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Cell Biology

by: Miss Moises Reinger
Miss Moises Reinger
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Class Notes
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This 7 page Class Notes was uploaded by Miss Moises Reinger on Monday September 7, 2015. The Class Notes belongs to BIOL 110 at University of California - Santa Cruz taught by Staff in Fall. Since its upload, it has received 48 views. For similar materials see /class/182299/biol-110-university-of-california-santa-cruz in Biology Molecular Cell & Dev at University of California - Santa Cruz.


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Date Created: 09/07/15
Biol110 Cell Biology Spring 2009 Problem SeT 4 QuesTion 1 You JusT Joined a laboraTory ThaT sTudies a novel Tumor suppressor39 proTein called p93 This proTein is a TranscripTion facTor and iTs nucleocyToplasmic TransporT is highly regulaTed by a sTeroid hormone p93 is localized To The plasma membrane in The absence of The hormone buT iT quickly enTers The nucleoplasm in The presence of The hormone which is made by a neighboring cell A member of your Team idenTified The gene ThaT encodes p93 iT encodes a 93 kDa proTein SuggesT a mechanism for The observed regulaTion of p93 localizaTion and describe key aspecTs of iTs Traffic from The plasma membrane inTo The nucleus How would you idenTify a nuclear localizaTion signal in p93 and demonsTraTe ThaT iT is necessary and sufficienT To TargeT a proTein To The nucleus How would you idenTify The karyopherin responsible for nuclear imporT of p93 Briefly menTion Two general sTraTegies DespiTe your besT aTTemst aT idenTifying The karyopherin in The above experimenT you ended up wiTh four candidaTe proTeins and any one of Them could be The imporTin you are looking for A colleague suggesTed ThaT you use purified RanGTP as a Tool To make a final disTincTion beTween The four candidaTes proTeins buT The phone conversaTion broke up before heshe had a chance To finish Telling you how To use iT How would you use purified RanGTP as a final TesT To idenTify The imporTin among The candidaTe proTeins Briefly explain your answer QuesTion 2 A new kind of eukaryoTic microorganism was found on skin lesions of a group of US army personnel The organism appears relaTed To yeasT buT feeds on skin cells raTher Than grapes by secreTing a proTease ThaT degrades collagen Soon afTer isolaTion of The organism a collecTion of 1000 TemperaTure sensiTive Ts sTrains was generaTed To sTudy The cell biology of The new organism Specifically each Ts sTrain carries a muTaTion in a single gene and The proTein producT of each muTanT gene funcTions properly aT 30 C buT ceases To funcTion aT 37 C Your research group has been assigned The Task of deTermining which of all The Ts muTanT sTrains are defecTive in The secreTory paThway in parTicular A If you were given a culTure conTaining a mixTure of all one Thousand differenT muTanTs how would you go abouT selecTing in baTch only Those muTanTs ThaT are defecTive in The secreTory paThway Explain The raTionale B WhaT would a block in differenT sTages of The secreTory paThway look like under The elecTron microscope in muTanTs afTer shifT To The nonpermissive TemperaTure Also how would each block affecT The glycosylaTion sTaTe of a marker secreTory proTein Block in proTein TranslocaTion inTo ER Morphology GlycosylaTion sTaTe of Tracer proTein Block in vesicle buddinq from The ER Morphology GlycosylaTion sTaTe of Tracer proTein Block in vesicle fusion wiTh The plasma membrane Morphology GlycosylaTion sTaTe of Tracer proTein Biol110 Cell Biology Spring 2009 Problem SeT 3 KEY QuesTion 1 The raTe of TransporT of small molecules across channel proTeins versus carrier proTeins is very differenT Which Type of TransporTer do you Think allows more rapid TransporT and why The channe proTeins aow more rapid TransporT because 1 Channe proTeins are ofTen open Though someTimes They aregaTed In conTrasT carrier proTeins spend Time opening and cosing Their channe To each side of The membrane When igand gaTed channes are open They aow rapid TransporT 2 Unike carrier proTeins channe proTeins do noT bind The moecue being TransporTed To The was of The channe39 Thus There is no inTerrupTion in movemenT inside The channe WhaT Type of carrier proTeins or proTein complex do you Think The miTochondriaI Translocons are eg passive or acTive uniporTer symporTer or anTiporTer plain channels or ligandacTivaTed channels Explain your answer Transocons are igandgaTed channes because signa sequences open Their conduiTs and The body of The TransocaTing chain does noT appear To make sTabe conTacTs wiTh The wa of The channe However They can aso be viewed as carrier proTeins because They bind The signa sequence aT enTry siTes exiT siTes and perhaps even inTerna siTes in The case of TransocaTion of muTipass membrane proTeins They are uniporTers because TransocaTion of nascenT poypepTide chains across is noT couped To