Yeast Two-Hybrid Screen. cDNAs encoding amino acids 4-82 (the Src unique domain) and amino acids 4-150 [the Src unique and Src homology 3 (SH3) domains] of murine n-Src were ligated into pEG202 (1) to create two expression vectors encoding in frame LexA fusions containing the Src unique domain. The bait constructs were then sequenced. Both baits were tested to ensure that the baits did not activate transcription of the reporters in the absence of prey and that both could enter the nucleus and bind to LexA operators. To create the selection strains for screening, each bait plasmid was individually transformed into the yeast strain EGY48. EGY48 has an integrated Leu-2 selectable marker regulated by six LexA operator repeats, and carries a reporter plasmid with a lacZ gene regulated by eight LexA operator repeats. Low-affinity bait-prey interactions result in activation of the Leu-2 reporter gene only, whereas high-affinity interactions activate both the Leu-2 and lacZ reporter genes, allowing for double selection of prey. The selection strain was transformed with a representative activation-tagged cDNA prey fusion library constructed by using ~1-kb EcoRI fragmented poly(A)(+) RNA from human fetal brain. Yeast transformed with the prey library (~1.1 × 10⁶ clones) were screened by double selection on 5-bromo-4-chloro-3-indolyl β-D-galactoside (X-Gal) Leu^- medium. Prey cDNAs encoding proteins that interacted with the bait were isolated and sequenced.

Src, Fyn, and NADH Dehydrogenase Subunit 2 (ND2) Recombinant Proteins. The cDNAs encoding the SH3 and SH2 domains of mouse n-Src and Fyn were PCR subcloned, ligated in frame into pGEX4T-1 (Amersham Pharmacia), and sequenced. These plasmids, as well as plasmids encoding the unique domains of Src and Fyn in pGEX2T’6, were transformed into BL21 bacteria, and GST fusion proteins were purified by glutathione affinity chromatography. To create the ND2.1, ND2.2, and ND2.3 GST fusion proteins, cDNAs encoding amino acids 239-321 (ND2.1-GST), amino acids 189-238 (ND2.2-GST), and amino acids 1-188 (ND2.3-GST) of human ND2 were PCR subcloned and ligated into pGEX4T-1. Using PCR-based single nucleotide mutagenesis, all cDNAs encoding ND2 fusion proteins were corrected for differences between mitochondrial and nuclear codons to prevent premature translation termination and protein truncation. All constructs were then sequenced. The plasmids were transformed into bacteria, and GST fusion proteins were purified as above.

In Vitro Binding Assays. To detect the binding of the Src unique domain with ND2 GST fusion proteins, the Src unique domain fusion protein was purified on glutathione Sepharose and removed by thrombin (Sigma) cleavage from the GST moiety. After chemical inactivation of thrombin, the fusion protein was biotinylated by incubating with Sulfo-NHS-Biotin (Pierce) for 30 min at room temperature. The biotinylation reaction was quenched by the addition of Tris·HCl (pH 8.0) to a final concentration of 20 mM before incubating with ND2.1-GST, ND2.2-GST, or ND2.3-GST on beads. The beads were washed three times with RIPA buffer [50 mM Tris, pH 7.6/150 mM NaCl/1 mM EDTA/1% Nonidet P-40/2.5 mg/ml NaDOC/1 mM Na₃VO₄/1 mM PMSF/2 m g/ml each of aprotinin, pepstatin A, and leupeptin (Sigma)], then resuspended in RIPA buffer plus SDS/PAGE sample buffer. After brief centrifugation, proteins were resolved by SDS/PAGE, transferred to nitrocellulose membranes, and probed by using a streptavidin-horseradish peroxidase (SA-HRP) conjugate (Sigma). Bound probe was then detected on film by using an enhanced chemiluminescence kit (Amersham Pharmacia). The same method was used to detect the binding of various biotinylated domains of Src and Fyn with ND2.1-GST bound on glutathione Sepharose beads. In competition experiments, Src40-58 or scrambled Src40-58 peptides (final concentration 30 m g/ml; synthesized by J. Bell, Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada) were preincubated with ND2.1-GST on beads before the addition of biotinylated Src or Fyn GST fusion proteins.

