Specific Interaction of Postsynaptic Densities With Membrane Rafts Isolated From Synaptic Plasma Membranes

Abstract

Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. We recently demonstrated the presence, at the electron microscopic level, of complexes consisting of membrane rafts and postsynaptic densities (PSDs) in detergent-resistant membranes (DRMs) prepared from synaptic plasma membranes (SPMs) (, J Neurochem, 119, 64–77). To further explore these complexes, here we investigated the nature of the binding between purified SPM-DRMs and PSDs in vitro. In binding experiments, we used SPM-DRMs prepared after treating SPMs with n-octyl-β-d-glucoside, because at concentrations of 1.0% or higher it completely separates SPM-DRMs and PSDs, providing substantially PSD-free unique SPM-DRMs as well as DRM-free PSDs. PSD binding to PSD-free DRMs was identified by mass spectrometry, Western blotting, and electron microscopy. PSD proteins were not incorporated into SPMs, and significantly less PSD proteins were incorporated into DRMs prepared from liver membranes, providing in vitro evidence that binding of PSDs to DRMs is specific and suggestion of the presence of specific interacting molecules. These specific interactions may have important roles in synaptic development, function, and plasticity in vivo. In addition, the binding system we developed may be a good tool to search for binding molecules and binding mechanisms between PSDs and rafts.

Keywords::

• detergent-resistant membranes
• DRM
• membrane rafts
• n-octyl-β-d-glucoside
• postsynaptic density
• PSD

ACKNOWLEDGMENTS

Anti-tubulin antibody was a kind gift from Dr. Toshihiro Fujii, Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This study was supported by National Institutes of Health grants DA021420 and DA032283 (W.-D.Y.) and National Center for Research Resources grant RR000168 (currently 5P51 OD011103 from the Office of Research Infrastructure Programs, Office of the Director).

Notice of correction:

The iFirst version of this article published online ahead of print on 25 March 2013 contained an error on page 4. The sentences “, but in the presence of iodoacetamide (2 mM)” and “and 2 mM iodoacetamide” should have appeared in the Preparation of SPMs, PSDs, DRMs and Liver Membranes (LMs) paragraph.