Studying the protein organization of the postsynaptic density by a novel solid phase- and chemical cross-linking-based technology.
Agarose beads carrying a cleavable, fluorescent, and photoreactive cross-linking reagent on the surface were synthesized and used to selectively pull out the proteins lining the surface of supramolecules. A quantitative comparison of the abundances of various proteins in the sample pulled out by the beads from supramolecules with their original abundances could provide information on the spatial arrangement of these proteins in the supramolecule. The usefulness of these synthetic beads was successfully verified by trials using a synthetic protein complex consisting of three layers of different proteins on glass coverslips. By using these beads, we determined the interior or superficial locations of five major and 19 minor constituent proteins in the postsynaptic density (PSD), a large protein complex and the landmark structure of asymmetric synapses in the mammalian central nervous system. The results indicate that alpha,beta-tubulins, dynein heavy chain, microtubule-associated protein 2, spectrin, neurofilament H and M subunits, an hsp70 protein, alpha-internexin, dynamin, and PSD-95 protein reside in the interior of the PSD. Dynein intermediate chain, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, kainate receptors, N-cadherin, beta-catenin, N-ethylmaleimide-sensitive factor, an hsc70 protein, and actin reside on the surface of the PSD. The results further suggest that the N-methyl-d-aspartate receptors and the alpha-subunits of calcium/calmodulin-dependent protein kinase II are likely to reside on the surface of the PSD although with unique local protein organizations. Based on our results and the known interactions between various PSD proteins from data mining, a model for the molecular organization of the PSD is proposed.
Liu
Szu-Heng
SH
Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan, Republic of China.
Cheng
Huei-Hsuan
HH
Huang
San-Yuan
SY
Yiu
Pei-Chun
PC
Chang
Yen-Chung
YC
D000818:Animals; D001386:Azides; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000737:chemistry / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D008863:Microspheres; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D012685:Sepharose; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides; D013552:Swine; D013569:Synapses / Q000737:chemistry*
D001386:Azides; D003432:Cross-Linking Reagents; D009419:Nerve Tissue Proteins; D013388:Succinimides; C033652:postsynaptic density proteins; C074882:sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate; D012685:Sepharose