This work was supported by grant no. 2016/23/D/NZ3/01314 from the National Science Centre (NCN), Krakow, Poland.

1. Generation of expression plasmids
<ol>
	<li>Examine membrane topology of the proteins of interest using a topology predicting tool.</li>
	<li>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&#160;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-t...

<ol>
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A., Coincon, M., Drew, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+delivery+of+activated+sialic+acid+into+Golgi+for+sialyation.">Structural basis for the delivery of activated sialic acid into Golgi for sialyation.</a> Nature Structural and Molecular Biology. 26 (6), 415-423 (2019).</li><li>Parker, J. L., Corey, R. A., Stansfeld, P. J., Newstead, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+substrate+specificity+and+regulation+of+nucleotide+sugar+transporters+in+the+lipid+bilayer.">Structural basis for substrate specificity and regulation of nucleotide sugar transporters in the lipid bilayer.</a> Nature Communications. 10 (1), 4657 (2019).</li><li>Wiertelak, W., Sosicka, P., Olczak, M., Maszczak-Seneczko, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+homologous+and+heterologous+interactions+between+UDP-galactose+transporter+and+beta-1,4-galactosyltransferase+1+using+NanoBiT.">Analysis of homologous and heterologous interactions between UDP-galactose transporter and beta-1,4-galactosyltransferase 1 using NanoBiT.</a> Analytical Biochemistry. 593, 113599 (2020).</li><li>Hong, K., Ma, D., Beverley, S. M., Turco, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Leishmania+GDP-mannose+transporter+is+an+autonomous,+multi-specific,+hexameric+complex+of+LPG2+subunits.">The Leishmania GDP-mannose transporter is an autonomous, multi-specific, hexameric complex of LPG2 subunits.</a> Biochemistry. 39 (8), 2013-2022 (2000).</li><li>Sosicka, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insight+into+the+orphan+nucleotide+sugar+transporter+SLC35A4.">An insight into the orphan nucleotide sugar transporter SLC35A4.</a> Biochimica et Biophysica Acta. Molecular Cell Research. 1864 (5), 825-838 (2017).</li><li>Sprong, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+Golgi+UDP-galactose+transporter+with+UDP-galactose:ceramide+galactosyltransferase+allows+UDP-galactose+import+in+the+endoplasmic+reticulum.">Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum.</a> Molecular Biology of the Cell. 14 (8), 3482-3493 (2003).</li><li>Maszczak-Seneczko, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UDP-galactose+(SLC35A2)+and+UDP-N-acetylglucosamine+(SLC35A3)+Transporters+Form+Glycosylation-related+Complexes+with+Mannoside+Acetylglucosaminyltransferases+(Mgats).">UDP-galactose (SLC35A2) and UDP-N-acetylglucosamine (SLC35A3) Transporters Form Glycosylation-related Complexes with Mannoside Acetylglucosaminyltransferases (Mgats).</a> Journal of Biological Chemistry. 290 (25), 15475-15486 (2015).</li><li>Khoder-Agha, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetylglucosaminyltransferases+and+nucleotide+sugar+transporters+form+multi-enzyme-multi-transporter+assemblies+in+golgi+membranes+in+vivo.">N-acetylglucosaminyltransferases and nucleotide sugar transporters form multi-enzyme-multi-transporter assemblies in golgi membranes in vivo.</a> Cellular and Molecular Life Sciences. 76 (9), 1821-1832 (2019).</li><li>Dixon, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoLuc+Complementation+Reporter+Optimized+for+Accurate+Measurement+of+Protein+Interactions+in+Cells.">NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.</a> ACS Chemical Biology. 11 (2), 400-408 (2016).</li><li>Tung, J. K., Berglund, K., Gutekunst, C. -. A., Hochgeschwender, U., Gross, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+in+live+cells+and+animals.">Bioluminescence imaging in live cells and animals.</a> Neurophotonics. 3 (2), 025001 (2016).</li><li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Molecular Cell. 9 (4), 789-798 (2002).</li><li>Inoue, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+G-Protein-Coupling+Selectivity+of+GPCRs.">Illuminating G-Protein-Coupling Selectivity of GPCRs.</a> Cell. 177 (7), 1933-1947 (2019).</li><li>White, C. W., Caspar, B., Vanyai, H. K., Pfleger, K. D. G., Hill, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Mediated+Protein+Tagging+with+Nanoluciferase+to+Investigate+Native+Chemokine+Receptor+Function+and+Conformational+Changes.">CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes.</a> Cell Chemical Biology. 27, 499-510 (2020).</li><li>Akinjiyan, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Luminescence-Based+High-Throuput+Approach+for+Cellular+Resolution+of+Protein+Ubiquitination+using+Tandem+Ubiquitin+Binding+Entities+(TUBEs).">A Novel Luminescence-Based High-Throuput Approach for Cellular Resolution of Protein Ubiquitination using Tandem Ubiquitin Binding Entities (TUBEs).</a> SLAS Discovery. 25 (4), 350-360 (2020).</li><li>Soave, M., Kellam, B., Woolard, J., Briddson, S. J., Hill, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoBiT+Complemetation+to+Monitor+Agonist-Induced+Adenosine+A1+Receptor+Internalization.">NanoBiT Complemetation to Monitor Agonist-Induced Adenosine A1 Receptor Internalization.</a> SLAS Discovery. 25 (2), 186-194 (2020).</li><li>Crowley, E., Leung, E., Reynisson, J., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+in+the+ATG5-ATG16L1+complex+following+nutrient+deprivation+measured+using+NanoLuc+Binary+Technlology+(NanoBiT).">Rapid changes in the ATG5-ATG16L1 complex following nutrient deprivation measured using NanoLuc Binary Technlology (NanoBiT).</a> FEBS Journal. , 15275 (2020).</li><li>Shetty, S. K., Walzem, R. L., Davies, B. S. 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The authors have nothing to disclose.

