Abstract
At the post-synaptic density (PSD), large protein complexes dynamically form and dissociate in response to synaptic activity, comprising the biophysical basis for learning and memory. The use of detergents to isolate the PSD and release its membrane-associated proteins complicates studies of these activity-dependent protein interaction networks, because detergents can simultaneously disrupt the very interactions under study. Despite widespread recognition that different detergents yield different experimental results, the effect of detergent on activity-dependent synaptic protein complexes has not been rigorously examined. Here, we characterize the effect of three detergents commonly used to study synaptic proteins on activity-dependent protein interactions. We first demonstrate that SynGAP-containing interactions are more abundant in 1% Deoxycholate (DOC), while Shank-, Homer- and mGluR5-containing interactions are more abundant in 1% NP-40 or Triton. All interactions were detected preferentially in high molecular weight complexes generated by size exclusion chromatography, although the detergent-specific abundance of proteins in high molecular weight fractions did not correlate with the abundance of detected interactions. Activity-dependent changes in protein complexes were consistent across detergent types, suggesting that detergents do not isolate distinct protein pools with unique behaviors. However, detection of activity-dependent changes is more or less feasible in different detergents due to baseline solubility. Collectively, our results demonstrate that detergents affect the solubility of individual proteins, but activity-dependent changes in protein interactions, when detectable, are consistent across detergent types.
Highlights
The postsynaptic density (PSD) is an electron dense specialization composed of multiprotein complexes, whose functions include mediating the apposition of pre- and post synaptic membranes, clustering glutamate receptors, and coupling the activation of receptors to downstream signaling cascades[1]
The relative amount of protein solubilized by these detergents varied based upon the protein of interest; NP-40 and Triton solubilized almost 90% of total mGluR5, but only 30–45% of GluR1, Homer[1], NMDAR1, and post-synaptic density (PSD)-95 and only 15% of SynGAP (Fig. 2C–H)
We characterized the effect of DOC, NP-40, and Triton on the solubility and co-association of multiprotein complexes comprised of synapse-associated proteins
Summary
The postsynaptic density (PSD) is an electron dense specialization composed of multiprotein complexes, whose functions include mediating the apposition of pre- and post synaptic membranes, clustering glutamate receptors, and coupling the activation of receptors to downstream signaling cascades[1]. Homer EVH1 domains can bind type 1 metabotropic glutamate receptor (mGluR)s, IP3 receptors, transient receptor potential channels, dynamin, and drebrin[14,15] Signaling enzymes such as Calcium/Calmodulin dependent Kinase II (CamKII) and the brain specific Ras GTPase, SynGAP are highly abundant[16,17], further increasing the complexity of the PSD Critically, the organization of the PSD is highly dynamic. We and others have demonstrated that synaptic stimulation elicits dissociation of mGluR5-Homer-Shank scaffolds[18,19] and dispersion of SynGAP from the PSD18,20–22, allowing for the recruitment and stabilization of AMPA receptors to the PSD and changes in synaptic strength[22,23]. The relative amount in lysate or pellet was determined by dividing the density of the band by the total. *p < 0.05 relative to DOC lysate; Wilcoxon-ranked sum test
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