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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Biology

Vækst-baserede Bestemmelse og biokemisk Bekræftelse af genetiske Krav til proteinnedbrydning i<em> Saccharomyces cerevisiae</em

Published: February 16, 2015
doi:
10.3791/52428
, , ,
1Department of Biology,Ball State University, 2Division of Nephrology,Cincinnati Children’s Hospital

Summary

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Abstract

Reguleret proteinnedbrydning er afgørende for stort set alle cellulære funktion. Meget af det, man ved om de molekylære mekanismer og genetiske krav til eukaryot protein nedbrydning blev oprindeligt etableret i Saccharomyces cerevisiae. Klassiske analyser af proteinnedbrydning har påberåbt sig biokemiske puls-chase og cycloheximid-chase metoder. Selv om disse teknikker giver følsomme midler til observation proteinnedbrydning, er de besværlige, tidskrævende, og lav-throughput. Disse metoder er ikke egnede til hurtig eller storstilet screening for mutationer, der forhindrer proteinnedbrydning. Her er en gær vækst-assay for facile identifikation af genetiske krav til proteinnedbrydning beskrevet. I dette assay er en reporter enzym, der kræves for vækst under specifikke selektive betingelser fusioneret til et ustabilt protein. Celler, som mangler det endogene reporterenzym men udtrykker fusionsproteinet kan vokse under Selective betingelser, når fusionsproteinet er stabiliseret (dvs. når proteinnedbrydning er kompromitteret). I vækstassay beskrevet her, er seriefortyndinger af vildtype og mutant gærceller indeholdende et plasmid, der koder for et fusionsprotein plettet på selektive og ikke-selektive medium. Vækst under selektive betingelser er i overensstemmelse med nedbrydning forringelse ved en given mutation. Øget protein overflod bør biokemisk bekræftes. En fremgangsmåde til hurtig ekstraktion af gærproteiner i en form egnet til elektroforese og Western blotting er også vist. En vækst-baseret udlæsning for proteinstabilitet, kombineret med en enkel protokol til proteinekstraktion til biokemisk analyse, muliggør hurtig identifikation af genetiske krav til proteinnedbrydning. Disse teknikker kan tilpasses til at overvåge nedbrydningen af ​​en række af kortlivede proteiner. I det præsenterede eksempel HIS3 enzym, som er nødvendig for histidin-biosyntese, blev fusionerettil Deg1 -Sec62. Deg1 -Sec62 er målrettet til nedbrydning efter det afvigende indgreb det endoplasmatiske reticulum translocon. Celler, der huser Deg1 -Sec62-His3 var i stand til at vokse under selektive betingelser, når proteinet var stabiliseret.

Introduction

Selektiv proteinnedbrydning er afgørende for eukaryot liv og ændret proteinnedbrydning bidrager til en række medicinske tilstande, herunder adskillige former for kræft, neurodegenerativ sygdom, cardiovaskulær sygdom og cystisk fibrose 1-5. Ubiquitin-proteasom-system (UPS), der katalyserer selektiv proteinnedbrydning, er en ny terapeutisk mål for disse betingelser 6-10. Ubiquitin-ligaser covalent vedhæfte polymerer af 76-aminosyre ubiquitin til proteiner 11. Proteiner, der er markeret med multiubiquitinkæder anerkendes og proteolyseret af ~ 2,5 megadalton 26S proteasom 12. Undersøgelser indledt i modellen eukaryote organisme Saccharomyces cerevisiae (spirende gær) har været grundlæggende i opklaringen af protein nedbrydningsmekanismer i eukaryote celler. Den første demonstreret fysiologiske substrat af UPS var gæren transskriptionsrepressor MATα2 13Blev 14, og mange højt konserverede dele af UPS først identificeret eller kendetegnet ved gær (fx 15-26). Opdagelser gjort i denne alsidige og genetisk medgørlig model organisme vil sandsynligvis fortsætte med at give vigtige indsigter i konserverede mekanismer ubiquitinmedieret nedbrydning.

Anerkendelse og nedbrydning af de fleste UPS substrater kræver samordnet indsats af flere proteiner. Derfor er et vigtigt mål i karakterisere det regulerede nedbrydning af en given ustabilt protein er at bestemme de genetiske krav til proteolyse. Klassiske tilgange (f.eks puls-chase og cycloheximid-chase eksperimenter 27) til overvågning proteinnedbrydning i mammale eller gærceller er besværlige og tidskrævende. Selv om disse typer af metode giver meget følsomme midler til detektering af proteinnedbrydning, er de ikke egnet til hurtig analyse af proteinnedbrydning eller storstilet flotationng for mutationer, der forhindrer proteinnedbrydning. Her er en gær vækst-assay for hurtig identifikation af genetiske krav til nedbrydning af ustabile proteiner præsenteres.