Coimmunoprecipitation of ND2 and Src and Immunoblotting. Total soluble protein was prepared from preweighed rat tissues by homogenization at 4°C in 0.25 M sucrose/10 mM Hepes-NaOH, 1 mM EDTA, pH 7.4 with 2 m g/ml each of aprotinin, pepstatin A, and leupeptin. After brief centrifugation of the samples at 4,000 ´ g, Nonidet P-40 was added to 1% (vol/vol) to the cleared supernatants. After incubation for 10 min, the protein concentration of the samples was determined by detergent compatible protein assay (BioRad Laboratories, Mississauga, Ontario, Canada) and equilibrated. The solubilized proteins were centrifuged briefly at 14,000 ´ g to remove insoluble material and then incubated with 5 m g of either anti-ND2 (rabbit polyclonal from R. F. Doolittle, University of California, San Diego; ref. 2), anti-Src (mouse monoclonal clone 327 from J. Bolen, DNAX, Palo Alto, CA), or control, nonspecific rabbit or mouse IgG (Sigma) overnight at 4°C. Immune complexes were isolated by the addition of 10 m l of protein G-Sepharose beads followed by incubation for 2 h at 4°C. Immunoprecipitates were then washed three times with RIPA buffer, resuspended in RIPA buffer plus SDS/PAGE sample buffer, and boiled for 5 min. The samples were resolved by SDS/PAGE, transferred to nitrocellulose membranes and analyzed by immunoblotting with anti-ND2, anti-Src, or anti-Fyn (mouse monoclonal clone 25, Pharmingen). Bound antibody was then detected on film by using appropriate secondary antibody/HRP conjugates and an enhanced chemiluminescence kit (Amersham Pharmacia). For control immunoprecipitations under denaturing conditions, SDS was added to the initial protein samples to a final concentration of 0.4% and the samples were boiled for 5 min and rapidly cooled to 4°C before addition of the antibodies used for immunoprecipitation.

Cellular Fractionation. Postsynaptic density proteins (3) were prepared from rat brain as described in ref. 4, with extraction using 1% Triton X-100 in the final step. Cellular fractionation of rat brain tissue into nuclear, heavy mitochondrial, light mitochondrial, microsomal, and cytosolic fractions was performed by differential centrifugation of tissue homogenate in 0.25 M sucrose/10 mM Hepes-NaOH, 1 mM EDTA, pH 7.4, with 2 m g each of aprotinin, pepstatin A, and leupeptin (Sigma) at 4°C. Nuclei were pelleted by centrifugation at 1,000 ´ g for 10 min, the supernatant was removed and spun at 3,000 ´ g for 10 min to obtain a heavy mitochondrial pellet. The supernatant was removed and spun at 16,000 ´ g for 15 min to obtain a light mitochondrial pellet. The supernatant was removed and spun at 100,000 ´ g for 1 h to obtain a microsomal pellet and the cytosolic fraction. All pellets were then resuspended in RIPA buffer. The light mitochondrial fraction was used in subsequent experiments. For immunoblots 50 m g of total protein was loaded per lane, resolved by SDS/PAGE, transferred to nitrocellulose membranes, and probed with anti-ND2, anti-Cyto1 and anti-ND4 (mouse monoclonals, Molecular Probes Inc., Eugene, OR), anti-PSD95 (mouse monoclonal clone 7E3-1B8, Oncogene Research Products, Cambridge, MA), anti-NR1 (mouse monoclonal clone 54.1, PharMingen, Mississauga, Ontario), anti-Src, or anti-synaptophysin (mouse monoclonal, Sigma). To estimate the proportion of ND2 found in the postsynaptic density (PSD) fraction, a total brain homogenate sample, samples from pellets and supernatants collected before obtaining the final PSD fraction, and a PSD sample were quantitated and concentrated using Amicon Centricon YM-30 centrifugal filter units (Millipore, Etobicoke, Ontario) as required. Equal amounts of protein were resuspended in RIPA buffer plus SDS/PAGE sample buffer and boiled for 5 min. The samples were resolved by SDS/PAGE, transferred to nitrocellulose membranes, and analyzed by immunoblotting with anti-ND2, anti-Cyto1 and anti-ND4. Preadsorption of the anti-ND2 antibody with antigenic peptide prevented antibody signal detection on immunoblots. Some experiments were repeated using a second rabbit polyclonal anti-ND2 antibody (anti-ND2.1). This antibody was generated against the ND2.1-GST fusion protein. Collected sera was precleared against GST, and the antibody was affinity purified by using ND2.1 before use.

Postembedding Immunogold Electron Microscopy. Sprague-Dawley rats were anesthetized and perfused with 4% paraformaldehyde/0.5% glutaraldehyde in 0.1 M phosphate buffer. Parasagittal sections of the hippocampus were cryoprotected in 30% glycerol and frozen in liquid propane. Frozen sections were immersed in 1.5% uranyl acetate in methanol at -90°C, infiltrated with Lowicryl HM-20 resin at -45°C, and polymerized with UV light. Sections were incubated in 0.1% sodium borohydride/50 mM glycine in TBS and 0.1% Triton X-100 (TBST), followed by 10% normal goat serum (NGS) in TBST, primary antibody in 1% NGS in TBST, and immunogold (10 nm; Amersham Pharmacia) in 1% NGS in TBST/0.5% polyethylene glycol. Finally, the sections were stained in uranyl acetate and lead citrate before analysis.

