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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 membran....

Protokół

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 ....

Wyniki

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.......

Dyskusje

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.......

Materiały

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