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Methods in Molecular Biology 2581 L. Maria Lois Marco Trujillo Editors Plant Proteostasis Methods and Protocols Second Edition M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Plant Proteostasis Methods and Protocols Second Edition Edited by L. Maria Lois Center for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Barcelona, Spain Marco Trujillo Faculty of Biology, University of Freiburg, Freiburg, Germany Editors L.MariaLois MarcoTrujillo CenterforResearchinAgricultural FacultyofBiology Genomics(CSIC-IRTA-UAB-UB) UniversityofFreiburg Barcelona,Spain Freiburg,Germany ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2783-9 ISBN978-1-0716-2784-6 (eBook) https://doi.org/10.1007/978-1-0716-2784-6 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface “Plant Proteostasis” Organismsdynamicallyregulatetheirproteomecompositioninordertorespondoradaptto environmental changes, while maintaining the needed concentration of each and every protein to maintain balanced cellular processes. The maintenance of protein homeostasis, orproteostasis,isdependentonvariouscellularpathwaysthatmediatebothproteinbiosyn- thesis and degradation. As sessile organisms, plants are exposed to a large variation in temperature,wateravailabilityandlightconditions,whileinteractingwithawidespectrum of beneficial and pathogenic microorganisms. Rapidly changing environmental conditions anddynamicpressurefrompathogensasaconsequenceoftheclimatecrisis,poseadditional challenges for plants to maintain proteostasis. Beyond adaptation to the environment, precise and balanced changes of proteome composition are crucial for plant development. Inordertodevelopstrategiestoincreasetheresilienceofplantsandenableenvironmentally friendlier agricultural practices, it is necessary to gain insight into molecular processes that safeguardproteostasis.Thus,thereisagrowingneedforstate-of-the-artmethodstostudy theseprocesses. Proteome imbalances can arise from a variety of conditions such as heat or during an infection,affectingbothbiosyntheticanddegradationpathways.However,degradationacts asabufferthatmediatesthedisposalofdamagedproteinsorevenorganelles.Inplants,two of the major degradation routes are the proteasomal and the vacuolar pathways. Both pathways are coordinated by post-translational modifications (PTM). PTMs comprise a widediversityrangingfromsmallmoleculestoproteins,andtheirattachmentandremoval is tightly regulated by specific enzymes. For instance, stress signals are relayed by kinase cascades that are in an intense crosstalk with degradation pathways via the ubiquitination cascade.Ubiquitinattachmentservesasasignalinbothproteasomaldegradation,aswellas vacuolarproteolysis,bymediatingvariousstepsofendocytosis,andduringselectiveautop- hagy. Autophagy itself, which together with the endovacuolar pathway transport cargo to the lytic vacuole, requires ATG8, an ubiquitin-like protein. An additional regulation of ubiquitin-dependent protein degradation is provided by prior modification of the target proteinbySUMO,anotherubiquitin-likeprotein.Thismechanisminvolvestherecognition of polySUMO chains by dedicated E3 ubiquitin ligases, the ubiquitination of the SUMO chainsanddegradationoftheSUMOylatedproteintarget. The increasing complexity of post-translational modifications, together with their highly dynamic nature, make them challenging to analyse. Hence, the need for custom- tailored and highly sensitive molecular tools. The difficulties to study these processes in plantsareexacerbatedbytheabsenceofwell-establishedcommercialtools,complexprepa- rationofplantsamplesrequiredforbiochemicalstudiesandlargegenefamilies.Theanalysis of protein homeostasis is even more complex in non-model plants since specific protocols and tools are poorly developed or unavailable. In this book, we have collected detailed protocols describing state-of-the-art approaches that will facilitate the analyses of proteos- tasisduringstressresponsesandplantdevelopment. InPartI,weprovideprotocolstoanalysetheattachmentofubiquitin,asignalmolecule key to degradation, but also able to convey changes in activity or localization of modified proteins. Chapter 1 describes a fluorescence polarization-based assay that is able to