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 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) assay^8,9,10,11,12, 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.

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 buffer (Table 1) in a glass homogenizer (~12 strokes) on ice.
2. Centrifuge lysates for 15 min at 1,400 x g at 4 °C. Transfer the supernatant to a new tube on ice. Resuspend the pellet (~6 strokes) in an equal volume of homogenization buffer and centrifuge at 710 x g for 10 min at 4 °C.
3. Combine the supernatants from step 1.2 and centrifuge at 40,000 x g for 20 min at 4 °C.
4. Discard the supernatant (cytosolic fraction) and resuspend the pelleted membrane (P2) fraction in homogenization buffer.
   NOTE: Samples can be flash frozen and stored at -80 °C for later use.
5. Perform a bicinchoninic acid (BCA) assay to quantitate protein levels. For the APEGS assay, begin with ~100−200 mg protein (maximum 250 mg) in a volume of 470 µL of buffer A (Table 1) in a 1.5 mL tube. Sonicate and centrifuge at >13,000 x g for 10 min at 25 °C to remove insoluble protein. Transfer solubilized protein to a new 1.5 mL tube.

2. Acyl-PEGyl exchange gel-shift (APEGS) assay

1. Disrupt disulfide bonds and block free cysteines.
   1. Disrupt disulfide bonds by incubating in 25 mM tris(2-carboxyethyl)phosphine (TCEP; 25 µL, added from a stock solution; Table 1) for 60 min at 55 °C.
   2. Block free cysteines with 50 mM N-ethylmaleimide (NEM; 12.5 µL, added from a stock solution; Table 1) at room temperature for 3 h.
      NOTE: The reaction can be extended overnight.
2. Perform chloroform methanol (CM) precipitation.
   1. Transfer the protein solution from 1.5 mL tube to a polypropylene or glass tube that can be centrifuged in a swinging bucket rotor at modest speed. A 5 mL tube is ideal. Add four volumes (2 mL) of methanol (MeOH) and vortex briefly.
   2. Add two volumes (1 mL) of chloroform and vortex briefly.
   3. Add three volumes (1.5 mL) of dH₂O and vortex briefly.
   4. Centrifuge the samples at 3,000 x g for 30 min at 25 °C in a swinging bucket rotor.
   5. Carefully remove and discard the upper phase.
   6. Add 3 volumes (1.5 mL) of MeOH and mix gently but thoroughly, being careful not to fragment the opaque protein pancake.
   7. Centrifuge at 3,000 x g for 10 min at 25 °C in a swinging bucket rotor.
   8. Using a glass serological pipet, remove as much of the top phase as possible without disturbing the protein pancake.
   9. Carefully rinse the pellet with 1 mL of MeOH.
   10. Air dry the pellet for at least 10 min.
       NOTE: Dried pellet can be stored at -20 °C.
3. Perform cleavage of palmitoyl-thioester linkages with hydroxylamine (NH₂OH, HAM).
   1. Resuspend protein precipitate in 125 µL of buffer A and sonicate briefly. Centrifuge at >13,000 x g for 10 min at 25 °C to remove insoluble material. Transfer solubilized protein to a new 1.5 mL tube.
   2. Add 375 µL of buffer H (HAM+; Table 1) or buffer T (HAM-; Table 1) and incubate samples for 60 min at 25 °C.
4. Repeat the CM precipitation described in step 2.2.
   NOTE: Dried pellet can be stored at -20 °C.
5. Add mPEG to unprotected cysteines.
   1. Resuspend pellet in 100 µL of buffer A containing 10 mM TCEP. Transfer to a 1.5 mL tube.
      NOTE: It is normal for significant loss of protein to have occurred during CM precipitation steps. All subsequent steps will be performed in a 1.5 mL tube, constraining the volume of protein to a maximum of ~120−130 µL. This will reduce protein loss during the final CM precipitation.
   2. Add 20 mM mPEG-5k (25 µL, added from a stock solution; Table 1) to protein sample and mix by pipetting. Incubate for 60 min at 25 °C with end-over-end rotation.
   3. To remove unincorporated mPEG-5k, perform CM precipitation as described in step 2.2 using these adjusted volumes: 4 volumes of MeOH (500 µL), 2 volumes of chloroform (250 µL), and 3 volumes of dH₂O (375 µL). Centrifuge at >13,000 x g for 10 min at 25 °C.
   4. Carefully remove the upper phase as before, avoiding the thick, flocculent pancake. Add 1 mL of MeOH and mix gently but thoroughly. Centrifuge at >13,000 x g for 10 min at 25 °C.
   5. Carefully remove the supernatant and rinse the pellet with 1 mL of MeOH. Centrifuge at >13,000 x g for 10 min at 25 °C. Air dry pellet.
      NOTE: Dried pellet can be stored at -20 °C.
   6. Resuspend the dried pellet in 50 µL of buffer A (without TCEP or any reducing agent). Reserve 5 µL for BCA protein quantitation. After quantitation, add an appropriate amount of 4x Laemmli sample buffer and, depending on the protein of interest, potentially heat the sample to 70−100 °C for 10 min.
      NOTE: Boiling may cause aggregation of some membrane proteins.

3. Western blot analysis of PEGylated proteins

1. Load the sample onto an sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel¹³.
   NOTE: In this protocol 10% polyacrylamide was used.
2. Use standard separation, transfer and detection protocols for Western blotting¹⁴.
3. Use densitometry¹⁴ to determine the relative amounts of PEGylated versus nonPEGylated proteins.

Results

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...

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

List of materials used in this article

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