Affinity purification of PSD-95-containing postsynaptic complexes.
A widely used method for the preparation of postsynaptic density (PSD) fractions consists of treatment of synaptosomal membranes with Triton X-100 and further purification by density gradient centrifugation. In the present study, the purity of this preparation was assessed by electron microscopic analysis. Thin-section and rotary shadow immuno-electron microscopy of the Triton X-100-derived PSD fraction shows many PSD-95-positive structures that resemble in situ PSDs in shape and size. However, the fraction also includes contaminants such as CaMKII clusters, spectrin filaments and neurofilaments. We used magnetic beads coated with an antibody against PSD-95 to further purify PSD-95-containing complexes from the Triton-derived PSD fraction. Biochemical analysis of the affinity-purified material shows a substantial reduction in the astrocytic marker glial fibrillary acidic protein and electron microscopic analysis shows mostly individual PSDs attached to magnetic beads. This preparation was used to assess the association of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptors with the PSD-95-containing complex. AMPA receptors are demonstrated by immunoblotting to be present in the complex, although they do not co-purify exclusively with PSD-95, suggesting the existence of two pools of receptors, one associated with the PSD-95 scaffold and the other not. Of the AMPA receptor-anchoring proteins tested, SAP-97 is present in the affinity-purified preparation whereas GRIP is found only in trace amounts. These results imply that a subpopulation of AMPA receptors is anchored to the PSD-95-containing scaffold through interaction of GluR1 with SAP-97.
Vinade
Lucia
L
Laboratory of Neurobiology, National Instutute of Neurological Disorders and Stroke/NIH, 9000 Rockville Pike, Building 36/2A21, Bethesda, MD 20892, USA. vinade@codon.nih.gov
Chang
Michael
M
Schlief
Michelle L
ML
Petersen
Jennifer D
JD
Reese
Thomas S
TS
Tao-Cheng
Jung-Hwa
JH
Dosemeci
Ayse
A
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001923:Brain Chemistry; D000076122:Disks Large Homolog 4 Protein; D015151:Immunoblotting; D018189:Immunomagnetic Separation / Q000379:methods*; D007163:Immunosorbent Techniques; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D016253:Microscopy, Immunoelectron; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D017830:Octoxynol / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000737:chemistry / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D017830:Octoxynol; D018091:Receptors, AMPA; C501119:Dlg1 protein, rat; C501449:Dlg4 protein, rat; C033652:postsynaptic density proteins