–58 or scrambled Src40–58 probed with anti-ND2 or stripped and reprobed with anti-Src. (F) Immunoblots of coimmunoprecipitates obtained from PSD proteins in the presence of either Src40–58 or scrambled Src40–58 (Left) or in the presence of ND2.1–GST fusion protein (Right) probed with anti-Src or anti-NR1. (G) Immunoblots of coimmunoprecipitates obtained from PSD proteins in the presence of either Src40–58 or scrambled Src40–58 probed with anti-ND2 or stripped and reprobed with anti-NR1.