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

Summary

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.

Abstract

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.

Introduction

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 function^2,3,4. Alterations in the levels of synaptic AMPARs in the....

Protocol

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

1. Decapitate mouse using a guillotine apparatus and dissect brain out rapidly (in <1 min, if possible, to minimize palmitoylation changes that may occur during dissection procedure). Homogenize immediately in 10 mL of homogenization bu.......

Discussion

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, .......

Materials

Name	Company	Catalog Number	Comments
Hydroxylamine (HAM)	ThermoFisher	26103
Methoxy-PEG-(CH2)3NHCO(CH2)2-MAL (mPEG)	NOF	ME-050MA	MW ~5000 kDa
Microfuge	Eppendorf	5415R	or equivalent equipment
N-ethylmalemide (NEM)	Calbiochem	34115	Highly toxic.
Optical imager for densitometry	Azure Biosystems	Sapphire Biomolecular Imager	or equivalent equipment
Polypropylene tubes with cap	Fisher Scientific	14-956-1D
Serological pipets (glass)	Fisher Scientific	13-678-27D
Table top centrifuge	Beckman	Allegra X-15R	or equivalent equipment
Tris(2-carboxyethyl) phosphine-hydrochloride (TCEP)	EMD Millipore	580560