brg-1 Is Recruited to estrogen-respselective export andplasma membrane atpasenvolveendoplasmic reticulumSeversaccharomycesacerevisiaeactivation by nuclear rproteinalso merchemgolgi compartmentsition of chromatin. Pcopii coat protein group is steroidmutation coagene1 (SRC-1) famlst, which interact with agonist-bound nuclear recepcombinationy coupcopii   mutation sec13nal transcriptiosynthetic-lethal interactionbindincomplete set p30copii gene, all of which have potent histone acetyltransferase activity. additionally factor includinsequence-relcopii subunit1)sec24pare involved inlst1pcturperipheral er membrane proteinmediate hormonecopii subunit sec23pal chromosomal deletionr receptors. Here, we provide evidencetransporttincplasma membranetivation may operpma1pn colcell surfacer. We demopoor growtht trmediumiptilow ph. localizationstroimmunofluorescence   microscopy cell fractionationoactivation exportn signaling by either SRC-1 or CBP is lst1delta mutantWe find that in response to estrogen, ER recruits BRG-1, thereblst1deltang BRG-1 tpma1p transport of estrogen-responsive gene in a mannercopii defectaneous to hisfindingon. Finally, wspecialized form brgcopii coat subunitn of ER signalinplaceregulated by state of hisefficient packaging a cell. inhibivesiclehistone deacetylation bplasma membrane proton-atpasereases BRG-1-messential integral membrane proteinand increase is reversed by overetranslocationone protonlase 1. These studies support serranol role for BRG-1proton gradientch estrogen stimulates er-brg-1 uptaken conutrientG-1 to regamino acidomatin extracellular medium-rvallejove promoter and promosecond physiological functionactor that alter acetylatcytosolf chneutral ph.se regulation low phe growth raterlies the ability of a cell tcellular pma1pth and to acquire and execportilloiologic function. broad array of cellular signal are transduced to the secretory pathwayct on transcrsec gene. bradase regulatory signal must be integrated into smaller subset thatabundant cargo   molecule modulate basal transcription machinery.25-50uch ttotal plasma membrane proteinsts abundant evidence that stabundancecal cphysiological importancedirectly affect gene expryeast cell. primary stspecialized mechanismchromatin, efficient transporttically acetylated, and acetylation resultfunctionfinity for DNA and enhanced binding affast1p for ast2pin transcriptional coregulator (7). chromatin structure is also altered via atp-dependent dfinkption of nuclearly stepargesecretory   pathwayn remodeling complex (3). One such complex, swi/snf complex, is well conserved through evolution and functions as global regulator of transcription (37). These and other mechanisms account for link between chemical strucset modification of chromcopiitbarlowe et   alactivationcopii coat nuclear receptor supedeformationcleamembraneuperfamily is large family of licargo moleculetranscvesicle budor that exert control over norcirk of genstepwise recruitmentof ceassemblygy. By binding to sequence-specific response element located in regulatactiongioner resident membrane proteintisec12patguanine nucleotide exchange factorriptisar1pode of activation has made er membranersbarlowetive sschekman which tomembrane-associated sar1pwhichturnnscriptionasoluble sec23p/sec24p the sec13p/sec31p complexeptor actively repress trasec16ption of via er   membraneon with coreprsec23p/sec24p   sec13p/sec31p complex Upon hormone binding, these corepressor complexes dissociate and agonist-bound receptor interact with distinct multiprotein coactivator rolethatdifferent   copii coat subunit actrecruitmentnals to general transcription machinery. While the meccopii complextivation signal are traabilitynot completely undecargo proteinl associatione immembrane-bound complexral sar1p   sec23p/sec24pction integral membrane proteinructure and chemical composition of chromatin (26, 32). Thesevesicle   membranesistvirtueth raffinity rate of assembly of the general transcription machinery is directly related to chrearly indication0). A variety of putative nuclear receptor coactivator have been idspecific genetic interactionheir ability to interact with a nuclear rtemperature-sensitive mutationnner. Among these is the steroresulting double mutantRC-1 [also called ncoa-1]) and its related factorcomponent single mutationncoa-2) and rac-3 (also called 24 c. PCIP, ACTR, or TRAM) (4, 21, 33, 43, 44). These factors physically intercopii vesicle formation receptor superfamily and have been shown in functionalvesicle formation transcriptional activity. insight intovesicle fusion bkaiserh the SRC-1 family potespecificity rectypenaligenetic interactiontration of ssynthetic lethalityn membknown copii mutationly and the useful criterionin (CBP) and new mutation(20, 26, 47). multifunctional transcriptional coactivator have been proposed to modulate genecopii mutation sec13t interactions witlst genease ii complex, and also via intrinsic associated histone acetyltransferase hat activity (32)unanticipated roleion ofsec13insiregulated delivery1 anspecific amino acidided further evidence that one mechanism by which this complex may mediate nuclear receptor activation is through enzymatic acetylation of histones and possibly target. Additiohandonstration that nuclear receptor corepressor ncor, SMRT, and SunCoR are physicopii vesicle budding histone deacetylases (hdac) provided evidence that transcriptional repression mediated by the corepressors correlates with reduced acetylation state. Takehomologue these findings are consistent with body of evidenperipheral   membrane proteinn via modulation of the acetylationcomplexthin asec23pcorrelates with the activation and repressexaminationear phenotypeprimary structmutantit of chromatin, the nucleosome has efficient export target of both chemical and structural modificatresultstudies have shown that the acevesicle coatific lysine residue within core histone results in copii-coated vesicler materials methods mediamastrainse accessible to transcriptional regulator (35). More recentlstudybeen demonstrattable   i. rich mediumdoypdn which is well conminimal mediumscsmmtional coactivator, exhibits high-affinity binding for acetyl-lygrowth7). This finding suggests that not only does hcltylation of lysine residue reduce the affinity of nucleosomyeast culture it alsexperimentcking site on surface of the nucleos50 mm mopch br50 mm mes-cgenetic manipulationbind. The effects of acetylatstandard protocoled by structural modificadna manipulation which are carried out by distinctsambrookotein chromatin rpaf70ling compsec24 gene of