Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Summary

The protocol presented here is intended to demonstrate the occurrence of heterologous interactions between Golgi-resident type III membrane proteins with cytoplasmically exposed N- and/or C-termini in live mammalian cells using the most recent variant of the split luciferase complementation assay.

Abstract

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous complexes formed by nucleotide sugar transporters (NSTs). These ER- and Golgi-resident multitransmembrane proteins carry the cytoplasmically synthesized nucleotide sugars across organelle membranes to supply enzymes that mediate glycosylation with their substrates. NSTs exist as dimers and/or higher oligomers. Heterologous interactions between different NSTs have also been reported. To verify whether the technique is suitable for studying the phenomenon of NST heteromerization, we tested it against a combination of the two Golgi-resident NSTs that have been previously shown to associate by several other means. The luciferase complementation assay appears to be particularly suitable for studying interactions between Golgi-resident membrane proteins, as it does not require high expression levels, which often trigger protein mislocalization and increase the risk of false positives.

Introduction

This manuscript describes a step-by-step protocol to check for the presence of heterologous interactions between Golgi-resident type III membrane proteins in transiently transfected human cells using the most recent variant of the split luciferase complementation assay. The procedure has been most extensively tested against nucleotide sugar transporters (NSTs) but we were also able to obtain positive results for other Golgi-resident type III membrane proteins whose N- and/or C-termini are facing the cytoplasm.

Our research group explores the role of NSTs in glycosylation of macromolecules. NSTs are Golgi- and/or ER-resident type III membrane proteins with N- and C-termini facing the cytoplasmic side of the organellar membrane¹. NSTs are thought to carry nucleotide-activated sugars across organelle membranes to supply glycosyltransferases with their substrates. NSTs form dimers and/or higher oligomers^{2,3,4,5,6,7,8,9,10}. Moreover, heterologous interactions between different NSTs have also been reported^6,11. NSTs were also demonstrated to form complexes with functionally related glycosylation enzymes^12,13,14. We sought for an alternative to the presently used technique, fluorescence lifetime imaging (FLIM)-based FRET approach, for studying interactions of NSTs and functionally related Golgi-resident proteins, so we decided to test the split luciferase complementation assay. It allowed us to identify a novel interaction between an NST and a functionally related glycosylation enzyme⁹.

The most recent modification of the split luciferase complementation assay, NanoBiT, is used in the protocol presented here¹⁵. It relies on the reconstitution of the luciferase enzyme (e.g., NanoLuc) from the two fragments - the large one, termed as large BiT or LgBiT, a 17.6 kDa protein, and the small one, composed of only 11 amino acids, termed as small BiT or SmBiT. The two proteins of interest are fused with the complementary fragments and transiently expressed in a human cell line. If the two fusion proteins interact, a luminescence is produced in situ upon addition of a cell-permeable substrate. These two fragments have been optimized so that they associate with minimum affinity unless being brought together by an interaction between the proteins of interest they are fused to.

In general, bioluminescence-based methods have some advantages over the ones based on fluorescence. Bioluminescent signals have a higher signal-to-noise ratio because the background luminescence is negligible compared to the luciferase-derived signal¹⁶. In contrast, fluorescence-based approaches usually suffer from a relatively high background caused by the phenomenon of autofluorescence. Besides, bioluminescence is less detrimental to the analyzed cells than fluorescence, as in the former case there is no need to excite the sample. For those reasons bioluminescent approaches to studying PPIs in vivo outcompete the commonly used fluorescent methods like Förster Resonance Energy Transfer (FRET) or Bimolecular Fluorescence Complementation (BiFC).

Our protocol relies on referring luminescence obtained for the protein combination of interest to luminescence obtained for the control combination. The latter includes the one of the tested proteins which is fused with a larger fragment and a control protein (e.g., HaloTag), fused with a smaller fragment. The latter is a protein of bacterial origin that is not expected to interact with any of mammalian proteins. Using this protein as a control poses limitations to the topology of the Golgi-resident pairs of proteins to be analyzed. Since in mammalian cells this protein is synthesized in the cytoplasm, both proteins of interest should have at least one cytoplasmic tail.

This approach can be particularly useful for initial screening of PPIs. It may become the method of choice when the fusion proteins of interest are expressed at levels that are simply insufficient for other approaches to be applied. Similarly, the split luciferase complementation assay can be the best option if the proteins of interest are expressed at high levels, but this adversely affects their subcellular localization or is known to force non-specific interactions. Since the smaller fragment has only 11 amino acids, the split luciferase complementation assay can be applied when using larger tags is impossible. Finally, it can be employed to further confirm data obtained using other techniques, as in the case presented here.

Protocol

1. Generation of expression plasmids

1. Examine membrane topology of the proteins of interest using a topology predicting tool.
2. Design the cloning strategy so that the larger and smaller fragments would face the cytoplasm once the fusion proteins have been inserted into Golgi membranes. If, as in the case presented here, both N- and C-termini of the proteins of interest are cytoplasmically oriented, tag the proteins in eight possible ways (see Figure 1B). If N- or C-terminus of one or both proteins of interest is luminally oriented, exclude it from tagging.
3. Subclone the genes of interest into appropriate expression vectors (see Table of Materials) by following standard cloning protocols.
   NOTE: Testing all the possible orientations is recommended, since some tagging options may not work due to insufficient proximity, suboptimal orientation, or spatial constraints.

