真菌子实体的形成需要.ppt

An eight-subunit COP 9 signalosome with an intact JAMM motif is required for fungal fruit body formationMen Xun Xiang Changji Zhuang QiangAn eight-subunit COP 9 signalosomeAn eight-subunit COP 9 signalosome with an intact JAMM motifwith an intact JAMM motif is required foris required for fungal fruit body formationfungal fruit body formation真菌子实体的形成需要一个八个亚基的COP9（组成型的光形态建成9）信号传导体带有一个完整的JAMM蛋白基序子实体子实体(fruitingbody)(fruitingbody)：当营养生活进行到一定时期时，真菌就开始转入繁殖阶段，形成的各种繁殖体。光形态建成：光形态建成：由光控制植物生长,发育和分化的过程。COP9COP9：一个光调控植物发育的分子开关。信号传导体：一类通过与细胞受体结合发挥作用，将信息传导到细胞核中，激活特定基因的物质。COP9 signalosomeCOP9 signalosome：简称为CSNJAMMJAMM蛋白基序：蛋白基序：来源于JAB1/MPN/MOV34 metalloenzyme，是CsnE上的结构域。Fruit body formation in filamentous fungi（丝状真菌）is a complex and yet hardly understood process.We show here that protein turn over（周转）control is crucial for Aspergillus nidulans（钩巢曲霉）development.Deletion of genes encoding COP9 signalosome(CSN)subunits 1,2,4,or 5 resulted in identical blocks in fruit body formation.The CSN multiprotein complex（多蛋白复合体）controls ubiquitin-dependent protein degradation（泛肽依赖性蛋白降解）in eukaryotes.ubiquitin-dependent protein degradation（泛肽依赖性蛋白降解）：有高度选择性的蛋白质降解途径。它通过调节功能蛋白质的周转(turn over)或降解不正常蛋白，实现对多种代谢过程的调节。Six CSN subunits interacted in a yeast two-hybrid analysis（酵母双杂交分析）,and the complete eight-subunit CSN was recruited（使恢复）by a functional tandem affinity purification（串联亲和纯化）tag（标记）fusion（融合）of subunit 5(CsnE).The tagged CsnE was unable to recruit any CSN subunit in a strain deleted for subunit 1 or subunit 4.yeast two-hybrid analysis（酵母双杂交分析）：（酵母双杂交分析）：通过报告基因的表达产物敏感地检测得到蛋白质之间微弱的、瞬间的作用，它是一种具有很高灵敏度的研究蛋白质之间关系的技术。tandem affinity purification（串联亲和纯化）：（串联亲和纯化）：是一种能快速研究体内蛋白质相互作用的新技术，经过两步特异性亲和纯化,可快速得到生理条件下与靶蛋白质存在真实相互作用的蛋白质。Mutations（突变）in the JAMM metalloprotease（金属蛋白酶）core of CsnE resulted in mutant phenotypes（显型）identical to those of csn deletion strains（缺失突变株系）.CsnE中心的JAMM金属蛋白酶发生突变导致与csn缺失突变株系同样的突变显型。（csn表示基因）We propose that a correctly assembled（装配）CSN including a functional JAMM links protein turnover to fungal sexual development.我们认为包括一个功能JAMM的CSN的正确装配联系到真菌有性繁殖的蛋白周转。metalloprotease（金属蛋白酶）：活性中心中含有金属离子的蛋白酶的总称。Fungal fruit bodies are sexual reproduction structures that generate meiotic spores（减数分裂的孢子）.The model mold Aspergillus nidulans（钩巢曲霉）develops a closed spherical fruit body（封闭的球状子实体）(cleistothecium（闭囊壳）)including different tissue types:Hlle cells surround and nurse the growing cleistothecium,pericarp cells develop the protecting wall,and inner ascogenous（产囊）cells mature into sexual spores.Massive reconstruction of vegetative hyphae（菌丝）is required to build the complex three-dimensional fruit body.The regulation of this development is hardly understood in any fungus.A genetic screen recently identified csnD and csnE resembling genes for subunits of the COP9 signalosome(CSN)of animals and plants to be essential for fruit body formation of A.nidulans.CSN is a multiprotein complex（多蛋白复合体）composed of proteins containing PCI and MPN interaction domains.Csn5/Jab1 is the only subunit conserved in all eukaryotes,and it carries an MPN+domain containing the JAMM motif conferring metalloprotease(deneddylation)activity.CSN controls by its MPN+domain the activity of cullin-RING E3 ligases（连接酶）by cleaving the ubiquitin-like protein Nedd8/Rub1 from the cullin（滞蛋白）.Neddylated E3 ubiquitin ligases are key mediators of posttranslational（转译后）labeling（标记）of proteins for the proteasome（蛋白酶体）.The CSN thus controls eukaryotic ubiquitin-dependent protein degradation.The complete eight-subunit CSN,composed of six PCI and two MPN domain proteins,was described for eukaryotes as humans,mice,plants,flies,and Dictyostelium.In fungi,definitive evidence for an eight-subunit CSN is lacking so far.CSN complex purification from