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Biologia

Cycloheximid Chase Analyse af proteinnedbrydning i<em> Saccharomyces cerevisiae</em

Published: April 18, 2016
doi:
10.3791/53975
, , ,
1Department of Biology,Ball State University, 2Bioproduct Research & Development,Eli Lilly and Company

Summary

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Abstract

Regulering af protein overflod er afgørende for næsten alle cellulære proces. Protein overflod afspejler integrationen af ​​satserne for proteinsyntese og proteinnedbrydning. Mange analyser rapportering på protein overflod (f.eks single-tidspunkt western blotting, flowcytometri, fluorescens mikroskopi, eller vækst-baserede reporter analyser) tillader ikke diskrimination af de relative effekter af oversættelse og proteolyse på protein niveauer. I denne artikel beskrives anvendelsen af cycloheximid chase efterfulgt af western blotting til specifikt at analysere proteinnedbrydning i modellen encellede eukaryot, Saccharomyces cerevisiae (knopskydende gær). I denne procedure, er gærceller inkuberes i nærvær af den translationelle inhibitor cycloheximid. Portioner af celler opsamles umiddelbart efter og ved specifikke tidspunkter efter tilsætning af cycloheximid. Cellerne lyseres, og lysaterne adskilles ved polyacrylamidgelelektroforese for Western blot-analyse af protein overflod på hvert tidspunkt. Proceduren for cycloheximid chase tillader visualisering af nedbrydningsprodukterne kinetik steady state population af en række cellulære proteiner. Proceduren kan anvendes til at undersøge de genetiske krav til og miljømæssige påvirkninger af proteinnedbrydning.

Introduction

Proteiner udfører livsvigtige funktioner i næsten alle cellulære proces. Mange fysiologiske processer kræver tilstedeværelsen af ​​et specifikt protein (eller proteiner) i en bestemt periode eller under særlige omstændigheder. Organismer derfor overvåge og regulere protein overflod at opfylde cellulære behov 1. For eksempel cycliner (proteiner, der styrer celledeling) er til stede på specifikke faser af cellecyklussen, og tabet af regulerede cyclin niveauer har været forbundet med malign tumordannelse 2. Ud over at regulere protein niveauer for at opfylde cellulære behov, celler ansætte nedbrydende kontrolmekanismer, som kan eliminere forkert foldede, ikke samlede eller på anden måde afvigende proteinmolekyler 3. Kontrol af protein overflod involverer regulering af både makromolekylær syntese (transskription og translation) og nedbrydning (RNA forfald og proteolyse). Nedsat eller overdreven protein nedbrydning bidrager til flere patologier, Herunder cancer, cystisk fibrose, neurodegenerative tilstande, og cardiovaskulære lidelser 4-8. Proteolytiske mekanismer udgør derfor lovende terapeutiske mål for en række sygdomme 9-12.

Analyse af proteiner på et enkelt tidspunkt (fx ved Western blot 13, flowcytometri 14, eller fluorescensmikroskopi 15) giver et øjebliksbillede af steady state-protein overflod uden at afsløre de relative bidrag af syntese eller nedbrydning. Ligeledes vækst-baserede reporter analyser afspejler steady state protein niveauer over en længere tidsperiode uden at forskelsbehandle de påvirkninger af syntese og nedbrydning 15-20. Det er muligt at udlede bidrag nedbrydende processer til steady state protein niveauer ved at sammenligne overflod før og efter at hæmme specifikke komponenter af nedbrydende mekanisme (fx ved farmakologisk inaktivere proteasome 21 eller banke ud et gen hypotese at være nødvendig for nedbrydning 13). En ændring i steady state protein niveauer efter at hæmme nedbrydningsveje giver stærke beviser for bidrag proteolyse til kontrol af protein overflod 13. Men en sådan analyse stadig ikke give oplysninger om kinetikken af ​​protein omsætning. Cycloheximid chase efterfulgt af western blotting overvinder disse svagheder ved at tillade forskerne at visualisere protein nedbrydning over tid 22-24. Da endvidere proteinpåvisning følgende cycloheximid chase typisk udføres ved western blotting, radioaktive isotoper og langvarige immunpræcipitation trin er ikke påkrævet for cycloheximid chase i modsætning til mange almindeligt anvendte puls chase teknikker, som også udføres for at visualisere proteinnedbrydning 25.