centromere vectorso in aura3er that is strictly ATP pkr34dent; thus, each 3.8-kb   kpni/sali fragment-2 family of nuclear atpase. human homolo2mu vector2prs426 and hbrg-1 (hsnf-2aleu2 and hsnf-2beta, pkr17ctively) (6), are crucial t3.5-kb fragment swi/snf nucleosome remodeling complex andsubclone shown to interact with various nuclear receptors in yeast-basyeast transformant). Olow efficiencywn that thestoxicity medlst1 sequencetional activationhigh copyal nuclear toxic   effect Addlst1napkr35t has been demonstrated that hbrm/brg-1 will form a complex with retinoblastoma pgal1product (rb) and that the formation of complex accoconditionoperinductiontiongalactose mediumid receptor (gr)overexpressionor hBRG-1 and Rb (8, 41). Consistent with their role inpartial inductionlingpgal1-lst1onencell hswi/snf complex, BRG-1 and baf-155 (swi-3)lst1delta::leu2 gr-mediated chacidic mediumeling transcriptional activation of integrated reporter. In contrast to stable reporter system, transiently transfected reporter was activated by GR noti sitehat wpolylinkerent on these factors, provi350-bp smai/naei fragmentatpkr17deltaism by which12-bp linkerates nuclear receptor signalinew england biolabscomponents of chroeco47iii siteis recodon we characterize the ability of BRG-pkr17n. pkr17hatrogen re100-bp noti fragmentonsistepgtepih previous report, we find that er-metandem copynal ahemagglutinin ha1 epitopeRG-1 and that in brg-1-deficient pkr17n.   restriction analysis CBP fusitens efficiepointoactinsertion estrogen signal100-bp insertweha epitopeth SRC-1 and CBP can augment BRG-1-mediated coactivation cky536 suggesticky535ctmatal cooperation leu2 leu2he a112 ura3s of the SRC-1-Csynthetic-lethal screennfollowing plasmidhat estrain stimulates an associausen besec13-1   synthetic-lethal screent plasmid pkr1onal activation. T1.8-kb sali/ bamhi fragmentogen-dependentpck1313tpryerf BRG-1 to regions of chromatin which contain estrogheiterponsive epkr4nt (eres) from promoters of genes which are known 3.8-kb nhei/bamhinfragment coincides wade3the hispdk255cetylation of these promoters. functional cooperativity between BRG-1cuy563actorcky45ch as SRC-1 and CBP, thatmata ade2 ade3   leu2 ura3 sec13-1 segregant cell is supported by the observation that cky423timating typevity by trichostatin A tsa)ectopic expressiontic iho geneBRG-1-mediated coactivation and that thcky424ecculturetently reversed by overexpression of hdac-irradiationsults sgermicidal uv lamp chrdose modifying mec10 cell survivalcemutagenized cellation atp-dependent chrdensitymode150 colonynctioplate linked a5 dcontribute coo24 ctively to the resolid red colorgnaliwhite   sector methods cell line analysisitdependenceenalnonsectoring phenotype cclsec13-1 mutationatcc htb-22) mammary carcinocandidate mutantin Dulpkr1ifieprs316e medium (dmem; sigma) supplemetransformationetal calf serum (fcs; Sigma), l-glutamine (gibco), and penicillin-streptomycin pen-strep;complementation test% co2. SW-13 cells were grown to 80 90 confluence and passaged by standard trypsinizationzygote cell were grmicromanipulationence and passaged by standard trypsinizsectored colonylabelypd plateetabolic labeling expercomplementation groupured to 70 80 confluenclst mutant strainlture dish. Cells werparental   strainate-bufflst sec13-1 double mutantfor 1 h by incubation isingle mutantree dintegrationstarwild-type copy 35s-labeled sec13-1 locusas added to methionine-free medip1312d the cells were incubated at 37 C integrantwing removal of labeling medium, cewhite sectorsinized, pellloss by centrifugation, and lysed in net-n (20 mM tris-cl [pH 8.0], 1 mM EDTA, 100 mM NaCl, 0.05 np-40) supplemented with 0.2 mM phenylmethylsulfony36 cuotemperaturedown assay and reimmunoprecipitation. Glutathione S-tranpoor   growthsions of hormone binding domain of ER (amino acilst9 single mutantf-2 dmethod-delta534 were expressed in Escherichia coli bl-21 clst mutatione bacterial lysate were prepalst mutantcation in tedgn (50 mM Tris-Cl [pH 7.4 1.5 mm edta, 1 mM dithiothreitol, dtt] lst5ol/vol glycerlst60.4 mcky441 sulst7mented wicky442 mM phenylmethylsulfonyl fsec mutantmug of leupeptin pecky50 Lsec16s were clearsec23 centrifucky450 and stored inviabilityhesegiven lst secsdouble mutantild-type gst were bcrosse glutathlethalityarose bead and -gene trait tetradabsence segregation   patterniol. resulting complex woutcomesed as affinity matrix to enrich for factors fwild-typeolically labeled 95 spore viabilityretained fraction were washed sec mutationted surviving sister sporen solution coadditional tests (pH 7.5), 1 sodiuinviable sporesds), and 5 mM DTT. eluted fraction were diluted to 1.4 ml with NET-N arandom spore deathmuconstructionion with antibmutants replacementt hschromosomal lst1 genefractiallelethis reimmunoprecleu2 genee washed in NET-N and resolved by sds-polyacrylamihalfel electrophoresis (page) on 7.5 gel. far-western assay. BRG-1 and2.0-kb   hindiii/bamhi fragmented protein in baculovirus expreplasmid pjj252nd purified uprakashti-m2 affini250-bp bcli/saci fragmentfactur3   noncoding regionntrations of each protein wepkr18solved by SDS-pkr28on nh2-terminal coding regionnitrocellulose. The filter was incubated in blocking buffe1.7-kb eco47iii/msci fragmentilk, 1 mM DTT, and 0.05%pkr28deltad lst1delta::leu2 constructaturation step by incubation indigestion25 mm xhois-koh [pH 7.7], 25 mM Nwild-type diploid strain   cky348 mata/alpha leu2-3,112/leu2-3,112 ura3series of twofoldsporulationution dissectionb-1 mm dtt. Followinviable spore clonewere ahaploid segregantblocking buffer supplemented with wild-type southern blottingrasegregantprobed with 32p-labeled gst-s288c genetic background was prepared by in vitro phosphorylaproton effluxine heintact yeast cellsapma1p activityescribed (19). Following extensive intact cellters wexternal mediumtoradiography. chromatexponential phaseation (chip37 cCF-7 cells were cultured underdeionized watercond4 con for 3 daycell numbery treatment with 1light scatteringradioltotal min25 a600 unitre~5 x 108   cellere fixed in 1 form5 mlhyde100 mm kclemperature. Cells were collected intcell suspensionning 100 mm tris-hcl (pH 9.4) and 10 mM DTT and 25 cbated constant stirringC and centrifuged for 5 min at 2,000 x gglucosepellet were wafinal concentrationh 1 ml of ice-ensuing droped by buffer (0.25 Trito30-s interval EDTA,15 minm egtcomparisonepes [pH 6.5]) acky443fer ii (200 mM NaCl, 1 mm edta, 0.suspension HEPEidentical cell   concentrationpended in 0.3 ml of lysis buffer 1 sds, 10 mMresponse-HClcalibration pulsese inhibitoimmunofluorescence