Name: demonstration of heterologous complexes formed by golgi-resident type iii membrane proteins using split luciferase complementation assay
Uploaded: 2020-09-10T15:30:00
Description: 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

Dette arbeidet ble støttet av tilskuddsnummer 2016/23/D/NZ3/01314 fra National Science Centre (NCN), Krakow, Polen.

1. Generering av uttrykksplasmider
<ol><li>Undersøk membrantopologien til proteinene av interesse ved hjelp av et topologiforutsigelsesverktøy.</li>
	<li>Design kloningsstrategien slik at de større og mindre fragmentene vil møte cytoplasma når fusjonsproteinene er satt inn i Golgi-membraner. Hvis, som i tilfellet presentert her, både N- og C-termini av proteinene av interesse er cytoplasmisk orientert, merke proteinene på åtte mulige måter (se figur 1B). Hvis N- eller C-terminus a...

<ol>
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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. 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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. 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Her gir vi en detaljert protokoll som muliggjør demonstrasjon av heteroologe komplekser dannet mellom Golgi-bosatte type III membranproteiner, som NSTs, ved hjelp av den delte luciferase komplementeringsanalysen. Den foreslåtte tilnærmingen til dataanalyse og tolkning innebærer å relatere luminescensen oppnådd for proteinkombinasjonen av interesse for luminescensen oppnådd for den tilsvarende kontrollkombinasjonen, som består av et av proteinene av interesse smeltet sammen med det større fragmentet og kontrollpr...

Dette manuskriptet beskriver en trinnvis protokoll for å se etter tilstedeværelsen av heterologe interaksjoner mellom Golgi-resident type III membranproteiner i forbigående transfekerte menneskelige celler ved hjelp av den nyeste varianten av den delte luciferase komplementeringsanalysen. Prosedyren har blitt mest omfattende testet mot nukleotid sukkertransportører (NSTs), men vi var også i stand til å oppnå positive resultater for andre Golgi-bosatte type III membranproteiner hvis N- og / eller C-termini står overfor cytoplasma.
Vår forskningsgruppe utforsker NSTs rolle i glykosylering av makromolekyler. NSTs er Golgi- og/eller ER-bosatte type III membranproteiner med N- og C-termini vendt mot den cytoplasmatiske siden av organellarmembranen1. NSTs antas å bære nukleotidaktiverte sukkerarter over organellemembraner for å levere glykosyltransferaser med sine substrater. NSTer danner dimers og/eller høyere oligomers2,3,4,5,6,7,8,9,10. Videre er det også rapportert heteroologe interaksjoner mellom ulike NST-er, 6,11. NSTs ble også demonstrert for å danne komplekser med funksjonelt relaterte glykosyleringsenzymer12,13,14. Vi søkte etter et alternativ til den nåværende brukte teknikken, fluorescens levetidsavbildning (FLIM)-basert FRET-tilnærming, for å studere interaksjoner mellom NSTs og funksjonelt relaterte Golgi-bosatte proteiner, så vi bestemte oss for å teste den splittede luciferase komplementeringsanalysen. Det tillot oss å identifisere et nytt samspill mellom en NST og et funksjonelt relatert glykosyleringsenzym9.
Den siste modifikasjonen av den delte luciferase komplementeringsanalysen, NanoBiT, brukes i protokollen som presenteres her15. Det er avhengig av rekonstituering av luciferase enzymet (f.eks. NanoLuc) fra de to fragmentene - den store, betegnet som stor BiT eller LgBiT, et 17,6 kDa protein, og den lille, bestående av bare 11 aminosyrer, betegnet som liten BiT eller SmBiT. De to proteinene av interesse er smeltet sammen med komplementære fragmenter og midlertidig uttrykt i en menneskelig cellelinje. Hvis de to fusjonsproteinene samhandler, produseres en luminescens in situ ved tilsetning av et cellegjennomtrengelig substrat. Disse to fragmentene er optimalisert slik at de forbinder med minimum affinitet med mindre de blir samlet av et samspill mellom proteinene av interesse de er smeltet sammen til.
Generelt har bioluminescensbaserte metoder noen fordeler i forhold til de som er basert på fluorescens. Bioluminescence-signaler har et høyere signal-til-støy-forhold fordi bakgrunnslysene er ubetydelige sammenlignet med det luciferase-avledede signalet16. I motsetning lider fluorescensbaserte tilnærminger vanligvis av en relativt høy bakgrunn forårsaket av fenomenet autofluorescens. Dessuten er bioluminescens mindre skadelig for de analyserte cellene enn fluorescens, som i det tidligere tilfellet er det ikke nødvendig å begeistre prøven. Av disse grunnene bioluminescent tilnærminger til å studere PPIs in vivo outkompetente de ofte brukte fluorescerende metoder som Förster Resonance Energy Transfer (FRET) eller Bimolecular Fluorescence Complementation (BiFC).
Vår protokoll er avhengig av å referere luminescens oppnådd for proteinkombinasjonen av interesse for luminescens oppnådd for kontrollkombinasjonen. Sistnevnte inkluderer et av de testede proteinene som er smeltet sammen med et større fragment og et kontrollprotein (f.eks. HaloTag), smeltet sammen med et mindre fragment. Sistnevnte er et protein av bakteriell opprinnelse som ikke forventes å samhandle med noen av pattedyrproteiner. Å bruke dette proteinet som en kontroll gir begrensninger for topologien til de Golgi-bosatte par proteinene som skal analyseres. Siden i pattedyrceller syntetiseres dette proteinet i cytoplasma, bør begge proteiner av interesse ha minst en cytoplasmatisk hale.
Denne tilnærmingen kan være spesielt nyttig for første screening av PPIer. Det kan bli den valgte metoden når fusjonsproteinene av interesse uttrykkes på nivåer som rett og slett ikke er tilstrekkelige for andre tilnærminger som skal brukes. På samme måte kan den delte luciferase komplementeringsanalysen være det beste alternativet hvis proteinene av interesse uttrykkes på høye nivåer, men dette påvirker deres subcellulære lokalisering negativt eller er kjent for å tvinge ikke-spesifikke interaksjoner. Siden det mindre fragmentet bare har 11 aminosyrer, kan den delte luciferase komplementeringsanalysen påføres når du bruker større koder er umulig. Til slutt kan den brukes til å bekrefte data som er innhentet ved hjelp av andre teknikker, som i tilfelle presentert her.

