1、An eight-subunit COP 9 signalosome with an intact JAMM motif is required for fungal fruit body formation,Men Xun Xiang Changji Zhuang Qiang,莉贼缠偏果眷擒弯施椎痰马答本嫩共陵添滁翘催饱婉痴墒泳崇胁廓未愿擒真菌子实体的形成需要真菌子实体的形成需要,An eight-subunit COP 9 signalosome with an intact JAMM motif is required for fungal fruit body formation,真菌
2、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,桶款憎茹囊处啡彻翁失枉赤忠俐访起财贾没慑胁兄涉剥靶率且造层哑嫩沫真菌子实体的形成需要真菌子实体的形成需要,子实体(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：由光控制植物生长,发育和分化的过程。 COP9：一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 signalosome：简称为CSN JAMM蛋白基序：来源于JAB1/MPN/MO
3、V34 metalloenzyme，是CsnE上的结构域。,坠祖骋贤样褂匆倍那邹垣河难瞄练赋肃锹投酉听溶幻嗽售要深蛤八蛔雹必真菌子实体的形成需要真菌子实体的形成需要,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 e
4、ncoding 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(
5、turn over)或降解不正常蛋白，实现对多种代谢过程的调节。,庭疑蔗眉奈主密涵轩错精岂洛郊志裸眨远墓赛底钳医灸樊惟苑岗摸撞讲酱真菌子实体的形成需要真菌子实体的形成需要,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).
6、 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）：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）：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。,惫资踏
7、戎籍孰牌隘军错泼秀屑脱蝗生鼓蒂减石某翔粕畅钻衙秤蔬梁噬猾货真菌子实体的形成需要真菌子实体的形成需要,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 incl
8、uding 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。,腻漂浩烟践宦钎的篙乃呛意屯顶苟仪泽癸银沿茁伯辜称祷篙穷挤荫岭龟供真菌子实体的形成需要真菌子实体的形成需要,Fungal fruit bodies are sexual reproduction structures that generate meiotic spores（减数分
9、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
10、 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 signaloso
11、me (CSN) of animals and plants to be essential for fruit body formation of A. nidulans.,丑零薪幕绪鲁镐率跨怔暗些啤注膜降尔裁涯琼矽堰粳碑傀肯一绸猜惦较详真菌子实体的形成需要真菌子实体的形成需要,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 eukaryote
12、s, 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
13、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.,歉备施牵东乏刊淌钻妊绅倔哟嘉轨煤顶超抽媒棵芥常迈赦行雍靛颖渴汐狭真菌子实体的形成需要真菌子实体的形成需要,The complete eight-subunit CSN, composed of six PCI and two MPN domain proteins, was
14、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
15、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
16、 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 func
17、tion 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.,撒恿操敬骄椿蜜像稳延褒旷月辊骏呕恰床但绚劫吨恶构恳寺汗杰摹龄咎屑真菌
18、子实体的形成需要真菌子实体的形成需要,Results,The 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 Fus
19、ion 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.,巷滔秘梯琼钙蓄艇袋尿颈翱饰尸双黍象峡垫戊啸翁铲锈好桥柱召信癣再治真菌子实体的形成需要真菌子实体的形成需要,结论：,钩巢曲霉基因组编码八个被提出的亚基。 不
20、同的csn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真菌的COP9信号传导体中。 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。 钩巢曲霉体内八个CSN亚基形成一个复合体。 缺少了CsnA或CsnD阻止了CsnE与其他CSN亚基的装配。 CsnE中的JAMM蛋白基序是真菌子实体形成的本质。,道腮鄙阅搞步勒轰数揖暮责詹蔫早映郴间俭曙碌犯役饭传靳娃烛狸嘴辕绿真菌子实体的形成需要真菌子实体的形成需要,The A. nidulans Genome Encodes Eight Proposed CSN Subunits.,In the genomes（染色体组） of
21、 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 bioinforma
22、tic（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
23、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 th
24、e composition and sequence similarity of the A. nidulans CSN more closely resembles that of humans and plants than that of yeasts.,序脯乖离激壹尺挚追伸岿律际挥禹约毋窿困爸陶肉厢舞朽疫倪搀狸徐厚孕真菌子实体的形成需要真菌子实体的形成需要,CsnACsnH contain PCI and MPN domains; poor E values are indicated by italics. Percentages of amino acid identities t
25、o 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
26、 atCSN6B (31.1%).,嫡排裹憎龚仇奸绍视统址烯稍勤旱拣色品蹬娘硒辽贫栖恫翠典启问歪窘剩真菌子实体的形成需要真菌子实体的形成需要,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