2. Transient transfection of the expression plasmids into the cells

1. Harvest the adherent HEK293T cell culture by trypsinization and resuspend the cells in a dedicated complete growth medium. Plate the cells (2 x 10⁴/100 µL/well) onto a clear bottom, white side 96-well plate. Adjust the total number of wells to accommodate all tested combinations and controls including replicates.
   NOTE: Attempt to use only the inner 60 wells of the plate to minimize thermal shifts and avoid overnight evaporation. Using poly-D-lysine-coated plates is highly recommended such as the ones indicated in the Table of Materials, otherwise cells may detach during the subsequent washing steps.
2. Culture the cells overnight in standard conditions (37 °C, 5% CO₂).
3. On the next day transfect the cells with the desired combinations of expression plasmids obtained in point 1.1.
   1. Dilute expression plasmids in a serum-free medium (see Table of Materials) to 6.25 ng/µL for each construct.
   2. Add the lipid-based transfection reagent at an appropriate lipid-to-DNA ratio and incubate according to the manufacturer’s instruction.
   3. Add 8 µL of lipid:DNA mixture to designated wells. Mix the content of the plate by gentle rotation. This results in transfection of both expression constructs at 50 ng/well.
4. Culture the cells for 20-24 h in standard conditions (37 °C, 5% CO₂).
   NOTE: Culturing the cells for a longer time may result in higher levels of fusion protein expression, which may promote a non-specific association between the fragments.

3. Medium exchange

1. On the next day replace the conditioned medium with 100 µL of a serum-free medium in each well. Make sure that the cells have not detached upon medium exchange.
   NOTE: This step should be done 2-3 h before the addition of the furimazine working solution. Serum withdrawal minimizes background caused by autoluminescence of furimazine.

4. Preparation of furimazine working solution

1. Just before the measurement, mix 1 volume of furimazine with 19 volumes of a dilution buffer (a 20-fold dilution).
   NOTE: The total volume of the furimazine working solution to be prepared depends on the number of individual wells to be analyzed (the furimazine working solution is added to the cell culture medium in a 1:5 ratio, therefore, to each well previously filled with 100 µL of a serum-free medium 25 µL of the furimazine working solution should be added).
2. Add the furimazine working solution to designated wells (25 µL/well). Gently mix the plate by hand or using an orbital shaker (e.g., 15 s at 300-500 rpm).

5. Measuring luminescence

1. Insert the plate into a luminescence microplate reader.
   1. For experiments that are to be performed at 37 °C equilibrate the plate for 10-15 min at the indicated temperature.
2. Select the wells to be analyzed.
3. Read luminescence with integration time of 0.3 s. Continue to monitor luminescence for up to 2 h when required.

6. Data analysis

1. Calculate mean values and standard deviations for all the tested and control combinations.
2. Analyze data using one-way ANOVA with multiple comparisons.
3. Calculate fold change values by dividing a mean luminescence obtained for combinations of interest by a mean luminescence obtained for the corresponding negative controls. Evaluate the results.
   NOTE: The approach to data analysis proposed here assumes that the interaction can be claimed if the luminescence obtained for the tested combination is statistically significantly higher than the luminescence obtained for the corresponding control combination and, at the same time, the ratio of these two values exceeds 10.

Results

To obtain the most reliable data in this approach all the possible combinations should be tested (see Figure 1). In parallel, positive and negative controls should be included. The positive control should consist of the two proteins that are known to interact, of which one is fused with the larger fragment and the other is fused with the smaller fragment. The negative control ideally should consist of the two non-interacting type III membrane proteins tagged likewise. However, establishing s...

Discussion

Here we provide a detailed protocol enabling the demonstration of heterologous complexes formed between Golgi-resident type III membrane proteins, such as NSTs, using the split luciferase complementation assay. The proposed approach to data analysis and interpretation involves relating the luminescence obtained for the protein combination of interest to the luminescence obtained for the corresponding control combination, which is composed of one of the proteins of interest fused with the larger fragment and the control p...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA solution
Adherent mammalian cell line
BioCoat Poly-D-Lysine 96-well White/Clear Flat Bottom TC-treated Microplate, with Lid	Corning	356651
Cell culture centrifuge
Cell culture supplements (heat-inactivated fetal bovine serum, L-glutamine, penicillin, streptamycin)
CO2 incubator
Expression plasmids encoding protein(s) of interest not tagged with NanoBiT fragments
FuGENE HD Transfection Reagent	Promega	E2311
GloMax Discover Microplate Reader (or a different luminescence microplate reader)	Promega	GM3000
Growth medium dedicated to the cell line used
Materials and reagents for standard molecular cloning (bacteria, thermostable polymerase, restriction enzymes, DNA ligase, materials and reagents for nucleic acid purification)
NanoBiT MCS Starter System	Promega	N2014	This kit contains vectors enabling tagging of the proteins of interest with NanoBiT fragments at different orientations as well as the control plasmid encoding HaloTag protein fused with SmBiT and a positive control plasmid pair.
Nano-Glo Live Cell Assay System	Promega	N2011	This kit contains furimazine, which is a substrate enabling detection of the NanoLuc activity in living cells, and a dedicated dilution buffer.
Opti-MEM I Reduced Serum Medium, no phenol red	Gibco	11058021
Oribital shaker
Software for data analysis (e.g. GraphPad Prism)
Thermocycler