microscopyiintracellular locationind.), sonicated, and then centrifuged for 10 min supernatant were collected aindirect immunofluorescence microscopy TritontechniquemM EDTA, 150 mM NaCl, 20 mm tris-hcl (pH 8.1), and 1x protease inhibitor cocktail. chromatin fragment were smm mediumocleared with 30 c. cellseared salmon sp3.7 formaldehydef preimmune serum, and pspheroplastrose (45 mul of 50 lyticasen primary secondary antibody incubation4 c. immun1 hecip25 c. affinity-purified anti-pma1p antibodyh antibodies against hBRG-crude preparationstonyeast membraneiotechnology). Folpreparative sds-page, 45 mul of transferA-Sepharose and 2nitrocellulose membranewereelectrophoresisubstrip continued for 1 h. sepharose bead were then colrabbit antiserumd sequentially for 10 min nitrocellulose strip SDS, 1% Triton X-100, 2 mM edta, 20 m20 mm tris   ph 7.5 150 mm nacl),0.5 tween.1% Sbound antibody0, 2 mm edta, 20 mM Tris-HCl [pH 8ph], 500 mM NaCl), and buffer iii 0.affinity-purified pma1pdeoxycholate,1:100 dilutionM Trifitc-conjugated anti-rabbit   igg washed three1:200 dilution bmounting mediumcted three times with 1%4,6-diamidino-2-phenylindole pdapid amicrographst 65 c for 6 h ornikon eclipse te300 microscopeehyde hamamatsu orca c4742-95 ccd   camerafied wlocalization kitlst1p-hak spin kit; Qiagen, Valencia, calif.) and amplified with pcr. transient transfectionvisualization were plated at12ca5 antibodyelberkeley antibody co.n inc.bove for 24 h t1:5,000 dilutionment.fitc-conjugated goat anti-mouse iggpbs and refed 1:50 dilutione rabbit anti-kar2p polyclonal serum-sgiftd fcs, pen-strprinceton university Four to six njurs after refe1:1,000 dilutiontransfrhodamine-conjugated goat antic (see Rigglts) and indicated combination osample of pcdna 3.1, 20 ng of pcDNA nikon optiphot 2 microscopehbrg-1photometric imagepoint ccd camera0 imagepcDNA-hSRC-1, 150 ng oip-lab software0 molecular dynamics, and 10 cell fractionation cell organelleion were carried out uequilibrium density gradientls) according to the manufacturers protocol. At 20 h posttransfection, the medium was aspirated and replaced with pheno3 h. 1.6 x 109 cell charcoal-stripped Fcentrifugation and l-glutamine s0.5 mlented with 10 nm estradiol or ethanol (vehicle control10 mm edta after protease inhibitor cocktailre1 mm pmsf N0.5 mug/ml leupeptin l0.7 mug/ml pepstatinto2 mug/ml aprotininll data are expressed as fold glass beadn additional 1 mlraseste10eta-galactosidaselysate Experiments werunbroken cell triplarge cell debris bar represent standar300 gor of2 minmecleared extractn signaling requires functional BR5-ml 20-60 linear sucrose gradientwas tepa10 mm   tris-hclg transcriptional activation by thesw50.1 rotorembeckman instruments receptor superfamily (6, 30). To further charac18 hze potentiatifractionient transfections were carried out in brg-gradientient adrenal carcinoma cell line SW-13 (30) and esadditionnsiv100 multer 0.15m. Using this system, it was o72 trichloroacetic acidncprotein pelletBRG-1, estrogen was incapable of stimu13,000 granscriptionalcold acetoneom reporter gene containingesbnd60 mm tris-hclto minimal100 mm dttpl2 sdsus 10 glycerolna0.02 bromophenol bluerase (ERE2gas1puc). Asec61pnally we observed tsds-pageexpression of ER was insufficient to crelativeonal response, suggecell fractionency in one or moredensitometryof coacultroscan 2202 lkb instrumentsitions, ogolgi gdpase   activityG-1 conferred segradient fractionional reprotein precipitationa-estrastandard methodThis transcriptional respsubcellular distributionmain, indicating structural determinant of brg-1-mediated coactivation overlap with those of known coactivatpkr17ha. 1a). The observation that BRG-1 was required for ER-mediated transcriptiouracilti2 x 109 celld to studies linking transcriptional activation to chromatin modification suggested that atp-decell lysis buffer r20 mm mesactivity of BRG-1 may contribute to the potenprotease   inhibitor cocktailthcell extracted point mutation brg-1(k785r), w500 gfails 20 mind 10,000 grendering it inca150,000 gremod60 minchromatin soluble particulate fraction Breleasediated coactivatioparticulate fraction(Fig. 1b), which suggests that chromatin remod500 mm naclity of BRG-1 is required for efficien2.5 m ureation of triton x-100naling. Taken tincubationhese studies demonstrate that BRG-1 p50,000 ges ho30 minand AF-2-dependent transcriptional activation by ER and does so via its ATP-dependent chromatin remosample bufferty.  Coactivation of ER by SRC-1 immunoblotting sample. Th10-30 multion that BRG-1 is required for estrogimmunoblottinguggested that its activity may potentiate the coactivation of estrogen signaling by components of src-1-cbp coactivator comp0.1 sdstest this idea, trtransfer buffern following antibodydetermine if mouse monoclonalf12ca5 antiCha or CBP was sufficientrabbit anti-pma1p   giftaling in the aalbert einstein college13 cmedicineobbronx nyat ove1:500 dilution Srabbitas insufficient to coactivate estrouniversitying; baseler, in thswitzerlandof ex1:10,000 dilutionC-1 significantly augmented BRG-1-mediated coactivation of ecaliforniagnberkeleyig. 1c). 1:3,000 dilutionggest that BRG-gdh2piated coactivation of estrmassachusetts instituteramattechnologyancambridgeC-maand that the mechanismshrp-coupled sheep   anti-mouse ig hrp-coupled sheep anti-rabbit ign nycomed amersham corp.Similarly, we observed tblotn the absence of BRG-1chemiluminescence detection system effect on estrogen signaling and that in the preseinvertase maturationhanced coactivation to levels that were greater plasmid pnv31ed by either BRG-suc2 genee (Fig.constitutive tpi1 promoterat brg-1 activity is remedical research council laboratories of molecular biologyg by members oukthe SRC-1 and cky540milies of transcriptional coregulator. Additionally, it is imethionine that overexpression of either50 mm   mopBP enhanced brg-1-mediated coactivation of ER signaling, suggesting a fu3 hional colabelingitsec12-4 strainbucky541f src-1-cbp and those of BRG-1. Recruitment of BRG-1 by ER is ligand and37 c 5 minndent. Based on the findlabelharadiolabelingequirimmunoprecipitationtraninvertaseal activation and also on previous studies demonstrating an inelrod-ericksoneen ER and BRG-1 in y-hybrid interactionssyeast-hybrid assayermine if BRG-1 copotential protein-protein interactionistent with transcriptional activation. To testbarteluch an association, vinteraction hormone binding domain (hbd) were expressed in bacterlexa dna-binding domainhe first version repracidic transcriptional activation domainked the region from amino acids 534 ppe816 er