Name: acyl-pegyl exchange gel shift assay for quantitative determination of palmitoylation of brain membrane proteins
Uploaded: 2020-03-29T14:00:00
Description: Watch this Scientific Journal Video about Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins at JoVE.com

The authors thank K. Woolfrey for advice and input on the APEGS assay. These studies were funded by research grants to E.D. from the Whitehall Foundation and the NIH-NIMH (1R01MH119347).

All animal procedures followed guidelines set forth by the National Institutes of Health (NIH) and have been approved by the Institutional Animal Care and Use Committee at the University of California, Davis.
1. Preparation of mouse brain membranes
<ol>
	<li>Decapitate mouse using a guillotine apparatus and dissect brain out rapidly (in &#60;1 min, if possible, to minimize palmitoylation changes that may occur during dissection procedure). Homogenize immediately in 10 mL of homogenization bu...

<ol>
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In our previous work, we utilized the ABE assay to demonstrate that SynDIG1 is palmitoylated at two conserved juxta-transmembrane Cys residues (found in all SynDIG proteins) in an activity-dependent manner to regulate stability, localization, and function6. A limitation is that the ABE assay requires affinity purification with agarose resins conjugated to avidin moieties as the final step in the procedure, resulting in significant loss of signal that complicate quantitative analysis. Furthermore, ...

S-palmitoylation is a reversible post-translational modification of target proteins that regulates stable membrane association, protein trafficking, and protein-protein interactions1. It involves addition of a 16-carbon palmitate moiety to cysteine residues via thioester linkage catalyzed by palmitoyl acyltransferase (PAT) enzymes. Many synaptic proteins in the brain are palmitoylated, including AMPA-Rs and PSD-95, in an activity-dependent manner to regulate stability, localization, and function2,3,4. Alterations in the levels of synaptic AMPARs in the PSD via interaction of auxiliary factors with synaptic scaffolds such as PSD-95 underlies synaptic plasticity; thus, methods to determine the palmitoylation state of synaptic proteins provides important insight into mechanisms of synaptic plasticity.
Previously, we identified the SynDIG family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs5. Overexpression or knock-down of SynDIG1 in dissociated rat hippocampal neurons increases or decreases, respectively, AMPA-R synapse size and number by ~50% as detected using immunocytochemistry and electrophysiology5. We utilized the acyl-biotin exchange (ABE) assay to demonstrate that SynDIG1 is palmitoylated at two conserved juxta-transmembrane Cys residues (found in all SynDIG proteins) in an activity-dependent manner to regulate stability, localization, and function6. The ABE assay relies on exchange of biotin on cysteines protected by modification and subsequent affinity purification7. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay8,9,10,11,12, which does not require affinity purification and instead utilizes changes in gel mobility to determine the number of modifications for a protein of interest. The protocol is described for investigation of endogenous membrane proteins from mouse brain for which suitable antibodies are available.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hydroxylamine (HAM)</td>
			<td>ThermoFisher</td>
			<td>26103</td>
			<td></td>
		</tr>
		<tr>
			<td>Methoxy-PEG-(CH2)3NHCO(CH2)2-MAL (mPEG)</td>
			<td>NOF</td>
			<td>ME-050MA</td>
			<td>MW ~5000 kDa</td>
		</tr>
		<tr>
			<td>Microfuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>N-ethylmalemide (NEM)</td>
			<td>Calbiochem</td>
			<td>34115</td>
			<td>Highly toxic.</td>
		</tr>
		<tr>
			<td>Optical imager for densitometry</td>
			<td>Azure Biosystems</td>
			<td>Sapphire Biomolecular Imager</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>Polypropylene tubes with cap</td>
			<td>Fisher Scientific</td>
			<td>14-956-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipets (glass)</td>
			<td>Fisher Scientific</td>
			<td>13-678-27D</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Beckman</td>
			<td>Allegra X-15R</td>
			<td>or equivalent equipment</td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl) phosphine-hydrochloride (TCEP)</td>
			<td>EMD Millipore</td>
			<td>580560</td>
			<td></td>
		</tr>
	</tbody>
</table>

acyl-pegyl exchange gel shift assay for quantitative determination of palmitoylation of brain membrane proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Immunoblotting with antibodies against the protein of interest reveals the palmitoylated state (non, singly, doubly, etc.) in mouse brain lysates as determined by mobility shift compared with samples in which HAM was not included. Previously, we had demonstrated that SynDIG1 was palmitoylated at two sites using the ABE assay6; however, we could not determine whether both sites were modified in brain tissue. Here we show that SynDIG1, SynDIG4/Prrt1, and Prrt2 are pa...

Watch this Scientific Journal Video about Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins at JoVE.com

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

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