I gærvækst-baserede metode til analyse af proteinnedbrydning, en ustabil protein af interesse (eller nedbrydning signal) er fusioneret i ramme til et protein, der er nødvendig for gær vækst under særlige omstændigheder. Resultatet er et kunstigt substrat, der kan tjene som et effektivt værktøj til at bestemme de genetiske krav proteinnedbrydning af den ustabile protein af interesse. Hensigtsmæssigt mest almindeligt anvendte laboratorium gærstammer huser et panel af mutationer i gener, der koder metaboliske enzymer involveret i biosyntesen af særlige aminosyrer eller nitrogenholdige baser (f.eks 20,28-30). Disse enzymer er essentielle for cellulær proliferation i fravær af eksogent leveret metabolitter i hvis syntese enzymerne deltage. Sådanmetaboliske enzymer kan således fungere som vækst-baserede reportere til nedbrydning af ustabile proteiner, hvortil de er fusioneret. De genetiske krav til proteinnedbrydning let kan belyses, da mutationer, der forhindrer proteolyse vil tillade celler, der huser nedbrydningen reporter til at vokse under selektive betingelser.

En vækst fordel er en indirekte indikation af, at en bestemt mutation øger overflod af proteinet af interesse. Dog er direkte biokemisk analyse forpligtet til at bekræfte, at en mutation tillader vækst gennem øget protein niveauer i stedet for via indirekte eller artefakt årsager. Effekten af ​​en mutation på protein overflod kan bekræftes ved Western blot analyse af steady-state proteinniveauer i celler, og andre ikke gør havn særlig mutation. En fremgangsmåde til hurtig og effektiv ekstraktion af gærproteiner (sekventiel inkubering af gærceller med natriumhydroxid og prøvepuffer) i en form egnettil analyse ved Western blotting præsenteres også 31. Tilsammen udgør disse eksperimenter lette en hurtig identifikation af kandidat regulatorer af protein nedbrydning.

Protocol

1. Gær Vækst Assay at identificere kandidatlandene Mutanter Defekte i proteinnedbrydning Omdan vildtype og mutante gærceller med et plasmid, der koder for et ustabilt protein fusioneret i ramme til en reporter metabolisk enzym. Podes transformanter i 5 ml syntetisk defineret (SD) minimalmedium, der er selektiv for celler, der huser plasmidmolekyler. Inkuber natten over ved 30 ° C, roterer. Mål den optiske densitet ved 600 nm (OD 600) for hver kultur dyrket natten over. <br …

Representative Results

For at illustrere denne metode har HIS3 enzymet er fusioneret til carboxyterminalen af modellen endoplasmatiske reticulum (ER) -associeret nedbrydning (ERAD) substrat Deg1 -Sec62 (figur 2A) for at skabe Deg1 -Sec62-His3 (figur 3) . Deg1 -Sec62 repræsenterer en af grundlæggerne af en ny klasse af ERAD substrater, der er målrettet efter vedvarende, afvigende tilknytning til translocon, kanalen har hovedansvaret for at flytte proteiner over ER membranen 3…

Discussion

Den metode, der præsenteres her giver mulighed for hurtig bestemmelse og biokemisk bekræftelse af genetiske krav til proteinnedbrydning i gærceller. Disse eksperimenter fremhæve nytte og magt gær som en model eukaryot organisme (flere fremragende anmeldelser af gær biologi og opsamlinger af protokoller for håndtering, opbevaring og manipulere gærceller (f.eks 41-44) er tilgængelige for efterforskerne nye for organismen). De teknikker kan let anvendes til at undersøge nedbrydning og overflod…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker nuværende og tidligere medlemmer af Rubenstein laboratorium for at give en støttende og entusiastisk forskningsmiljø. Vi takker Ryan T. Gibson om bistand i protokol optimering. Vi takker Mark Hochstrasser (Yale University) og Dieter Wolf (Universität Stuttgart) for gærstammer og plasmider. Vi takker vores anonyme korrekturlæsere for deres hjælp til at forbedre klarhed og nytten af ​​dette manuskript. Dette arbejde blev støttet af en forskningspris fra Ball State University kapitel i Sigma Xi til SGW, en National Institutes of Health tilskud (R15 GM111713) til EMR, en Ball State University aspire forskningspris til EMR, og midler fra Ball State University Provost kontor og Biologisk Institut.