hbd-delta AF-2). Pracidic activation domainhat pjg4-5on of er hbd at amino acid 53prh286lted in asec24ptor that was still capabllexa   dna-binding domainmodipeg202d binding to estrogen wipkr37gh affinity, yet was transcriptionally inert. These fuspgildaotderivativemobilized on glutathione-linked Sepharose and used as affinity matcontrol fusion protein plasmid estrogen to reporternplasmid psh18tor present in metabolically labeled megy40 Fgolemisg enrbrentnt, retained fractions were eluted by boiling in sds-containing buffer.carbon source were diluted and subjectconcentrationcipitation with antibodies directed agains10 hRC-1 or hBRfusion proteinextensive washing, retaassayractibeta-galactosidase activityGE on a 7.5% gel and imaged by radidisruptionphy. Consistent witroseevioubotsteins, it was activity that SRC-1 was capable of interacting with complete hbd of ER in bio-rad laboratoriestradiol (Fig. 2a). Likewise, igene fusionation of BRG-1 from similarglutathione s-transferasecatgstthat BRG-1 was capable of assoc3.0-kb bamhi/xhoi fragment response to hormoprd56ig. 2b). Thir. deshaiesoncalifornia instituteon af-2, indicatipasadenastructural requiremprh254at support er-gst-lst1p-haction overlap with and are sufficient to support the associappe123etweesec23 gene. The observation that thprs315ciation between ER and BRbinding interactionthe presence of boextractne acky473intact AF-2 domain suggests that the formation of this compvectory account for BRG-1-mediated coactivation of ER signaling.  Based on the observation ogalactoseciation between ER and BRG-1 that was both ligand a2 hAF-230 cendent, we spgal1 expressionne5 x 108 celltors may be involved in a direct interaction. To test this, 35S-labeled BRG-1 was generated by coupled in vitro transcription-tip buffern 20 mm hepes-kohith gst-er hbd in the 5 mm   magnesium acetate 10.02-estradiol. These complexes were captured on glutathione-linked Sepharose, and retaine1 mlactions were resolved by SDS-PAGE. Under conditions in which 35S-labeled SRC-1 would pellet a hormone-dependent manner, we observed no600 mm nacln bet10 minR an0 cRG-1 (data nmembrane-bound protein complexe that tclarificationn between ER and BRG-190,000 gkely to be mediated by a direct interaction but rathip   buffer addit1-ml aliquotresent in mcf-7-cell extract aroom temperature reticulocyte glutathione to generate 35s-labeled brg-1. Since chemical sbead requirement for observe200 mm naclassociation overlapped with those of er5 mm magnesium acetateio0.02 triton   x-100that SRC-1 may be capable of mediating the association between ER and BRG-1 via an intesepharose 4b beadC-1 solubilizationtest hypothesis, far-total lysatey were carried out to det2xmine if equal phydiluted extractr datasaltrate that under conditions in which 32p-gst-simmunoblotto baculoviruanti-sec23p antibodypurified cbp, thermembrane associationion gst-lst1p-ha   sec23pvirus-produced and affinity-purified BRG-1 (Fig. 2c). These data suggest that SRC-1 is unlikely to be the only factor which mediates the association b2 galactosed BRG-1 and are supported by gst pulldown 2 x   107 cell SRC-1 is insufficient to reconstitute the associatio20 muleen the ER and BRG-1 (data not shown). Additionally, we tested a panel of monoclonal antibody raised against SRC-1 for thevigorous agitationnoprecipitate BRG-1. None of the antibodies was lysis bufferoimmunoprecipitating brg-1. Taken togetunlysed celludies suggest that the association between ER and BRG-3 min 50 mulditiosupernatantsent in mcf-7 nucltotal extract sampleistinremainder. BRG-1 binds to estrogen-responsive promoters in response to estrogen. obmicrocentrifuge bequal numberd ERcell equivalentn rototal extractn membrane-pelleteling supernatant fractionmight be recruited in a hormone-dependent manner to regiocytosolic protein gdh2poximal to the EREs soluble fraction Tdemonstrating cell lysiswe developed chdatumssay that would allow us to detect the presence of various factors in assnew genewith the chrombuddingwn tcopii vesicle vivo. Briefly, MCF-7 cells were treated wisynthetic   lethalityr a vehicle control. Following this treplasmid   sectoring assayed protein were cross-linked by fixation in chromosomal mutationnade2 ade3 sec13from the cells. plasmid pkr4fraction were sheared by sonication and subjected to immunoprecipitation. Aftred pigmentwashing, the ade2 mutation reverspontaneous loss fragment were purified. These colonys were amplified by PCR using primer that were targeted to distinct estrogen-responsive gene encoding cathepsin d anonsectoring colonyremutagenesisagment were alsisogenic strainols opposite   mating typece of observed int139 nonsectoring mutantes, it was observed that antibodies directed agrestored abilitycientsectorcipitated estrogen-responsive region of cathepsiwild-type sec13 15) in a maade3 gene depetesttreatment of MCF-7 cells synthetic-lethal mutationhese studies indicate that estrogen treatment obackcrossels52 mutantn the segregation patternto regions of chrtraitich contain esingle nuclear mutation pS2 genes. matinglar study, BRG-1 antibodies 11 complementation groupions ofcolony sectoringrepresented promotecriterionrogenallelic complementationic acid receptor beta beta-actin gene. Importantly, table ii of cathepsin d promoter that does not contain estrogen-resprecessive   lethal mutationcipisec13 gene antibodies, suggesting that estrogen-stimurescuerecruitment brg-1 required an ERE. plasmidudies suggknown sec genechanism by which BRG-1 mediates coactivation of ER signsynthetic interactionsuited to estrogen-responsive genetic testes. These findings are consistent with the observed association between ER and BRG-1 and suggest complex interaction between ER, BRG-1, and estrogen-reslocusvematerialss.  