Cycloheximid blev først identificeret som en forbindelse med anti-fungal korrektbånd fremstillet af gram-positiv bakterie Streptomyces griseus 26,27. Det er en celle-gennemtrængelig molekyle, der specifikt hæmmer eukaryote cytosol (men ikke organellar) oversættelse ved at ændre ribosomal translokation 28-31. I et cycloheximid chase eksperiment cycloheximid tilsat til cellerne, og alikvoter af celler opsamles straks og ved specifikke tidspunkter efter tilsætning af forbindelse 22. Cellerne lyseres, og protein overflod ved hvert tidspunkt analyseres, typisk ved western blot. Fald i protein overflod efter tilsætning af cycloheximid kan trygt tilskrives proteinnedbrydning. En ustabil protein vil falde i overflod med tiden, mens et relativt stabilt protein vil udvise en lille ændring i overflod.

Mekanismer af selektiv proteinnedbrydning er blevet stærkt bevaret hele Eukarya. Meget af hvad man ved om protein nedbrydning blev først lært imodellen encellede eukaryot, Saccharomyces cerevisiae (knopskydende gær) 25,32-36. Undersøgelser med gær sandsynligvis fortsat levere nye og vigtige indsigt i protein nedbrydning. Fremgangsmåde til cycloheximid chase i gærceller, efterfulgt af Western blot-analyse af protein overflod præsenteres her.

Protocol

1. Vækst og Harvest af gærceller Hvis ikke analysere nedbrydningskinetik af et endogent gærprotein, transformere ønskede gærstamme (er) med et plasmid der koder for proteinet af interesse. Pålidelige metoder til gærtransformation er tidligere blevet beskrevet 37. Pode gær i 5 ml passende medium (f.eks selektiv syntetiske defineret (SD) medium til plasmid vedligeholdelse af transformerede celler eller ikke-selektiv gærekstrakt-pepton-dextrose (YPD) medium til ikke-transfo…

Representative Results

For at illustrere cycloheximid chase metode blev stabiliteten af Deg1 -Sec62 (figur 1), en model gær endoplasmatiske reticulum (ER) associeret nedbrydning (ERAD) substrat, analyseres 42-44. I ERAD, kvalitetskontrol ubiquitin ligaseenzymer covalent vedhæfte kæder af lille protein ubiquitin til afvigende proteiner lokaliseret til ER-membranen. Sådanne polyubiquitylated proteiner fjernes efterfølgende fra ER og nedbrydes af proteasomet, et stort, cy…

Discussion

I dette papir, er en fremgangsmåde til at analysere protein nedbrydningskinetik fremlagt. Denne teknik kan let påføres til en række proteiner nedbrydes af en række mekanismer. Det er vigtigt at bemærke, at cycloheximid chase rapporterer om nedbrydningskinetik af steady state pulje af et givet protein. Andre teknikker kan anvendes til at analysere nedbrydningskinetik specifikke populationer af proteiner. For eksempel kan nedbrydende skæbne nascente polypeptider spores af pulse chase analyse 55. I sådan…

Declarações

The authors have nothing to disclose.

Acknowledgements

The authors thank current and former members of the Rubenstein lab for providing a supportive and enthusiastic research environment. The authors thank Mark Hochstrasser (Yale University) for sharing reagents and expertise. E.M.R. thanks Stefan Kreft (University of Konstanz) and Jennifer Bruns (University of Pittsburgh) for sharing invaluable expertise in kinetic analysis of proteins. This work was supported by a National Institutes of Health grant (R15 GM111713) to E.M.R., a Ball State University ASPiRE research award to E.M.R, a research award from the Ball State University chapter of Sigma Xi to S.M.E., and funds from the Ball State University Provost’s Office and Department of Biology.

Materials

Desired yeast strains, plasmids, standard medium and buffer components
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 A tube roller is recommended to maintain overnight  starter cultures of yeast cells in suspension. Alternatively, if a tube roller is unavailable, a platform shaker in a temperature-controlled incubator may be used for overnight starter cultures. 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
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- 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 Heaters Fisher Scientific 1172011AQ It is recomended that two heaters are available (one for incubating cells during cycloheximide treatment and one for boiling lysates to denature proteins). Alternatively, 30 °C water bath may be used for incubation of cells in the presence of cycloheximide. Boiling water bath with hot plate may altertnatively be used to denature proteins.
Heating block for 12 x 15-ml conical tubes Fisher Scientific 11-473-70 For use with Dri-Bath Heater during incubation of cells in the presence of cycloheximide.
Heating block for 20 x 1.5-ml conical tubes Fisher Scientific 11-718-9Q For use with Dri-Bath Heater to boil lysates for protein denaturation.
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

Keywords: Protein DegradationSaccharomyces CerevisiaeCycloheximide ChaseEndogenous ProteinPlasmid-expressed ProteinOptical DensityMid-logarithmic GrowthHeat BlockStop MixCell LysisProtein Denaturation
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Buchanan, B. W., Lloyd, M. E., Engle, S. M., Rubenstein, E. M. Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae. J. Vis. Exp. (110), e53975, doi:10.3791/53975 (2016).
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