Materials

Desired yeast strains, plasmids, standard medium and buffer components Yeast strains with desired mutations may be generated in the investigator's laboratory. Wild-type yeast and a variety of mutants are also commercially available (e.g. from GE Healthcare). Plasmids encoding fusion proteins may be generated in the investigator's laboratory.
3-amino-1H-1,2,4-triazole Fisher Scientific AC264571000 Competitive inhibitor of His3 enzyme. May be included in medium to increase stringency of growth assay using His3 reporter constructs
Endoglycosidase H (recombinant form from Streptomyces plicatus) Roche 11088726001 May be used to assess N-glycosylation of proteins; compatible with SDS and beta-mercaptoethanol concentrations found in 1X Laemmli sample buffer
Disposable borosilicate glass tubes Fisher Scientific 14-961-32 Available from a variety of manufacturers
Temperature-regulated incubator (e.g. Heratherm Incubator Model IMH180) Dot Scientific 51028068 Available from a variety of manufacturers
New Brunswick Interchangeable Drum for 18 mm tubes (tube roller) New Brunswick M1053-0450 Tube roller is recommended to maintain overnight yeast starter cultures of yeast cells in suspension. A platform shaker or tube roller may be used to maintain larger cultures in suspension.
New Brunswick TC-7 Roller Drum 120V 50/60 H New Brunswick M1053-4004 For use with tube roller
SmartSpec Plus Spectrophotometer Bio-Rad 170-2525 Available from a variety of manufacturers
Sterile 96-well flat bottom microtest plates with lid individually wrapped Sarstedt 82.1581.001 Available from a variety of manufacturers
Pipetman Neo P8x200N, 20-200 μl Gilson F14403 Single-channel and multichannel pipettors are used at various stages of the protocol. While multichannel pipettors reduce the pipetting burden at several steps, single-channel pipettors may be used throughout the entire protocol. Available from a variety of manufacturers
Pipetman Neo P8x20N, 2-20 μl Gilson F14401 Available from a variety of manufacturers
Plate imaging system (e.g.
Gel Doc XR+ System)		 Bio-Rad 170-8195 A variety of systems may be used to image plates, including sophisticated imaging systems, computer scanners, and camera phones.
Centrifuge 5430 Eppendorf 5427 000.216  Rotor that is sold with unit holds 1.5 and 2.0 mL microcentrifuge tubes. Rotor may be swapped for one that holds 15 ml and 50 ml conical tubes
Fixed-Angle Rotor F-35-6-30 with Lid and Adapters for Centrifuge Model 5430/R, 15/50 mL Conical Tubes, 6-Place Eppendorf F-35-6-30
15 ml screen printed screw cap tube 17 x 20 mm conical, polypropylene Sarstedt 62.554.205 Available from a variety of manufacturers
1.5 ml flex-tube, PCR clean, Natural microcentrifuge tubes Eppendorf 22364120 Available from a variety of manufacturers
Analog Dri-Bath Heater Fisher Scientific 1172011AQ Boiling water bath with hot plate may also be used to denature proteins
SDS-PAGE running and transfer apparatuses, power supplies, and imaging equipment or darkrooms for SDS-PAGE and transfer to membrane Will vary by lab and application
Western blot imaging system (e.g. Li-Cor Odyssey CLx scanner and Image Studio Software) Li-Cor 9140-01 Will vary by lab and application
EMD Millipore Immobilon PVDF Transfer Membranes Fisher Scientific IPFL00010 Will vary by lab and application
Primary antibodies (e.g. Phosphoglycerate Kinase (Pgk1) Monoclonal antibody, mouse (clone 22C5D8)) Life Technologies 459250 Will vary by lab and application
Secondary antibodies (e.g. Alexa-Fluor 680 Rabbit Anti-Mouse IgG (H+L)) Life Technologies A-21065 Will vary by lab and application
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Tags

Protein DegradationSaccharomyces CerevisiaeGrowth-based AssayReporter EnzymeDeg1-Sec62-His3 FusionGenetic RequirementsBiochemical ConfirmationProtein ExtractionWestern BlottingEndoplasmic Reticulum Translocon
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Watts, S. G., Crowder, J. J., Coffey, S. Z., Rubenstein, E. M. Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae. J. Vis. Exp. (96), e52428, doi:10.3791/52428 (2015).
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