Activity-dependent changes in synaptic protein complex composition are consistent in different detergents despite differential solubility

AbstractAt 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 both isolate the PSD fraction 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 (HMW) complexes generated by size exclusion chromatography, although the detergent-specific abundance of proteins in HMW 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.

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Chlorophyll-protein complex composition and photochemical activity in developing chloroplasts from greening barley seedlings

Photosynthesis Research ◽

10.1007/bf00024184 ◽

1986 ◽

Vol 10 (1-2) ◽

pp. 37-49 ◽

Cited By ~ 11

Author(s):

Kent O. Burkey

Keyword(s):

Protein Complex ◽

Photochemical Activity ◽

Complex Composition ◽

Chlorophyll Protein

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NMDAR-activated PP1 dephosphorylates GluN2B to modulate NMDAR synaptic content

10.1101/520882 ◽

2019 ◽

Author(s):

Andrew M. Chiu ◽

Jiejie Wang ◽

Michael P. Fiske ◽

Pavla Hubalkova ◽

Levi Barse ◽

...

Keyword(s):

Protein Complex ◽

Lateral Diffusion ◽

List Type ◽

Dependent Manner ◽

Critical Determinant ◽

Nmdar Activation ◽

Mature Neurons ◽

Synaptic Neurotransmission ◽

Intracellular Cascades ◽

Activity Dependent

SUMMARYIn mature neurons, postsynaptic NMDARs are segregated into two populations, synaptic and extrasynaptic, which differ in localization, function, and associated intracellular cascades. These two pools are connected via lateral diffusion, and receptor exchange between them modulates synaptic NMDAR content. Here, we identify the phosphorylation of the PDZ-ligand of the GluN2B subunit of NMDARs (at S1480) as a critical determinant in dynamically controlling NMDAR synaptic content. We find that phosphorylation of GluN2B at S1480 maintains NMDARs at extrasynaptic membranes as part of a protein complex containing Protein Phosphatase 1 (PP1). Global activation of NMDARs leads to the activation of PP1, which mediates dephosphorylation of GluN2B at S1480 to promote an increase in synaptic NMDAR content. Thus, PP1-mediated dephosphorylation of the GluN2B PDZ-ligand modulates the synaptic expression of NMDARs in mature neurons in an activity-dependent manner, a process with profound consequences for synaptic and structural plasticity, metaplasticity, and synaptic neurotransmission.HIGHLIGHTSPhosphorylation of the PDZ-ligand of the GluN2B subunit of NMDARs (GluN2B-pS1480) maintains NMDARs at extrasynaptic sites.Extrasynaptic NMDARs form a stable protein complex containing PP1.Global NMDAR activation increases NMDAR synaptic content by promoting PP1-mediated dephosphorylation of GluN2B-pS1480.GluN2B-pS1480 dephosphorylation is mediated by a PP1 subpopulation not involved in LTD and is enhanced by age.

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A Co-Fractionation Mass Spectrometry-based Prediction of Protein Complex Assemblies in the Developing Rice Aleurone-subaleurone

10.1101/2021.06.16.448567 ◽

2021 ◽

Author(s):

Youngwoo Lee ◽

Thomas W Okita ◽

Daniel B Szymanski

Keyword(s):

Mass Spectrometry ◽

Protein Complex ◽

Large Scale ◽

Rna Binding ◽

Seed Quality ◽

Protein Complexes ◽

Early Stage ◽

Practical Significance ◽

Rice Seed ◽

Complex Composition

Multiprotein complexes execute and coordinate diverse cellular processes such as organelle biogenesis, vesicle trafficking, cell signaling, and metabolism. Knowledge about their composition and localization provides useful clues about the mechanisms of cellular homeostasis and systems-level control. This is of great biological importance and practical significance in heterotrophic rice endosperm and aleurone-subaleurone tissues that are a primary source of seed vitamins and stored energy. Dozens of protein complexes have been implicated in the synthesis, transport, and storage of seed proteins, lipids, vitamins, and minerals. Mutations in protein complexes that control RNA transport result in aberrant endosperm with shrunken and floury phenotypes, significantly reducing seed yield and quality. The purpose of this research is to broadly predict protein complex composition in the aleurone-subaleurone layers of developing rice seeds using co-fractionation mass spectrometry. Following orthogonal chromatographic separations of biological replicates, thousands of protein elution profiles were subjected to distance-based clustering to enable a large-scale determination of multimerization state and complex composition. Predictions included evolutionarily conserved proteins across diverse functional categories, including novel heteromeric RNA binding protein complexes that influence seed quality. This effective and open-ended proteomics pipeline provides useful clues about systems-level controls in the early stage of rice seed development.

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