Neurospora crassa（粗糙脉孢霉）revealed subunits 17,but subunit 8 was identified neither in the purification experiment nor in the genome sequence by bioinformatics means.In fission yeast subunits 6 and 8 have not been identified yet,and in the CSN-related complex of Saccharomyces cerevisiae only subunit Csn5(yeast Rri1p)is well conserved.The fungal CSN complexes known to date are not essential for viability but are involved in cellular processes like circadian clock（生物钟）regulation,cell cycle progression,and the pheromone（信息素）response.In contrast,CSN dysfunction leads to severe defects in cell proliferation of Dictyostelium discoideum and embryonic lethality of mice,plants,and flies,indicating a function of CSN in regulation of basal developmental processes.Here we demonstrate the existence of the first complete fungal eight-subunit CSN.In A.nidulans,CSN complex formation and a functional JAMM deneddylase motif are critical for development of fruit bodies.ResultsThe A.nidulans Genome Encodes Eight Proposed CSN Subunits.Different csn Strains Share the Same Phenotypes.PCI Domain Proteins Form a Core Interaction Cluster in the Fungal CSN.An Adapted Tandem Affinity Purification(TAP)Tag Enables Expression of a Functional CsnE Fusion Protein.The Eight CSN Subunits of A.nidulans Form a Complex in Vivo.Absence of CsnA or CsnD Prevents the Assembly of CsnE with Other CSN Subunits.The JAMM Motif of CsnE Is Essential for Fungal Fruit Body Formation.结论：钩巢曲霉基因组编码八个被提出的亚基。钩巢曲霉基因组编码八个被提出的亚基。不同的不同的csn株系表现着相同的显型。株系表现着相同的显型。PCI功能结构域蛋白源自一个核心交感集群在功能结构域蛋白源自一个核心交感集群在真菌的真菌的COP9信号传导体中。信号传导体中。采用合适的串联亲和纯化能够使有功能的采用合适的串联亲和纯化能够使有功能的CsnE融合蛋白表达。融合蛋白表达。钩巢曲霉体内八个钩巢曲霉体内八个CSN亚基形成一个复合体。亚基形成一个复合体。缺少了缺少了CsnA或或CsnD阻止了阻止了CsnE与其他与其他CSN亚亚基的装配。基的装配。CsnE中的中的JAMM蛋白基序是真菌子实体形成蛋白基序是真菌子实体形成的本质。的本质。The The A.nidulansA.nidulans Genome Encodes Eight Proposed Genome Encodes Eight Proposed CSN Subunits.CSN Subunits.In the genomes（染色体组）of three aspergilli we identified genes for 18 PCI,five MPN,and three MPN+domain proteins.Surprisingly,we found eight subunits for CSN,which were designated csnAH(Table 1),and which characterize Aspergillus（曲霉菌）as the first fungus where all eight subunits are clearly recognizable by bioinformatic（生物信息学）means on the genome level.The other genes encode putative subunits of the LID of the proteasome（蛋白酶体）,of translation factor eIF3,the AMSH-like ubiquitin isopeptidase,and the Prp8-like splicing factor see supporting information(SI)Table 3).Only A.nidulans csnD,csnE,and csnG/acoB had been previously described.We amplified all csn cDNAs from a vegetative cDNA library to verify that all eight genes are transcribed(data not shown).Intron positions and lengths were determined by comparison with the corresponding genomic sequences(Table 1).In silico analyses revealed that the composition and sequence similarity of the A.nidulans CSN more closely resembles that of humans and plants than that of yeasts.CsnACsnH contain PCI and MPN domains;poor E values are indicated by italics.Percentages of amino acid identities to the sequences of Homo sapiens(hs),Arabidopsis thaliana(at),Schizosaccharomyces pombe(sp),and S.cerevisiae(sc)(26)are given(sequence IDs are in SI Table 3).Positions of PCI/MPN(blue)and introns(red)within coding regions are indicated.