Abstract
Postsynaptic density (PSD) is a protein supramolecule lying underneath the postsynaptic membrane of excitatory synapses and has been implicated to play important roles in synaptic structure and function in mammalian central nervous system. Here, PSDs were isolated from two distinct regions of porcine brain, cerebral cortex and cerebellum. SDS-PAGE and Western blotting analyses indicated that cerebral and cerebellar PSDs consisted of a similar set of proteins with noticeable differences in the abundance of various proteins between these samples. Subsequently, protein localization in these PSDs was analyzed by using the Nano-Depth-Tagging method. This method involved the use of three synthetic reagents, as agarose beads whose surface was covalently linked with a fluorescent, photoactivable, and cleavable chemical crosslinker by spacers of varied lengths. After its application was verified by using a synthetic complex consisting of four layers of different proteins, the Nano-Depth-Tagging method was used here to yield information concerning the depth distribution of various proteins in the PSD. The results indicated that in both cerebral and cerebellar PSDs, glutamate receptors, actin, and actin binding proteins resided in the peripheral regions within ∼ 10 nm deep from the surface and that scaffold proteins, tubulin subunits, microtubule-binding proteins, and membrane cytoskeleton proteins found in mammalian erythrocytes resided in the interiors deeper than 10 nm from the surface in the PSD. Finally, by using the immunoabsorption method, binding partner proteins of two proteins residing in the interiors, PSD-95 and α-tubulin, and those of two proteins residing in the peripheral regions, elongation factor-1α and calcium, calmodulin-dependent protein kinase II α subunit, of cerebral and cerebellar PSDs were identified. Overall, the results indicate a striking similarity in protein organization between the PSDs isolated from porcine cerebral cortex and cerebellum. A model of the molecular structure of the PSD has also been proposed here.
Highlights
Postsynaptic density (PSD)1 is a disk-shaped protein complex lying underneath the postsynaptic membrane and a landmark subcellular structure of most excitatory synapses in mammalian central nervous system [1]
An earlier study has indicated that the PSD isolated from dog cerebral cortex differs from the PSD isolated from dog cerebellum in protein composition, protein phosphorylation, and morphology [17]
The results further indicated that 14, 11, 5, and 3 of cerebral PSD proteins were co-immunoabsorbed with PSD-95, ␣-tubulin, CaMKII␣, and EF-1␣, respectively (Fig. 7A) and that 10, 19, 3, and 1 of cerebellar PSD proteins were co-immunoabsorbed with PSD95, ␣-tubulin, CaMKII␣, and EF-1␣, respectively (Fig. 7B)
Summary
Postsynaptic density (PSD) is a disk-shaped protein complex lying underneath the postsynaptic membrane and a landmark subcellular structure of most excitatory synapses in mammalian central nervous system [1]. A proteomic study has further delineated the molecular heterogeneity between the PSDs isolated from rat forebrain and cerebellum in a quantitative manner [29] It still remains unclear whether the PSDs in different brain regions share a similar protein organization or not. The abovementioned solid-phase and chemical crosslinking-based methodology, called Nano-Depth-Tagging (NDT), has been developed for identifying proteins residing at different depths from the surface of protein supramolecules. This methodology involves the use of several synthetic reagents, called NDT reagents. Because the functional azido groups on the SAED of various NDT reagents are at different distances from the surface of the bead (Fig. 1A), they could reach into and interact with proteins residing at different depths in a supramolecule. A model of the protein organization of the PSD has been proposed here
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