<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

Målet med denne protokollen er å utforske anvendelsen av den nyeste varianten av delt luciferase komplementering for å demonstrere heteroologe komplekser dannet av nukleotid sukkertransportører (NSTs). Disse ER- og Golgi-bosatte multitransmembrane proteinene bærer cytoplasmatisk syntetiserte nukleotidsukker på tvers av organellemembraner for å levere enzymer som formidler glykosylering med deres substrater. NSTer finnes som dimers og/eller høyere oligomerer. Det er også rapportert om heterologe interaksjoner mellom ulike NST-er. For å verifisere om teknikken er egnet for å studere fenomenet NST-hetermerisering, testet vi den mot en kombinasjon av de to Golgi-bosatte NSTene som tidligere har vist seg å assosiere på flere andre måter. Luciferase komplementeringsanalysen ser ut til å være spesielt egnet for å studere interaksjoner mellom Golgi-bosatte membranproteiner, da det ikke krever høye uttrykksnivåer, som ofte utløser protein feillokalisering og øker risikoen for falske positiver.

For å oppnå de mest pålitelige dataene i denne tilnærmingen bør alle mulige kombinasjoner testes (se figur 1). Parallelt bør positive og negative kontroller inkluderes. Den positive kontrollen skal bestå av de to proteinene som er kjent for å samhandle, hvorav den ene er smeltet sammen med det større fragmentet og den andre er smeltet sammen med det mindre fragmentet. Den negative kontrollen bør ideelt sett bestå av de to ikke-interagerende type III membranproteinene merket på sa...

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

Protokollen som presenteres her er ment å demonstrere forekomsten av heteroologe interaksjoner mellom Golgi-bosatte type III membranproteiner med cytoplasmatisk eksponert N- og / eller C-termini i levende pattedyrceller ved hjelp av den nyeste varianten av den delte luciferase komplementeringsanalysen.

Demonstrasjon av heteroologe komplekser dannet av Golgi-Resident Type III Membranproteiner ved hjelp av 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.

Research

JoVE Journal

Biology

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

High-throughput krystallisering av membran proteiner Bruke Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Bruk av en robot for High-throughput Krystallisering av membran proteiner i Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

Høsting og Cryo-kjøling Krystaller av membran proteiner Grown i Lipidic Mesophases for strukturbestemmelse av Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

Høy gjennomstrømming Funksjonell Screening ved hjelp av en hjemmelaget Dual-glød Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...