The enTry or exiT of oTher moecues Through The same channe If you added an ionophore To isoIaTed miTochondria and aTTempTed To reconsTiTuTe proTein TranslocaTion inTo iTs maTrix which parTicuIar sTep in The TranslocaTion paThway would be affecTed and why An ionophore woud disrupT The inTegriTy of The inner miTochondria membrane making permeabe To ions IT woud aso incorporaTe in The ouTer membrane wiThouT effecT because ThaT membrane aready has arge porins and Thus no ionic gradienTs The TransocaTion of The poypepTide across TIM woud be affecTed because ThaT sTep requires an inTacT eecTrochemica membrane poTenTia To drive The inserTion of The presequence from The inTermembrane space To The maTrix side of The TIM How would you prove your model experimen rally I woud conduc139 an in vi139ro mi139ochondria pro139ein 139ransoca139ion assay using isoa139ed mi139ochondria an ATP regenera139ion sys139em and a cefree 139ransa139ion ex139rac139 con139aining radioabeed precursor I woud expec139 139ha139 ionophore 139rea139ed mi139ochondria coud bind 139he precursor and 139ransoca139e i139 par139iay across 139he TOM The presequence woud be abe 139o bind TIM bu139 woud be unabe 139o proceed pas139 TIM 139o 139he ma139rix side In 139ha139 case 139he radioabeed precursor wi cosedimen139 wi139h mi139ochondria a139 ow speed cen139rifuga139ion bu139 139he nascen139 chain wi be danging from 139he mi139ochondria surface par139iay exposed 139o exogenous pro139eases 0ny a sma N139ermina fragmen139 of 139he precursor may be pro139ec139ed from pro139einase K for exampe As ceavage of 139he signa pep139ide presequence occurs in 139he ma139rix successfu en139ry of 139he unfoded chain in139o 139he ma139rix can be diagnosed by a reduc139ion in size of 139he precursor in SOSPA 6E ges A precursor missing 139he presequence wi have a smaer size in such ges Par139ia 139ransoca139ion can be resoved from no 139ransoca139ion via cen139rifuga139ion if 139he precursor does no139 bind TOM i139 wi no139 sedimen139 a139 ow speed 139oge139her wi139h mi139ochondria QuesTion 2 The mitochondria 2007 midTerm A human disease can be explained by The absence of only singlepass Transmembrane proTein ThaT is normally presenT in The miTochondrial ouTer membrane of healThy individuals The missing proTein is called OMPI for ouTermembrane proTein 1 All oTher ouTer miTochondrial membrane proTeins are presenT in The sick individual39s cells When you examine The faTe of OMPI in muTanT cells ie where is iT using a fusion of OMPI wiTh The green fluorescenT proTein you discover ThaT iT is synThesized normally in The cyToplasm buT cannoT be TargeTed To miTochondria in The muTanT cells insTead iTs locaTion is dispersed across The cyToplasm In conTrasT in normal cells There is liTTle if any 0MP dispersed across The cyToplasm and when Tagged wiTh GFP iT can be seen shining brigthy in The ouTer miTochondrial membrane Describe a simple in vivo assay To TesT unequivocally wheTher The OMP GFP mislocalizaTion in The muTanT cells is due To a defecT in OMPI proTein iTself eg a muTaTion in iTs leader pepTide or signal sequence or is due To a defecT in one of The membranecomponenTs of The muTanT cell39s miTochondrial Translocon machinery 5 st Here are Three possible answers 1 Take cyTosol from The muTanT cell make a crude cell exTracT and microinjecT iT inTo normal cells The 0MP 6FP in The muTanT exTracT will now have a chance of being TransocaTed inTo miTochondria of a wild Type cell If 0MP 6FP does noT incorporaTe inTo The wild Type miTochondrial membrane Then The 0MP is defecTive if The 0MP 6FP does geT incorporaTed Then The defecT was likely wiTh The muTanT cell mi Tochondrial TOMs To de TecT TransocaTion score for GFP fluorescence around miTochondria and confirm Topology using a proTease proTecTion assay of a digiTonin permeabilized in jecTed cell 2 A heTerokaryon can also be used To mix The cell conTenTs insTead of microinjecTion may have To rule ouT The possibiiTy ThaT miTochondria from The Two cells fuse To differenTiaTe defecT in membrane or cyTosoic componenT InTerpreT daTa as above 3 Could obTain gene for 0MP in muTanT and wild Type and could express iT as a fluorescenT molecule in The converse cell InTerpreT daTa as above 4 Exchanging The signal sequence from The muTanT cell s 0MP wi Th a known funcTional signal sequence If This new hybrid proTein is associaTed wi Th The miTochondria The defecT was in The original signal sequence Upon very careful examinaTion of The miTochondrial membrane of muTanT cells under The besT fluorescence microscope you acTually see a Tiny liTTle biT of OMP GFP decoraTing The miTochondrial ouTer membrane To follow up on This observaTion you decide To isolaTe miTochondria from The muTanT cells and examine The Topology of The observed OMPGFP on The miTochondrial ouTer membrane How would you TesT wheTher The OMPlGFP proTein bound To isolaTed miTochondria from The muTanT cells is associaTed via ionic inTeracTions eg bound To a TOM recepTor or is