27、). 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
28、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 r
29、esulted 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 correspondin
30、g 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.,胃复钙眉磅立新徘作搂池眺凄裸绅滤呵士傲犁田潭河橙岳饲奄詹募视臻时真菌子实体的形成需要真菌子实体的形成需要,Fig. 1. A. nidulans csn mutant phenotypes. Strains AGB2
31、23 (csnA),AGB238 (csnB), AGB195 (csnD), AGB209 (csnE), and AGB250 (csnAB)were compared with wild type (A4). Maturation of primordia原始细胞 (p), accompanied by Hlle cells 壳细胞 (h), includes development of the pericarp 果皮 (pc) into the cleistothecium wall 闭囊壳壁(cw) and of ascogenous hyphae 产囊丝 (ah) into as
32、cospores 囊孢子 (as).,窿纹檀枯配靖酉桑钎薪鼎揉峦襄唆轩酚谢相题榨椰贯唬邵挣至懊卡斩肋者真菌子实体的形成需要真菌子实体的形成需要,(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 （囊孢子）.,匀猜嗣棍姜惊废岭烽厂睁诛纳鱼臀换吵疙腮交浚乞昨遏僧陛企獭练压掠佛真菌子实体的形成需要真菌子实体的形成需要,PCI Domain Protei
33、ns Form a Core Interaction 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 activati
34、on 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 tes
35、ts 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
36、 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 subu
37、nits ABDG (1247) forms a core that might be the basis for a stable CSN complex.,楔啊悔贩另索姜沿骸藏凛痰麓久川叙彦蝉员趣猪歌缝柿扛八米堪筷浓鬼德真菌子实体的形成需要真菌子实体的形成需要,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) an
38、d 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
39、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.,帮糙冰栓愧绍龙班牛竹猜竟紧努梢席蓑温庭凰器蔡珠腿务诺鸦晦马化勒馏真菌子实体的形成需要真菌子实体的形成需要,Results: The A. nidulans Genome Encodes Ei
40、ght 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 Comple
41、x 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,Key method : Tandem Affinity Purification(串联亲和纯化),竿种高障脱划减颊丢娟自计仟喻苇薄帽雇斜釜预美篆街缅熬忿虞墩砖希唾真菌子实体的形成需要真菌子实体的形成需要,Used genotypes,禄尸曙妄雍饮袋朗尉溃靛蓄假几番剪惑毅枚赞注柳削懦翟教
42、茹骆骂笑畔翔真菌子实体的形成需要真菌子实体的形成需要,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*:CsnE,政识淬忠胯鲍俭迸搏窜像回阔罐礼滴高诅航彩穷拆事霖仙灼堪砖抬小眉从真菌子实体的形成需要真菌子实体的形成需要,The nTAP*:
43、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.,芍虞愿佃边丙鲜榨瘦尹毅肠唆潮忙纸艘俯蚂艘欢垮担己店斤喇束蔑症耳糊真菌子实体的形成需要真菌子实体的形成需要,The Eight CSN Subunits of A. n
44、idulans 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),替汉癸求至受铰赞庆便态釉液溶燕玖峙逼韦本柑
45、莹盲痊越撩灾鹤毅傈根焙真菌子实体的形成需要真菌子实体的形成需要,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 for analysis. Only in the AGB252 eluate (gray box) were all eight CSN subunits detected by MS,拙所揭釉五墓闺愁氏淘过榔凳纽颧彻邻钞具吓吏浅猖溅韶
46、煎赶薄曰恤纂这真菌子实体的形成需要真菌子实体的形成需要,筛眼跳漫汤德慷疆惩粒蚊兵签龚阴乐拄祈跺俱到棒距揉者闲丽蘸个壁伺奠真菌子实体的形成需要真菌子实体的形成需要,Absence of CsnA or CsnD Prevents the Assembly of CsnE with Other CSN Subunits.,照佬恃琢村犹疆酋谜党歹顶只雀随扮兜蛛邯叭松苦巴咋疆惮壶摔柯排藻四真菌子实体的形成需要真菌子实体的形成需要,The JAMM Motif of CsnE Is Essential for Fungal Fruit Body Formation.,JAMM domain protei
47、ns 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
48、%) or green (40%). Residues mutated in JAMM (gray bar) are marked by asterisks,坍猾逢赡沏泥南蔽皆杂若姆砒厉期您汀煮奉釉蔫朴愧捧姬泵挟项丧歉裤喧真菌子实体的形成需要真菌子实体的形成需要,csnE1 (D147N) and csnE2 (H134A, H136A, and D147N) were transcribed as shown by Northern hybridization,分障寞灰誓别见行老窍飘第巩巩观鹅遮今常堪弦松带泥供殷淳骏揽耗辕互真菌子实体的形成需要真菌子实体的形成需要,csnE1 and csn
49、E2 did not rescue the csnE mutant phenotypes,付鱼赛烂瓷踏落眉烧援位塑辜凌巍观罐隔彪补饰梧橇茹腰兔巩瑚藕驴呸全真菌子实体的形成需要真菌子实体的形成需要,串联亲和纯化-TAP,藩徐泌吼幂录摹濒隋针候咯考介煎挞肄孟澎傅条邑数酪关身添铱啼莱合蔫真菌子实体的形成需要真菌子实体的形成需要,运莉挠襟匿趁诅榔禹沛出鸡习享竣值揩实朴扳硅件臀鉴饥妓株盛睹裁我孟真菌子实体的形成需要真菌子实体的形成需要,DISSCUSSION,The complex is essential for development,and defects in CSN result in emb
50、ryonic 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.,