track v vi Preface“PlantProteostasis” ubiquitin conjugation and deconjugation in real time based upon changes in molecular weight. Because the E2 enzyme mediates ubiquitin attachment and determine to a large extent the type of ubiquitin signal produced, Chap. 2 provides a pipeline to identify and characterisephysiologicalE2-E3pairs.Chapter3providesmethodsforanalysingtheinvivo dynamicsofCullins,whichactasscaffoldsforCullin-RING-Ligases(CRLs).Aspecialtype ofCRLsuseaso-calledF-boxasasubstrateadaptor,manyofwhichhavebeenshowntoact as plant hormone receptors. Chapter 4 describes a protocol to study hormone-triggered SCF-substrateinteraction.SomeE3ligasescooperatecloselywiththeproteasometoensure substratedegradation,andChap.5providesanapproachtoanalyseproteasome-associated E3s.Ubiquitinationisahighlydynamicprocessduetoubiquitin-specificproteases(deubi- quitinases) that cleave ubiquitin chains, and Chap. 6 (Isono) focuses on an approach to studydeubiquitinasesandubiquitinremoval. Part II focuses on the study of the post-translational modification with SUMO, an ubiquitin-like protein, and includes protocols to analyse the in vitro formation of SUMO chains (Chap. 7), the kinetic analysis of SUMO conjugation (Chap. 8), and the in vitro analysis of SUMO proteases involved in SUMO maturation and SUMO removal from substrates(Chap.9). Autophagy is dependent on ATG8 ubiquitin-like proteins, and PART III provides protocols to monitor the autophagic flux in the microalga Chlamydomonas reinhardtii andtheanalysisofautophagybyfluorescencemicroscopyinChaps.10and11,respectively. To study mechanisms of cargo selection, Chap. 12 provides biophysical methods to study theassociationbetweenATG8sandinteractingproteins. A fundamental aspect for the study of proteostasis are assays that allow to determine protein degradation rates. In Chaps. 13 and 14 of Part IV, different and complementary approaches are presented to determine protein degradation using metabolic labelling and tandem fluorescent timers, respectively. A common response to protein stress is the accu- mulationofproteinsinaggregates.Thesecanbedetectedandquantifiedusingtheprotocol described in Chap. 15. Small changes in the amino acid sequence can have dramatic consequences on proteinstability and, consequently, protein activity. Chapter 16describes anapproachtoevaluatetheeffectofsequencevariationsonproteinstability. Theidentificationofkeyplayersinproteostasisandtheposttranslationalmodifications involved in their regulation through techniques such as mass spectrometry, are pivotal to understandplantresilience.InPartV,protocolsareprovidedtoenrichbothubiquitinated (Chap. 17) and phosphorylated proteins (Chap. 18), as well as to isolate proteins from chloroplastmembranes(Chap.19)andfromchromatin(Chap.20).Chapter21describesa protocolforproximitylabellingtoisolateinteractorswithweakprotein-proteininteractions. Tandem mass tag (TMT) labelling allows the quantitation of phosphopeptides and non-phosphopeptidesfromthesamesamplesbymassspectrometrydescribedinChap.22. Proteases catalyse the breaking down of proteins into smaller polypeptides or single amino acids. They do so to degrade proteins, as in the case of the proteasome, or mediate proteolyticprocessing,whicharetakenupinPARTVI.Forinstance,peptidehormonesare generatedaspropeptides,andChap.23describeshowtocharacterisethecleavagesite,while Chap. 24 provides a protocol to improve their identification. While Chap. 25 presents a pipelinetomonitor theproteasome. Finally, PartVII encompassesprotocols for the insilico analysis ofdifferent aspects of proteostasis.Chapter23describestheuseofbioinformaticstoolsfordatamining,focusing Preface“PlantProteostasis” vii on the SUMO gene network, while Chap. 27 describes how to analyse transcription net- works during ER stress. Last, but not least, Chap. 28 provides approaches to identify intrinsicallydisordereddomains,whichplaykeyrolesintheactivityandstabilityofproteins. Together,thisbookhighlightstheroleofproteostasisinplantbiologyanditsrelevance in providing solutions to future challenges. It provides state-of-the-art protocols to study proteostasis and to gain insight that holds promise for the development of more resilient cropvarietiesbasedonproteostasis. We are thankful to the authors who have shared their expertise to make this book possible, which is intended as a reference for the proteostasis community and its development. Barcelona,Spain L.MariaLois Freiburg,Germany MarcoTrujillo Contents Preface“PlantProteostasis” .................................................... v Contributors................................................................. xiii PART I UBIQUITIN CONJUGATION AND DECONJUGATION ANALYSIS 1 ObservingReal-TimeUbiquitinationinHighThroughput withFluorescencePolarization ...... ....... ....... ........ ....... ........ 3 TylerG.FranklinandJonathanN.Pruneda 2 IdentificationandCharacterizationofPhysiologicalPairingofE2 Ubiquitin-ConjugatingEnzymesandE3UbiquitinLigases.......... ........ 13 CarlaBrilladaandMarcoTrujillo 3 ImmunoprecipitationofCullin–RingLigases(CRLs)inArabidopsis thalianaSeedlings ......... ........ ....... ....... ........ ....... ........ 31 FedericaCasagrandeandGiovannaSerino 4 AnInvitroAssaytoRecapitulateHormone-Triggered andSCF-MediatedProteinUbiquitylation....... ... ........ ....... ........ 43 MichaelNiemeyer,JhonnyOscarFigueroaParra, andLuzIrinaA.Calder(cid:1)onVillalobos 5 AnalysisofProteasome-AssociatedUbiquitinLigaseActivity ......... ........ 57 ZhishuoWang,BeatrizOrosa-Puente,andStevenH.Spoel 6 MeasuringtheDUBActivityofArabidopsis DeubiquitylatingEnzymes.......... ....... ....... ........ ....... ........ 69 KarinVogel,Marie-KristinNagel,andErikaIsono PART II SUMO CONJUGATION AND DECONJUGATION 7 SUMOConjugationandSUMOChainFormation byPlantEnzymes.......... .... .... ...... ........ ........ ....... ........ 83 KonstantinTomanov,JoseJulian,IonidaZiba,andAndreasBachmair 8 KineticAnalysisofPlantSUMOConjugationMachinery..... ....... ........ 93 LauraCastanõ-MiquelandL.MariaLois 9 Expression,Purification,andEnzymaticAnalysisofPlant SUMOProteases .......... ........ ....... ....... ........ ....... ........ 109 PrakashKumarBhagat,DipanRoy,andAriSadanandom PART III ANALYSIS OF AUTOPHAGY 10 MonitoringAutophagicFluxintheModelSingle-CelledMicroalga Chlamydomonasreinhardtii .... ..... ....... ....... ........ ....... ........ 123 Jose´L.CrespoandMarıáEstherPe´rez-Pe´rez ix x Contents 11 DetectionofAutophagyinPlantsbyFluorescenceMicroscopy....... ........ 135 YuntingPuandDianeC.Bassham 12 CharacterizationofATG8-FamilyInteractorsbyIsothermal TitrationCalorimetry ...... ........ ....... ....... ........ ....... ........ 149 LorenzoPicchianti,ArthurSedivy,andYasinDagdas PART IV PROTEIN TURNOVER AND STABILITY 13 ProtocolsforStudyingProteinStabilityinanArabidopsisProtoplast TransientExpressionSystem ........ ....... ....... ........ ....... ........ 179 Se´verinePlanchais,LaurentCamborde,andIsabelleJupin 14 RelativeProteinLifetimeMeasurementinPlantsUsingTandem FluorescentProteinTimers ....... .. ...... ....... ......... ....... ........ 201 HongtaoZhang,EricLinster,MarkusWirtz,andFredericaL.Theodoulou 15 DetectionandQuantificationofProteinAggregatesinPlants........ ........ 221 UjjalJyotiPhukan,SimonStael,AmandaGonçalves, FrankVanBreusegem,andNu´riaS.Coll 16 UsingIntrinsicFluorescencetoMeasureProteinStabilityUpon ThermalandChemicalDenaturation........ ....... ........ ....... ........ 229 NathaliaVarejaõandDavidReverter PART V PROTEIN ISOLATION AND MASS SPECTROMETRIC ANALYSIS 17 PurificationandDetectionofUbiquitinatedPlantProteins UsingTandemUbiquitinBindingEntities.......... ....... ....... ...... ... 245 DongHyukLeeandGittaCoaker 18 TitaniumOxide-BasedPhosphopeptideEnrichment fromArabidopsisSeedlings.......... ....... ....... ........ ....... ........ 255 SharonC.MithoeandFrankL.H.Menke 19 ChloroplastEnvelopeMembraneSubfractionation fromArabidopsisandPea ... ........ ....... ....... ........ ....... ........ 267 AnnabelDischingerandSerenaSchwenkert 20 ChromatinEnrichmentforProteomicsinPlants(ChEP-P)..... ..... ........ 285 IsabelCristinaVe´lez-Bermu´dezandWolfgangSchmidt 21 Proximity-DependentInVivoBiotinLabelingforInteractome MappinginMarchantiapolymorpha.. ....... ....... ........ ....... ........ 295 KatharinaMelkonian,SaraChristinaStolze,AnneHarzen, andHirofumiNakagami 22 TandemMassTag-BasedPhosphoproteomicsinPlants....... ....... ........ 309 IsabelCristinaVe´lez-Bermu´dez,DharmeshJain,AryaRavindran, Chin-WenChen,Chuan-ChihHsu,andWolfgangSchmidt