Detection of Detergent-sensitive Interactions Between Membrane Proteins

Summary

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Abstract

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

Introduction

GLUT4 is a glucose transporter protein which is expressed predominantly in fat and skeletal muscle cells where it mediates the effect of insulin on post-prandial blood glucose clearance¹. Being a very stable protein, GLUT4 is regulated at a post-translational level. In the absence of insulin, GLUT4 is largely excluded from the plasma membrane (hence low basal permeability for glucose) and is localized mainly inside the cell in small insulin-responsive vesicles (IRVs) and trans-Golgi network (TGN) that is likely to represent the IRV donor compartment. Upon insulin administration, the IRVs fuse with the plasma membrane and deliver GLUT4 to the si....

Protocol

1. Handling of the Peptide

1. Design and ordering of the peptide
   1. Choose the desired sequence of the peptide, and add a tag, such as myc epitope (EQKLISEED) at its either N- or C-terminus.
   2. Check the predicted solubility of the peptide in water, using peptide solubility calculator http://pepcalc.com/peptide-solubility-calculator.php. If the solubility is low, try to add another water-soluble tag to change the charge balance.
      NOTE: We used the peptide corresponding to the first luminal loop (fll) of GLUT4 tagged with the myc epitope (bold font) at the N terminus (Myc-fll-GLUT4): EQKLISEEDLNAPQKVIEQSYNATWLGRQGPGGPSS....

Results

Lysates were prepared from 3T3 L1 cells stably transfected with Sortilin-myc/His⁵ and from WT 3T3 L1 cells, used as a negative control. Both lysates were incubated with cobalt beads at pH 6 or pH 8 and thoroughly washed. Beads with immobilized proteins were then incubated in the solution of Myc-fll-Glut4. After careful washes, proteins bound to the beads were eluted with 0.25 M Imidazole. Samples were subjected, along with the original lysates, to SDS-PAGE in a 10-.......

Discussion

Sortilin is an evolutionary conserved multi-ligand protein receptor that is involved in both signaling at the plasma membrane and in intracellular sorting events^6,7. However, the search for the authentic sortilin's ligands (some of which are luminal or integral membrane proteins) is complicated as the interaction of sortilin with some of its binding partners appears to be sensitive to detergents. Therefore, an easy and a widespread approach for studying prote.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Protease inhibitor cocktail	Sigma	P8849	for use in purification of histidine tag protein
Phosphate-Buffered Saline	Corning	21-040-CV	PBS
1mL Insulin Syringe U-100	BD	329652	26G x 1/2
BCA Protein Assay Kit	Pierce	23228
Wash buffer	Boston BioProducts	BP-234	For His-tag protein purification
HisPur Cobalt Resin	Thermo Scientific	89964
Tricine sample Buffer	Bio-Rad	161-0739	with SDS, bMercaptaethanol should be added
Elution  Buffer	Boston BioProducts	BP-236	For His-tag protein purificationwith 250mM Imidazole
Mini-Protean Tris-Tricine precast gels 10-20%	Bio-Rad	456-3115	For seperation of peptide and small protein
Tris/Tricine/SDS running buffer	Bio-Rad	161-0744
Transfer Buffer	Boston BioProducts	BP-190	Add 20% methanol
Nitrocellulose membrane  0.45 mm	Bio-Rad	1620115
Bovine Serum Albumin	Sigma	A9647	BSA
Anti Myc antibody	Cell Signaling	2272	Rabbit
Peptide	Genscipt		customize ordering
Precision Plus Protein Dual color Standarts	BioRad	161-0374