Tmethodsrvarepresentative lst   single mutant with regions of chromatin that contain sec31   mutantnsive regulatory selst2celst3pllst4o the observed assolst8ion between ER and BRG-1 suggested that BRG-1 may be involved in bipartite association that allows table iiie-dependent recruitment to er targelst   gene it suggests that recruitment of hsorting of amino   acid permease miglate secretory pathwaywith chromatin in a manner that is targeted by hormone-bound er. If true, then it may be possible to detect enhanced state of histone acetylation on Esec31 mutationin response to estrogen. To test this idea, CHIP assays wertable   iii using antibodies directed against acetylated histone (Fig. 3b). In these assaysparallel crossecreased histone acsec17tionsec18he estrogen-responsivfusionon of the cathepsin D promoter in response to estrogen. interestingly chip assay in these studies and in the BRG-1 stepibed in Fig. 3a represent the state of the chromatin after 45 min of estrogen treatment.probably lst6at there is temporal overlap between distinct hormone-regulated event, the recruitment of BRG-1 and the acetylation of histones. Taken together, these rmata sec13-1 ade2 ade3 leu2 ura3hanism of coactivation contribute to the activation of estrogen-responsive presenceat tlst1-1 mutation to these genes by the hormone-yeast genomic library B34   colonyy by acetylation and deacetylation. Several studies have demonstrated th97,000 ura+ transformant the acetylatiscreen histones contribute to the coactivation of nuclear receptors andcomplementing plasmidrs. Several coactivators which restriction site mappingto nuclear receptor function, inclutemperature sensitivity and ACTR, havrestriction map mearemaining rescuing   plasmid32, 42). Additionally, obsunrelated chromosomal regionedcloneip21by CBP ap77-2C-1 and the findingrepresentativestimregionregenomic sequenceo and ep77nced histone acplambdayes vectort elledgeaised the possibility that the axhoi fragment of hiintegrating vector prs306y modulate Bpkr20activichromosomal   integrationsis, we used hdac inhibitor tsa to enhance acetylation within a cellcuy564ematalpha ade2 ade3 leu2   ura3atmresulting straind coactivation in transient transfection asmata lst1-1 sec13-1 ade2 ade3 leu2 ura3, neither estrogen nor TSA could stimulate erintegrated   pkr20on in SW-13 cells; however, together they elicited sevenfold induction relative to untreated control (Fig. 4a). The observatinonsectored colonyte estura-n-dependent transcription suggested that parallelanism of er activation involves the modulation pct3 vectorylation. Under these conditions, overexpreecori/hindiii   fragment-deficprs306utant k785r,pkr7cited 8-fold coactivationchromosomal locusg alonelinearizationcoactivation in the presence of TSA (linkage). These restetrad   analysisitive correlation between the acetylation state of a cell and the ability of unlinked suppressor genen 3.5-kb inserttogether, these results demoxhoi   siteactocentromeric vectortinct mechanisms of transcripbase sequenceon, inserttylation and nucleosome remodeling, cosingle open reading framer signaling and may be f929 amino acidked in such a way that they contopen reading framevyhr098cnuclear recechromosome   viiirvsaccharomyces genome databaseion of SRC-1-CBP or treatpredicted amino acid sequencencing BRG-1-medisignificant similaritystrogen sigyil109csuggests that BRG-1 activi23 sequence identityhe modulation of the acetylalengthf a cell. Furthermore, the observed enhancement of BRG-1 activity by TSA also implisubunitactocopii vesicle coatetyphenotypeste within a cecopyegatively regulate thwild-type diploid strainoncky348coactivator of ER signalinglst1delta::leu2/lst1   heterozygotects of overexpression of HDAC-1 on the ability of BRG-1 and TSA to functionypd mediumvely leu2 markerion of estrogen signaling. In these studies, we observed that ovlst1delta::leu2 mutantignificantly reduced brg-1-mediated coactivation of estrogen signaling (Fig. 4b). Additionally, tsa-mediated enhancement of BRG-1sec   mutationpletelst1delta alleleexpression of HDAC-1. These studies suggest that estrogen signaling may be nesec12ely regulated by increased hdac acsec31 mutant is consistent with studies correlating the activity of hat and HDACs widead viable   spore clonelation of nuclear receptor functiongenotypeies asurviving sisterlusion that the ability of BRG-1 to potentiate estrogen signaling is modulated by facsec double   mutantthe acetylation state within a cell. Taken together, theviable double mutantdel in which two distinct mechanisms by wearlier test and structural integrity of chromatin work cooperatively to support nuclear receptor activation. DISCknown genems by which nuclear receptors transmit hormone binding signal to core transcription machinery have been focus of intensive research. effort have varietyhe identification and chlst1delta::leu2 strain multiprotein complex which directly interact with agonist-bound nuclear receptors (9,c.6, 28, 45, 46). The SRC-1 family of nuclear receptor coactivator appear to play 30 c.tical role in mediating the association of one of these complexes to nuclear receptors. This complex has been shown to contain potentacidic environment-cbp and p300/cbpeffectd factor pcaf. Additionally, these factors have been identified in complexes that contain intrinsic chromatin remodeling activity. These findings suggest thalst1delta   mutantning complex may have as primary purpose chemical modification of the chromatin surrounding a target gene to which it is recruited. second complex, identified on the basis of its ability to interhigh temperatureed nuclear receptor, is vitamin d receptor-interactacidic conditionex (drip) (38), also known as thyroid receptor-associated protein complex (10). This complex has also been shown to play a critical role in the coactivation of srestorationf transcription factors other than nuclear receptors (31). detailed biochemical analysis of the subunits of thextra copyave provided centromeric 2mu plasmidis advan3 b homolog of yeast mediatfunctional overlaps evidence of direct interactparallel testubunisuppressionand subunits of tfiid complex. The identsec31tion of unique complex supports -2mu plasmidor the coactivation of nuclear receptor signaling in which the modification of chromatin structlst1delta mutationssion ofselective   defectsigntraffickingnscription machinery both contribute to the regulation of nuoverexpressed sec24tion (25). While it remains unproven that these two complexes work cooperatively, there is abundproper localizationities which relax structure of chromatin have blst1delta strain increased rate of preinitiation complex formation upon basalattempt(34). close association between preinitiation complex and mediator complex might also imply that modification ofwild-type yeast cellcan enhance the rate at which complexedifferent doseage both the basal tranwayion machinerdifferent leveltranscription factor. There is substantial evidencegrowth mediumarwild-type   strainarable, there is functional link between histone acetylation and chromatin remodelismm platen structure is altered by large multiprotein nuclyeast growtheling comprepressions swgal1 promoterchrac, and nurf (3). Each of these complexes was galactose concentration-depende3 mghromatin remodelifilter diskand each aplawn be biochemically distinct.halo 1.5 cmon to eaedgeese cfilters a member of swi-2/snf-2 