*Isoforms:hsCSN7B(27.8%),atCSN5B(53.2%),and atCSN6B(31.1%).Different csn Strains Share the Same Phenotypes.Different csn Strains Share the Same Phenotypes.We found previously that A.nidulans strains deleted for csnD(AGB195)or csnE(AGB209)stop fruit body formation at the level of primordia and produce an aberrant red dye (Fig.1A).To survey whether the CSN mutant phenotypes are restricted to these subunits,we used the csnA strain AGB223 and deleted the complete coding sequences of csnB(strain AGB238).Additionally,we constructed a double deletion strain lacking subunits csnA and csnB(strain AGB250).The five different csn deletion strains were able to initiate fruit body formation by production of Hlle cells and primordia like the wild-type(Fig.1B).However,further maturation of primordia was aborted,and mature cleistothecia were never observed.Therefore,like in higher eukaryotes,deletion of any csn gene resulted in early developmental defects.Additionally,the csn strains produced an aberrant red color within distinct hyphae after 48 h of growth on an airmedium interface(data not shown).The mutant phenotypes were complemented in the csn strains(Table 2)by ectopic integration of the corresponding csn wildtype alleles,respectively(data not shown).These data suggest that the integrity of the fungal CSN complex is more important for function than unique roles of individual subunits.Fig.1.A.nidulans csn mutant phenotypes.Strains AGB223(csnA),AGB238(csnB),AGB195(csnD),AGB209(csnE),and AGB250(csnAB)were compared with wild type(A4).(A)Maturation of primordia原始细胞原始细胞(p),accompanied by Hlle cells 壳细胞壳细胞(h),includes development of the pericarp 果皮果皮(pc)into the cleistothecium wall 闭囊壳壁闭囊壳壁(cw)and of ascogenous hyphae 产囊丝产囊丝(ah)(A)into ascospores 囊孢子囊孢子(as).(B)csn deletion strains developed sexual cell types on an air-limited liquid medium surface(96 h at 37C),but not mature fruit bodies and ascospores（囊（囊孢孢子）子）.PCI Domain Proteins Form a Core Interaction PCI Domain Proteins Form a Core Interaction Cluster in the Fungal CSN.Cluster in the Fungal CSN.CSN complex integrity is presumably mediated by subunit interactions based on the characteristic PCI and MPN motifs.Therefore,we analyzed binary protein interactions of CSN subunits.A.nidulans csn cDNAs were fused reciprocally to the activation domains and DNA binding domains(DBD)of the yeast two-hybrid plasmids pEG202 and pJG4-5.Interactions were tested by two reporter systems based on leucine prototrophy and galactosidase activity(Fig.2A).The CsnB:DBD construct conferred strong growth on its own;therefore,only the activity tests were taken into account for this construct(Fig.2B).Six proteinprotein interactions were shown by both complementary bait/prey pairs between CSN subunits AB,AD,BD,DG,EF,and FG,leading to a minimum binary interaction map(Fig.2C).Subunits CsnC and CsnH,showing least overall similarities and lowest e values for PCI domain prediction(Table 1),did not interact in this experimental setup,indicating that they might need other subunits or specific posttranslational modifications for stable interactions.Our results suggest that the interaction cluster between the four PCI domain subunits ABDG(1247)forms a core that might be the basis for a stable CSN complex.Fig.2.A.nidulans CSN yeast two-hybrid interactions.(A)Yeasts with all plasmid combinations of A.nidulans csn cDNAs(lanes AH)and empty DNA binding domain(d)and activation domain(a)control vectors(0)were viable on SC plates with leu.Interactions were monitored as leu prototrophy and-galactosidase activity.(B)Readouts of growth(red)and activity(blue)were evaluated as strong(bold+),weak(regular+),or absent(-),and combined readouts overall evaluated as positive were highlighted by gray boxes.