TeThered To The ouTer membrane lipid bilayer via inserTion of iTs Transmembrane domain 4 st 0 A simple exTracTion wiTh salT aT high concenTraTion 1M NaCl would suffice To remove i T if iT is peripherally bound ExTracTion wiTh mild nonionic deTergenT such as TXIOO would be needed if iT were inserTed in The membrane One could score for release from The membrane by wesTern bloTTing pelleT and supernaTanT fracTions for OMP afTer medium speed cen TrifugaTion ProTease proTecTion assay would compeTey degrade an ionicaly bound OMP buT noT The Transmembrane domain of a TeThered OMP Urea and high pH soluTions can dissociaTe an ionic bond buT noT a TeThered pro Tein Assume you find ThaT OMPlGFP is sTuck bound To The ouTer miTochondrial membrane via an ionic inTeracTion wiTh a TOM proTein in The muTanT cells Among The candidaTe TOM proTeins ThaT could mediaTe The inTeracTion There are seven differenT TOM recepTors in Two physically separable complexes ie one TOM complex is a heTeroTeTramer and The oTher is a heTeroTrimer In addiTion There is The TOM41 proTein which iTself forms The TranslocaTion channel How could you idenTify The specific TOM proTeins ThaT inTeracT direchy wiTh OMPlGFP during iTs TranslocaTion inTo miTochondria in normal cells 5 st 0 I would aTTach a phoToacTivaTabe crossinker To OMP before presenTing iT To a miTochondrial membrane in viTro AfTer allowing iT To bind I would acTivaTe The crossinker wiTh lighT flashes and would proceed To isolaTe The OMP via anTibody affiniTy purificaTion Any proTein ThaT coimmunoprecipiTaTe afTer 505 denaTuring The samples would be a good candidaTes for being The recepTor FRET combinaTions beTween subuniTs of TOM and The OMP may idenTify which TOM proTein OMP mosT closely inTeracTs wiTh 6eneTic deleTions of individual TOM proTeins may abolish binding beTween OMP and TOM WiThouT doing any more experimenTs can you guess correchy which parTicular proTein carries The molecular defecT The muTaTion in The sick paTienTs In your answer assume you find one of The TOM proTeins as making a direcT inTeracTion wiTh OMPlGFP in muTanT cells Which parTicular sTep in The TranslocaTion paThway do you Think is being affecTed by The muTaTion explain your answer 5 st The OMP proTein has The defecT likely in iTs signal sequence because iT is The only proTein missing from The ouTer membrane in The muTanT cells For example if a TOM proTein was defecTive Then you would expecT To find more Than one Type of ouTer membrane proTein missing as TOMs mediaTe The membrane incorporaTion of many proTeins QuesTion 3 The Ran GTPase Taken from 2007 midTerm When cells undergo miTosis The nuclear envelope breaks down and all cyToplasmic and nucleoplasmic proTeins are free To mingle AfTer The nuclear envelope reassembles how is The 4000fold RanGTP concenTraTion difference ie The RanGTP gradienT reesTablished across The nuclear envelope In your answer menTion The cellular locaTion where RanGTP is produced 10 st AfTer assembly of The nuclear envelope wi Th funcTional NPC s The RanGTP gradienT would be reesTabished by relocalizing The RanGEF and RanGAP To opposiTe sides of The nuclear envelope 0 Since RanGEF binds To chromaTin iT may already be parle or mosTy included in The nucleus during assembly serving as a landmark for The nuclear environmenT RanGEF bound To chromaTin would sTimuaTe local producTion of RanGTP by sTimuaTing nucleopasmic Ran To exchange GDP for GTP This is aided by The 101 raTio of GTP GDP in cells 0 CyTopasmic RanGAP would converT any RanGTP in The cyToplasm To RanGDP 0 Upon diffusion or exporT of RanGTP in The cyToplasm The RanGAP would immediaTey converT i T To RanGDP 0 Upon diffusion or imporT of RanGDP inTo The nucleus The RanGEF would immediaTey converT i T To RanGTP 0 Any RanGAP caughT in The nucleus upon nuclear membrane reassembly would have To be exporTed from The nucleus likely be an exporTin 0 Any RanGEF lefT ouT in The cyToplasm would need To be reimporTed inTo The nucleus Besides simple diffusion across The nuclear pore complex Ran is small enough To diffuse across The NPC whaT oTher mechanisms do you Think Ran uTilizes To shuTTle beTween The nucleus and The cyToplasm of cells In your answer menTion The form iT is likely in ie GTP bound GDP bound or empTy form when moving Through The NPC by These oTher mechanisms Explain your answer 5 st 0 Pan is exporTed ouT of The nucleus bound To exporTins ie iT is a necessary cofacTor for exporTins 0 Pan piggybacks iTs way ouT of The nucleus on imporTins as They recycle back To The cyToplasm for anoTher round of imporT 0 Pan is exporTed in iTs GTP bound form because ThaT is The only form recognized by exporTins and imporTins excepT for The imporTin NTf2 0 Pan is imporTed inTo The nucleus bound To iTs imporTin NTf2 or p10 This is a commiTTed imporTin for RanGDP 0 Pan is exporTed in iTs GDP bound form because ThaT is The form recognized by NTf2 and is The form of Pan presenT in The cyToplasm


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