Immunoprecipitations from PSD Preparations. PSD proteins (400 m g) were solubilized in RIPA buffer for 10 min at 4°C. Following this procedure, we estimate the amount of solubilization of NMDAR subunits to be 15%. Solubilized PSD proteins were centrifuged briefly at 14,000 ´ g to remove insoluble material and then incubated with 5 m g of either anti-ND2, anti-Src, anti-NR1, anti-GluR2, anti-GABA_ARa , anti-GABA_ARb 2/3 (from Y. Wang, University of Toronto), anti-Kv3.1, or nonspecific rabbit or mouse IgG overnight at 4°C. Immune complexes were isolated by the addition of 10 m l of protein G-Sepharose beads followed by incubation for 2 h at 4°C. Immunoprecipitates were then washed three times with RIPA buffer, resuspended in RIPA buffer plus SDS/PAGE sample buffer, and boiled for 5 min. The samples were resolved by SDS/PAGE, transferred to nitrocellulose membranes, and probed with anti-ND2, anti-Src, anti-NR1, or anti-phosphotyrosine (clone 4G10, Upstate Biotechnology, Lake Placid, NY). Blots were also stripped and reprobed with the antibody that was used for immunoprecipitation. In competition experiments, Src40-58 or scrambled Src40-58 peptides (final concentration 30 m g/ml) were preincubated with the starting material before the addition of the antibodies used for immunoprecipitation. Some experiments were repeated using a second anti-ND2 antibody (anti-ND2.1; see above).

Dot Blotting. Src40-58 and scrambled Src40-58 peptides were biotinylated by incubating with Sulfo-NHS-Biotin (Pierce) for 30 min at room temperature. The biotinylation reaction was then quenched by the addition of Tris-HCl (pH 8.0) to a final concentration of 20 mM. Purified recombinant fusion proteins (~20 m g each) were dotted onto nitrocellulose and dried overnight. Membranes were blocked with 5% BSA in PBS (pH 7.5) for 1 h, after which biotinylated peptides (30 m g/ml) diluted 1:1,000 in fresh 5% BSA in PBS were added. The membranes were incubated with the peptides for 1 h, washed, and probed with streptavidin-HRP conjugate. Bound probe was then detected on film by using an ECL kit.

Pull Down Assays. Solubilized proteins (250 m g) from PSD preparations were centrifuged briefly at 14,000 ´ g to remove insoluble material and then incubated with ~20 m g of ND2.1-GST, ND2.2-GST, ND2.3-GST, or GST alone on glutathione Sepharose beads. The beads were then washed three times with RIPA buffer, resuspended in RIPA buffer plus SDS/PAGE sample buffer, and boiled for 5 min. The samples were resolved by SDS/PAGE, transferred to a nitrocellulose membrane, and probed with anti-Src or anti-GST (mouse monoclonal clone Z-5, Santa Cruz Biotechnology).

Electrophysiological Recording from Cultured Hippocampal Neurons. Fetal hippocampal neurons were prepared, cultured, and used for electrophysiological recordings 12-17 days after plating. Methods for whole cell recordings are described in ref. 4. Briefly, cultures were bathed in extracellular solution containing 140 mM NaCl, 5.4 mM KCl, 25 mM Hepes, 1.3 mM CaCl₂, 33 mM glucose, pH 7.35 (310-320 mOsm) supplemented with tetrodotoxin (0.5 m M), strychnine (10 m M), bicuculline methiodide (10 m M), and glycine (1 m M). Patch electrodes (4-7 MW ) were filled with intracellular solution containing 140 mM CsCl, 10 mM Hepes, 10 mM BAPTA, 2 mM Mg-ATP, pH 7.25 (300-310 mOsm). The EPQ(pY)EEIPIA, Src40-58, and scrambled Src40-58 peptides were administered intracellularly by inclusion in the patch pipette. All recordings were performed at room temperature (22-24°C). For acute chloramphenicol treatment, media was removed from the culture dishes, and the neurons were rinsed and then bathed in extracellular solution supplemented with 50 m g/ml chloramphenicol (Sigma). For neurons treated for 48 h, growth media was supplemented with 50 m g/ml chloramphenicol 48 h before electrophysiological recording. Miniature excitatory postsynaptic currents (mEPSCs) were recorded immediately upon formation of the whole cell configuration and continuously monitored for 20-30 min thereafter. mEPSCs were filtered at 2 kHz and acquired online with a personal computer using STRATHCLYDE software (courtesy of J. Dempster) with the detection level set to approximately three times higher than the baseline noise. False events and traces with more than one event per 200-ms recording period were eliminated by subsequent inspection of the raw data. Averages were created using a minimum of 50 traces aligned by their rising edges.