(C)Arrows indicate a minimum binary interaction map,and solid lines indicate that both bait/prey combinations of a given protein pair were positive.Results:Results:The A.nidulans Genome Encodes Eight Proposed CSN SubunitsThe A.nidulans Genome Encodes Eight Proposed CSN SubunitsDifferent Different csn Strains Share the Same Phenotypescsn Strains Share the Same PhenotypesPCI Domain Proteins Form a Core Interaction Cluster in the Fungal CSNPCI Domain Proteins Form a Core Interaction Cluster in the Fungal CSNAn Adapted Tandem Affinity Purification(TAP)Tag Enables An Adapted Tandem Affinity Purification(TAP)Tag Enables Expression of a Functional CsnE Fusion Protein Expression of a Functional CsnE Fusion Protein The Eight CSN Subunits of A.nidulans Form a Complex in VivoThe Eight CSN Subunits of A.nidulans Form a Complex in VivoAbsence of CsnA or CsnD Prevents the Assembly of CsnE with Absence of CsnA or CsnD Prevents the Assembly of CsnE with Other CSN SubunitsOther CSN SubunitsThe JAMM Motif of CsnE Is Essential for Fungal Fruit Body The JAMM Motif of CsnE Is Essential for Fungal Fruit Body FormationFormationKey method:Tandem Affinity Purification(串联亲和纯化)Used genotypes An Adapted Tandem Affinity Purification(TAP)Tag Enables Expression of a Functional CsnE Fusion Protein Western experiments with anti-calmodulin and 20 g of protein crude extract show expression of nTAP*:CsnEThe nTAP*:csnE fusion of AGB252(csn)complements mutant phenotypes of AGB209.Deletion of csnA or csnD in AGB252 resulted in csn phenotypes in AGB253(A)and AGB254(D).nTAP*:csnE was analyzed in 20-h-old vegetative mycelia.The Eight CSN Subunits of A.nidulans Form a Complex in Vivo.nTAP*:CsnE enrichment from 150 mg of crude protein of AGB252 was monitored by silver stain(50)and Western experiments using anti-calmodulin:crude extract(1),flow-through after binding to IgG(2)and calmodulin(3)beads,and final eluate(4)Final eluates from 950 mg of crude proteins were separated on a gradient Final eluates from 950 mg of crude proteins were separated on a gradient SDS gel and stained by EZBlue,and the complete lane was cut into 18 pieces SDS gel and stained by EZBlue,and the complete lane was cut into 18 pieces for analysis.Only in the AGB252 eluate(gray box)were all eight CSN for analysis.Only in the AGB252 eluate(gray box)were all eight CSN subunits detected by MSsubunits detected by MSAbsence of CsnA or CsnD Prevents the Assembly of CsnE with Other CSN Subunits.The JAMM Motif of CsnE Is Essential for Fungal Fruit Body Formation.JAMM domain proteins CSN subunit 5,26S proteasome LID subunit RPN11/PSMD14,and AMSH of A.nidulans(an),H.sapiens(hs),A.thaliana(at),S.pombe(sp),or S.cerevisiae(sc)were aligned by ClustalW with the archaea JAMM protein AF2198.JAMM is highlighted in black,and additional conserved residues are red(100%)or green(40%).Residues mutated in JAMM(gray bar)are marked by asteriskscsnE1(DcsnE1(D147147N)and csnE2(HN)and csnE2(H134134A,HA,H136136A,and DA,and D147147N)were N)were transcribed as shown by Northern hybridizationtranscribed as shown by Northern hybridizationcsnE1 and csnE2 did not rescue the csnE mutant csnE1 and csnE2 did not rescue the csnE mutant phenotypesphenotypes串联亲和纯化串联亲和纯化-TAPDISSCUSSIONThe complex is essential for development,and defects in CSN result in embryonic death of these multicellular organisms The fifth subunit(CsnE/Jab1)of CSN includes the only known enzyme activity of the complex,a metalloprotease that binds zinc ions by its conserved JAMM motif.In the fungal system,CsnE/Jab1 is unable to recruit any other CSN subunit and is less stable when CSN subunits 1 or 4 are absent.This argues against stable CsnE subcomplexes in fungi,although it does not exclude the formation of CsnE oligomers or unstable CsnE-containing subcomplexes.The A.nidulans genome reveals that all major genes for putative substrates of the CSN deneddylase activity are present.These include a proposed Nedd8 encoding sequence and the three putative cullins Cul1,Cul3,und Cul4.Cullins are part of cullin-RING E3 ligase cores and are known to assemble with various specific substrate-adaptors like F-box proteinsthe control of specific E3 ubiquitin ligases by CSN seems to be less critical for hyphal growth itself t