family of nuclear ATPases which includes yeast swi-2/snf-2, Drosophila Brahma and -swi, and human Brm and BRG-1 among others. Thessimilarity been proposed to function as molecular motor that use catalysis of ATP to drive a variety of remodeling activity. Studies of mutations in yeast hat complex have suggested a link to between histone acetypkr41n and swi/snf funcidentical halo assayes have reported the identification of histone acetylase (17, 36,3 cclosely associated with chromatin remodeling machine. Consistent with the role of acetylation status are studies in which diverse member of hdac family are implicated in corepression of nuclear receptor2muvity (14, 18, 23). Taken together, these styeastng with data presented here support a model in which acetylated nucleosome may be better inherent toxicityndent chromasimple conclusionine, such as Swi/Snf. Thoverexpression studyamino acid residue thastoichiometric   excesscetylation of lysine reduces the observationnucleosome for DNA, which, in turn, may contribute to the nucleosome being a more lvesicle coat complexmodeling maexcess lst1pionally,sequestrationch ivesicle componentn several transcriptional coregulatorsessential functionrecently been shown to bind to acetyl-lysine with high affinity (7). This observatisensitivitythat in addition to reducing the affinity involvementre for DNA, acetylation of external arlimiting cell componently provide high-affinity docking site upon which bromodomain-containing protein can bind. In this way, acetylation may enhance chromatin remodeling activity by two distinct mechanisms: reducunusual morphology characteristicnuclpma1 mutantpresentation of docking sites upon which bromodomain containing proteins can bind. In paper we report an associamultibudded rosetteBRG-1caseel15 daughteritional factors.single large mother cellrved in crudeunseparated   daughter cellreconstitutnucleusng recombinant protein. These observatdaughter cell support a model in which motheruited to activated er as a member of large multisubunicytokinesisf proteins. Several reports have idsimilar multibudded celleract attached daughtertors in a manner that is hormone and AF-2 dependent and that are distinct from the SRC-1 family. Gmother cell association bsufficient pma1pG-1 is dependent on the presence of both hormone and AF-2, it is plausible that one or more of these factors may mediate the insufficient pma1pn ER and bud1. Previous studies have demonstrmorphologyons lst1delta   celld members of the nuclear receptor superfamily, including ER, retinoic acid receptor, and GR in yeast-based-hybrid system (6, 30). There are possibldirect testapparent conflict. While -hybrid system are designed to measure dircapacityionsmutant cellmeric protein, it is difficult to control fowild-type   lst1delta strain endogenous factor. Equally possible is that the interaction between ER and BRG-1 in yeast istarvationnd maprolonged incubationistiwaterechanisms by which heterologweakly buffered medium proton effluxreceptors is mediated in yeast (13). Thisdropbiliextracellular ph.y intrwild-type lst1delta strain no obvious SRC-1 family homolosharp declineeast genome an30-s lagest that the SRC-1 family members e~5 min as dyn4 bc platform upon coactivator cowild-type lst1delta cell may bqualitatively similar proton effluxtion ilst1delta cellnts global mechanism 5 minal activation that has beenrated bychangers such as the SRC-1 family in higher eukaryote. The findings presented in this report are consistent with a model in which BRG-1 is required for transcriptional activation by twild-type cell suggest that upon hormone binding, the ER associates with BRG-1, thereby recruiting BRreduced pma1p activityen-responsive chromatin. similar cdefecty using antibodies directed against SRC-1 and acetyl-lysine have suggested that the associawild-type lst1delta mutant cellve pimmunofluorescence   microscopyby the interaction of SRC-1 and CBP (Y. Shang et alpossible secondary effecton). Coactivation of estrogen signaling by members of the SRC-1 family or members of cbp family is alnuclear peripheryctions ocellular rimtudies also suggeslarge proportionch enhance the acetylation state withinpatternit corresponding enhancement of BRG-1-medsurface localizationtrogen signaling. enhancement may be accounted for by increased access of BRG-1 to acetylated chromatin, which in turn might render an acetylated nucleosome a better substrate for the remodelingcell fractionation. Additionally, the presence of a bromodomain at c terminus of BRsucrose density gradientr conditions of enhanceder plasma membranesociate via recently idbasisintebuoyant densitydomain acetyl-lysine. Such mechanisms may acdense   fractionorative effect of BRpeakd factors that promote histone acetylationgpi-linked plasma membrane proteinignaling. Figures and tables contrast required total   pma1p and for coactivation by SRC-1 and CBP. The BRG-1- and Brm-deficiengas1p proteincinommajoritywas plated at 50,000 cells per well in 24-well dishes and transfected using FuGene according to the manufacturers protocol. All samples were similarly processed, and datdifferent   densityld of inductiaccumulationll dpma1p segregates   er membrane estrosubdomainndenrelatively high low   density(a) ER signaling is repressed in SW-13 cells ier peake ofhigher densitypression of BRG-1 elicited a sevenfold induction in response to estrogen. This induction was dependent similar increaseact af-2. b brgportioned coactivation of er transcriptional activitypma1dependent on atpase activity of BRG-1. Transient tranfact assays in sw-13 cells demonstrate that a point mutation in Bdeletiont abolishes ATP binding also abolishes BRG-1-mediated coactivation of ER signaling. c overexpressabsence1 ingeneral protein secretion defectR signaling but can enhance BRG-1-medinormal growth of estrogen signaling, suggesting that SRC-1 activdoubling timetionalst1delta wild-typeof CB1.75 hs to coactivate ER sinormal rate13 cexpansioncan enhance BRG-1-mediated cspecific teststrogen signaling, sugggolgi transports pulse- chase experiment functional BRG-1. All experiments werematurationut in triplicate; ecoresglycosylated er   formrd egolgihe mean. brg-1formcidelay ER invertase transport both hormone and AF-2 dependent. (a) MCF-7 cells were metabolically labeled, and crude-cell lysate was prepared. wild-type wt gst and er hbd fusion protein depicted were immcarboxypeptidase yhione-er formarose (gsh-ag) and vacuolar formty menzymen the presence and absence of estrogen, to enrich for factpossibilityradiolabeled lysate. Retained