Protein Extraction From Cultured Hippocampal Neurons and immunoblotting. After electrophysiological recording, the extracellular solution was removed and the neurons were scraped from the dishes in cell lysis buffer (50 mM Tris, pH 8.0/150 mM NaCl/5 mM EDTA/0.5% Nonidet P-40/1% Triton X-100/0.5 mM PMSF) and incubated at 4°C for 1 h. The supernatant was cleared by centrifugation at 4,000 ´ g and NaDOC was added to a final concentration of 0.5%. After a 10-min incubation, the supernatant was cleared by centrifugation at 14,000 ´ g for 90 min. Soluble protein was quantitated by detergent compatible protein assay, and SDS/PAGE sample buffer was added to the equilibrated samples. Samples were resolved by SDS/PAGE, transferred to nitrocellulose membranes, and probed by using anti-ND2, anti-NR1, and anti-Src.

Luciferin-Luciferase Assay to Determine ATP Levels. Cultured hippocampal neurons (12-17 days in vitro), either untreated or treated with 50 m g/ml chloramphenicol for 48 h, were trypsinized from the culture dishes, and cell density was determined by using a hemocytometer. The cells were washed once with ice-cold PBS, spun down at 3,500 ´ g for 5 min at 4°C, and resuspended in an appropriate volume of ice-cold PBS to equilibrate cell density. The cells were then sonicated on ice, and the samples were centrifuged briefly at 10,000 ´ g at 4°C. Twenty-five microliters of the supernatant was added to luciferin-luciferase assay solution (250 mM glycylglycine, pH 7.4/2 mM EGTA/2 mM MgCl₂/0.4 g of BSA per liter) containing luciferin (15 m M; Sigma) and luciferase (15 m M; Sigma), and read immediately by using a photomultiplier tube (Photon Technologies, London, Ontario, Canada) set to read emission at 533 nm in single photon counting mode. The output from the photomultiplier tube was sampled by a computer equipped with hardware and software from Photon Technologies. Photon counts were obtained every 0.2 s for 120 s. All light intensity measurements were analyzed off-line. After background subtraction for each sample, values over a continuous 100-s interval were averaged, and the ATP concentration in the sample was calculated by using a standard curve of known ATP concentrations. Equilibration of cell density was also assessed post hoc by quantitation of the total protein content in each sample.

Assessment of Mitochondrial Membrane Potential (D y _M) in Cultured Neurons. Mitochondrial membrane potential was monitored with the potentiometric fluorescent cationic dye tetramethylrhodamine methyl ester (TMRM, Molecular Probes). Cultured neurons (12-17 days in vitro), either untreated or treated with 50 m g/ml chloramphenicol for 48 h, were loaded in culture media with TMRM (0.5 m M) for 20 min at 37°C. The neurons were washed once and bathed in extracellular recording solution containing 140 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl₂, 33 mM glucose, 25 mM Hepes, pH 7.35 (310-320 mOsm); supplemented with 0.5% BSA. Fluorescence levels within individual neurons were measured by determining single photon counts using an inverted microscope equipped with a fluorescent imaging system and a photomultiplier tube detector (Photon Technologies). The excitation wavelength was set to 488 nm, and fluorescence emission was detected at 578 nm. To assess the response of untreated or chloramphenicol-treated neurons to D y _M collapse, the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, Sigma) was bath applied at a final concentration of 2 m M. All fluorescence intensity measurements were analyzed off-line. After background subtraction, fluorescence intensity values over a continuous 3 min interval after FCCP application were averaged.

1. Gyuris, J., Golemis, E., Chertkov, H. & Brent, R. (1993) Cell 75, 791-803.

2. Mariottini, P., Chomyn, A., Riley, M., Cottrell, B., Doolittle, R. F. & Attardi, G. (1986) Proc. Natl. Acad. Sci. USA 83, 1563-1567.

3. Kennedy, M. B., Bennett, M. K. & Erondu, N. E. (1983) Proc. Natl. Acad. Sci. USA 80, 7357-7361.

4. Pelkey, K. A., Askalan, R., Paul, S., Kalia, L. V., Nguyen, T. H., Pitcher, G. M., Salter, M. W. & Lombroso, P. J. (2002) Neuron 34, 127-138.