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...

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&#160;and/or higher oligomers2,3,4,5,6,7,8,9,10. Moreover, heterologous interactions between&#160;different NSTs have also been reported6,11. NSTs were also demonstrated to form complexes with functionally related glycosylation enzymes12,13,14. 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&#160;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 F&#246;rster 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&#160;protein (e.g., HaloTag),&#160;fused with a smaller fragment. The latter&#160;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&#160;presented here.

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

demonstration of heterologous complexes formed by golgi-resident type iii membrane proteins using split luciferase complementation assay

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&#160;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&#160;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.

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...

Watch this Scientific Journal Video about Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay at JoVE.com

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

The&#160;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.

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous ...

Our protocol allows to identify and analyze interactions between multi transmembrane proteins of the endoplasmic reticulum and golgi complex in living cells at low expression levels. The main advantage of this technique is it's to special sensitivity, which allows to avoid high over expression levels that are often detrimental to cellular health and may disturb protein subcellular localization. Harvest the adherent HEK293 T-cell culture by trypsinization and re-suspend the cells in a dedicated complete growth medium.Plate the cells onto a clear bottom, white side 96-well plate. Adjust the total number of wells to accommodate all tested combinations and controls, including replicates. Then, culture the cells overnight in standard conditions.On the next day, transfect the cells with the desired combinations of expression plasmids. Dilute the plasmids in a serum-free medium to 6.25 nanograms per microliter for each construct and add the lipid-based transfection reagent. Add an appropriate lipid to DNA ratio.Incubate the plasmids according to manufacturer's instruction. Then, add eight microliters of the lipid DNA mixture to the designated wells. Mix the content of the plate with gentle rotation and culture the cells for 20 24 hours in standard conditions.On the next day, replace the conditioned medium in each well with 100 microliters of a serum-free medium, making sure that the cells do not detach upon medium exchange. Just before measuring luminescence, mix one volume of furimazine with 19 volumes of a dilution buffer. Add 25 microliters of the furimazine working solution into each well, and gently mix the plate by hand or with a orbital shaker.Insert the plate into a luminescence micro plate reader and equilibrate the plate at 37 degrees Celsius for 10 15 minutes. Select the wells to be analyzed and read luminescence with an integration time of 0.3 seconds. Continue to monitor luminescence for up to two hours when required.This protocol was tested on two nucleotide sugar transporters, SLC35A2 and SLC35A3, which are golgi-resident type three membrane proteins with N and C termini facing the cytoplasm. There were eight possible tagging options and the resulting fusion proteins could be set together in eight possible ways. Mean relative luminescence units or RLU values corresponding to all the tested and control combinations are shown here.The RLU values obtained for three of the combinations were significantly higher than those obtained for the corresponding control combination. To obtain ratio values for the combinations of interest, the RLU values were divided by the RLU values obtained for the respective controls. Ratios between 10 and 1000 are highly indicative of specific interactions.There were two such combinations supporting the idea that SLC35A2 and SLC35A3 interact. To confirm this data, HA tagged SLC35A2 or SLC35A3 were co-expressed with the combination that resulted in the highest relative luminescence. Varying amounts of the plasmids and coding HA tagged NST variants were used for co-transfection, resulting in a statistically significant dose dependent decrease in RLU values.The most critical thing is to tackle your protein self interest in all possible ways and to assay all possible combination, as some tagging orientation may produce false negative results. Once the best performing combination has been selected, the dynamics of the corresponding interaction can be monitored to, for example identify agents or conditions that disrupt your complex of interest over time.

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2.2K ビュー.  University of Wroclaw. Our protocol allows to identify and analyze interactions between multi transmembrane proteins of the endoplasmic reticulum and golgi complex in living cells at low expression levels. The main advantage of this technique is it's to special sensitivity, which allows to avoid high over expression levels that are often detrimental to cellular health and may disturb protein subcellular localization. Harvest the adherent HEK293 T-cell culture by trypsinization and re-suspend the cells in a dedicate...