fractions were boiled in an Ssubtle defectfer, and the eluted fraction was diluted and subjected to immunoprecipitation (ip) with mouse mosec24-1 sec31-2 mutant cell hsrsemipermissive temperatureacti28 c.ere resolved by SDS-PAGE on a 7.5% gel and detected by radiofluorography. size are indicatimein kiloda2.9 h (b) similar gst2.4 hdown-reimmunoprecipitation ex1.7 hent were done using rabbit polyclonal raised against hBRG-1perinuclear   regionirectly with CBP but not with BRG-1. flag-tagged cbp and flag-tagged brg-1 partial defectin bcopii   functionm and purified byextensive accumulationgraphy. Both proteins were resolved by SDS-PAGE on a 7.5% gel, transferred to solid support, and probed with 32p-gst-src-1(381-copii gene mutationrn assay. rrasv, recognition sequence for heart muscle kinase. BRG-1 binds pma1p exittaining chromatin fragment in a hormone-dependent mannercargo   proteine deprived of estrogen for 72 h andintracellular distributionnM 17beta-eepitope-tagged derivativentrol. At 45 min posttreatment, cells were ha10-amino acid ha 1% fonh2 terminuslting in theha-tagged lst1chromatin-associated factor to DNA. fixed cell were sonicated, and fragmsectoring assayas subjected to immunoprecipitation with an antibody directed against BRG-1. antibody-antigen complex were captured on protein A-linked Sepharose and washed extensively. The retained fraction were incubated at 60 c overnight, resulting in libesignal retained DNA fragments. Theseuntagged lst1pe purified and suorigin PCRstaining   pattern the presence or absence of indicated promoter region. nt, nucleotides. er marker kar2pon of chromatisubtle differencedirected against acetylhistone indicakar2phat estrogen-responsive genes undergo ennuclear   peripheryresponse tlst1p-ha stainingent. punctate appearance as above, using antibody directed acetylated hiparticular region-h; Upstate Biotechnologyweak   punctate stainingid receptor beta. BRG-1-mcell bodyivation of estrogen signaling is positively and negatcell peripherythe acetylation state of a cell. Transient transfection assays wsubcellular fractionationly as for Fig. 1. (a) Inhibition of HDAC activity dramatically augments BRG-1-mediacell lysate of estrogen signadifferential centrifugationn of SW-13 cells was performed as described in text, and cells were treated with a vehicle soluble marker protein   gdh2piol e2), 150 nM TSA, or 10 nM 17beta-estradiol plus 150 nM TSA (Both). b overexpression of complete cell lysisrg-1-mediated activation of ER signalilst1p proteincooperasedimenting fraction1 activity observchemical   treatmentperiments were carried out in triplicate; 50,000 g.   incubationthe standard error of the mean. reference characterization of proximal estrogen-responsive elemnaclof human cathepsin d gene The histone acetylase PCAF is nuclear receptor coactivator ch9 batipartial dissociationmilar motor, ulterior motive nuclear receptor cagentctr is novel histone acetyperipheral membrane proteinimeric activation complex with p/caf and lst1p binds sec23p sec24pl co-repressor that interacts with nuclear horm400-kd   complexn homologue of Saccharomyces cerevisiae SWI2/SNF2 and drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and rpotential interactionstructure and ligand of histone acetyltransferase bromodomain retinoblastoma protein and brg1 form a complex and cooperate to induce cell cycle arrest coregulatory protein in nuclear hormone receptor action unliganded thyactivationne rlacz reporter genemation of functbeta-galactosidasen complex: implicatlst1 sec23 fusionhuman histone acetyltransferase gcn5 exists in stable macromollevelmplex lacking adapter ada2 chromatin remodelling by the glucocorticoid receptor requires brg1 complex genetic dissection of signaling domain of mammalian steroid receptor in yeast Aberrant recruitment of nuclear receptor corepressor-histoneyeast cell extractx coding sequenced lelst-1-hausion partner ETO Chromatin structure of the regulatory regionspgal1   promoterin d gene in hormone-dependent -independent breast cancer cell line nuclear receptor coaintracellular   membranetion in10 b multisubunit complexes to acetylate nucleosomal sec23p gst-lst1p-ha ada complex and saga spt/ada complex core smrt corprotein complexcontaining hdac3 andsalt extractt protein linked deafness estrogen receptor-associated protein: possfinal   concentrationne-induced transcription p300 is a component of estrogen receptor coactivatorglutathione   sepharose beadprotein that serves as a transcriptionalgst-lst1ptor in yeast forcontrol extractding domains of steroid receptor ligand-independent repression by thyroid hormone receptor mediated by nuclear receptor co-repressor nuclear receptor corepressor partner with class ii histone deacetylase in sin3-independent repression pathway ligand-dependent interaction between the estrogen receptor and trecovery homologues of SWI2/SNF2 Steroid receptor induction of gene transclst1p/sec23p complexel cbp integrator complex mediates transcriptional activation and ap-1 inhibition by nuclear receptors BRG1 contains conserved domain of swi2/snf2 family necessary for normal mitotic growth and transcription nuclear receptor, their coactivators and modulation of transcription atp-dependent chromatin remodelling:er membrane protein job A human homologue of Sacchamaterialcerevisiae snf2/swi2 and drosophila brm gene 60 sucrose transcriptional activation by the glucocorticoid receptor compositgst- lst1p-haC mediates chromatin-directed trer resident   membrane proteinscriptional coactivators p300 and CBP are45 sucroseetyltransferase sequence and characterization of a coactivator for steroid hormone receptor superfamily Role of chromatin structure in the regulation of transcription by rna polymerase ii hmg17 is chromatin-specific transcripstrain overexpressing sec23pses efficiency of t< 20cription initiation mutations in chromatin component suppress a defect of gcn5 protein in Saccharomyces cerevisiae characterization of yeast swi1 swi2 swi3 gene, which encode global activoverexpressed proteinel protein complex that interacts with vitamin d3 receptor in ligand-dependent manner and enhances vdr transactivation in cell-free system essential functional interaction of saga, a Saccharomyces cerevisiae colst1p/ sec23p complexn5 proteins, with snf/swi srb/mediscussionplex swi-snf complex participation in trasynthetic-lethal   genetic interactionequent to activator binding role for retinoblastoma protein in psubsequent genetic testnal activation by glucocorticoid receptor steroid receptor coactivator-1 is histone acetyltransferase tram-1, a novel 16sec12   sec13hormone receptor activator molecule, exhibits distinct property from steroid receptor coactivator-1 The transcriptional co-activator p/cip binds CBP and mediates nuclear-receptor function nuclear receptor coactivator coactivator and corepressor complexes in nucprocessor function nuclear hormone receptexpectationr SRC-1 is specific target of p300 cloning and characterizatproductrefollowing observationonent of nuclear hormone receptor reprcopii-like vesicle coatlasmic reticulumpla90-kd proteinane h(+)-atpaseplasma mecopii-coat subunit sec24pxpressionreduced expressio23 amino acid identityn1upstentire lengthcoii lst1pta-binding proteinrna polymerase ii ctdmultisubunit complexpma2 atpaseintracellularcell   fractionationtiiii lst1p manipulationyeast host   strainshuffle vectorsystemcopii-hybrid interaction2k+-kaffinity purificationudding yeast plasma membraneyeast coat piv assemblysionsec23p-lst1p complexamino-acid permeasecontrolmembrane proteinvitro plst1p   sec23pnyeast cytosolic sec13p complexer-vesicle proteinaxl2psite-directed mutagenesis transportactive siteyeast   plasma-membrane h+-atpase gene growth control strengthdominant lethal mutationcharacterizationsaccharomyces cerevisiaegas1 protesecretionosphmarker proteingdeterminantnitrogen pronounced accumulationamate dehydrogenasechemically-defselective defectotropic plgolgi traffic atpase mutationpurified coat proteinyeast copii-coated vesicle formationnovel cspecialized copii coat complexser-derived transportvitro copii-cargo interaction direct proteinpackaginger resident membrphenotypic hallmarkry padeficiencyacid permeasetransport vesicle formationsecgenepolylinker distinct setvacuolar formationemblmultibudded cellyces cdecreased rateble markeryeast endoplasmic   reticulum proteinyeast vacuolar h+-atpase complex vma12 encodenovel endoplasmic reticulum- associated proteinvacuolar h+-atpase complex vma22pdi-lysine motifyeast membrane proteinendoplasmic reticulum immunofluorescenceing protein trdensity cell fractionationovel 105-kda protein functionmating-type switching sec23ppractical usesaccharomyces ce37 c. exportma membrane h+-atpase genecdk2 dominant lethal mutationhuman g1 s phase protein phosphatasednasec16p fused protein domainadjacent regionbindvesicblockion pma1p   transportit interactionyeast e~35 rettotal   pma1p fractionateicle coat proteinvesicle budding mutation yeast sec16genesuppressorfidelitycomplementationisolationmultifunctional cdna expression vectorplshiftatpase gene lambda yesyeast saccharomyces cerevisiaeplasma membrane h(+)-atpasenull muburdentional complementationphysiological consequencegalactose-dependent promoteratpase genepromotervacuole replacementmutant atpaseast1preventsnovel geneintracellular transport tarcompensationorylationyeast plasma membraneyeast secretory pathway maturationorganelexpressiony plasma membrane protein-htransport defectent localizationendoplasmic reticulum analyzing protein-protein interactionsec proteinmembrane coater copiitransport vesicleguanine-nucleotide- exchange factorcopii budding machinerygolgi-like vesicle cargo selectionlumenal guanosine dipgeneral assaydp mantrafficking defectsylationminimal medium ypd rich medium refidentical ratesec-thirteen pma1p plasma membrane proton-atpase smmpaper dapi 4,6-diamidino-2-phenylindole gst glutathione s-transferase haabbreviationsec23p prplasma   membranesionttotal cleared lysatealiqessential plasma   membrane proteinctionpellet p2   mincell debriscell   lysate-sixthlaneglutathione beadsepharose beadlst1p/-sec23p complexaffidifferenceionnmol/mg x min         lst1p/-sec23p complgeneral defectnt tprotein transport machinerytransparticular concernion domainanti-ha antibody50,000 g.   lst1p-hab cell lysates150,000 g supernatant fractionp150,000 g pellet fractionsequential centrifugation step1 mumdicdifferential interfestandard marker proteini stainingnusignificant   effectnjugated anti-rabbit antibodyantimouseleu2-3,112 ura3immunolocalizationcore glycopii componentionlabeled extractunlabeled methionineexcesscysteinesec12-4 cky541 strainconstitutive ptpi1-suc2 fusioncky542evidencessaygolgi compartment markergdpasesec61p ersemipermissive   temperaturekerrelative level20-60 sucrosedensity gradient3 h. cell lysaessential proteinratedecrease37 c.   cellsproton   pumpingb reducedlst1 mutantmumlst1delta culturetype comprisnonessential proteinbarplasmid pkr34genotypically wild-type cellmifactornelvector prs316montagepanelnucspecific membrane   proteinted secondary   antibodyanumberpurified anti-pma1shr3 genemaldehydeer resident protein mg galactose solutioncm2800 celltransformantplasmid pkr41prs425-lst1wild-type strainccentromeric plasmidcky552lst1delta::leu2   strainb2 d.acidic ypd mediumfunctional relkuehnshipoalrall amino acid   ier proteined linesolvma21peidentivma22p4 sequencendordernd   nd growthnd   nd   lstintegral membrane subunitlst1lst sec11   latpase13hill    gene   numbersecond   sjacksonaringstevensng132 nonsectoring cmutational studynonsectoring solismall er   membrane proteincerv14pde2 ade3ade2 ade3 leu2 ura3 sec13colony-sectplasma membrane proteinhamatalst1delta::leu2 lpowers112 ura3pnv31     cky542matasec12pkr17ha     cky535matalst8-1 sec13-1::[sec13 ura352     cky436alphacky78cky58   matalpha sec18-1 his4sec17-1 his4-619 ura3-52   kaiser lab collectioncky54   matalphakaiser lab collectioquestionalpha sec13-1 his4-619 ura3-52   kaiser lab collection   cky50   matalpha svesicular transportmatalrespective cargo moleculeemiscornell universitymataade2-101 ade3-24 leu2-3,112 ura3-52   influence proteinnce    cuy563figuredifferent quality control mechanismpacitycopii-like vesiclewide vcomponentp sec24p homologuepossible combinationmixed coatcargodirect roleferent   determinantyhr035wuncharacterizefoldingading framesaccharomyces genodominant pma1 mutationily membergreater combinatormutant wild-type   pma1pl hoproliferated ermharrisnsec23p/ sec24p complexclasssec23p/lst1p complexcoatsec24p howild-type pma1pcationdirect bindinginterestsepma2/lisoformex   efficientplant plasma membrane proton-atpasepurified cytosolic componentvvillalbading reactionfuture experimentspecific akerchoveonvesicle fractionhigh bacproposalle lst1p-haspecial factore quantityvitro   budding reactioner-derived vesicleinformatmannertionplanemembrrequirementteincshr3pespecific binding sitesec23p/sec24p complexesupplyental systemconcspecific rolemembranemammalian sec23p/sec24pmammalian microsomesparallel experimentamino acid permeasepurified copidominant formderived microsofuturetudymechanismlst1p overexpresidea