Global identifications and analyses of the proteins that interact with chitin synthases of filamentous fungi.

与丝状真菌几丁质合酶相互作用的蛋白质的整体鉴定和分析。

• 批准号: 15380057
• 负责人: HORIUCHI Hiroyuki
• 金额: $ 9.98万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15380057/
• 关键词: chitin synthase; Aspergillus; myosin; septum; actin

We have cloned five chitin synthase genes from filamentous fungus Aspergillus nidulans and investigated their functions. In this project, we constructed the strains that produced functional epitope-tagged chitin synthases to clarify the localization of their gene products. HA epitope-tagged ChsA (HA-ChsA) mainly localized to forming septa, while GFP or FLAG epitope-tagged ChsB (GFP-ChsB or FLAG-ChsB) and FLAG epitope-tagged ChsC (FLAG-ChsC) mainly localized to forming septa and tips of hyphae. We next constructed the strain that produced both HA-ChsA and FLAG-ChsC and investigated their co-localization. The localizations of HA-ChsA and FLAG-ChsC were partly overlapped. Western blot analyses against the cell lysates of the strains expressing HA-ChsA, FLAG-ChsC, and FLAG-ChsB showed that each of them was present as several bands in the cell, suggesting that they underwent post-transcriptional modifications. We showed by phosphatase treatment that a portion of FLAG-ChsB were phosphorylate … More d. We also constructed chsA chsC chsD and chsA chsC csmA triple deletion mutants and indicated that csmA played crucial role(s) for the growth of the chsA chsC double mutant.Next, we constructed the strains that expressed the CsmA tagged with nine copies of HA eppitope (CsmA-HA) instead of wild-type CsmA. CsmA-HA localized at hyphal tips and forming sepia. We showed that the myosin motor-like domain of CsmA was able to bind to actin in vivo and in vitro and that this binding is indispensable for the correct localization and function of CsmA. We isolated a paralog of csmA, csmB, from A. nidulans and characterized its function. Growth and morphological defects of csmB deletion mutants were partly similar to those of the csmA deletion mutant. We showed that deletions of csmA and csmB were synthetically lethal. We next constructed the strains that produced both CsmA-HA and CsmB tagged with three copies of FLAG epitope at its C-terminus and investigated their co-localization. Their localizations were partly overlapped. Less

我们从丝状真菌nidulans中克隆了5个甲壳素合成酶基因，并对它们的功能进行了研究。在本项目中，我们构建了产生功能表位标记的甲壳素合成酶的菌株，以阐明其基因产物的定位。HA表位标记的CHSA(HA-CHSA)主要定位于形成菌丝隔膜，而GFP或FLAG表位标记的CHsB(GFP-ChsB或FLAG-ChsB)和FLAG表位标记的CHSC(FLAG-CHSC)主要定位于形成菌丝的隔膜和菌丝顶端。接下来，我们构建了同时产生HA-CHSA和FLAG-CHSC的菌株，并研究了它们的共定位。HA-CHSA和FLAG-CHSC的定位部分重叠。对表达HA-CHSA、FLAG-CHSC和FLAG-ChsB的菌株的细胞裂解产物进行的Western印迹分析表明，它们在细胞中都以几条带的形式存在，这表明它们经历了转录后修饰。我们通过磷酸酶处理表明，部分FLAG-ChsB是磷酸化的…构建了CHSA-CHSC-CHSD和CHSA-CHSC-CSMA三重缺失突变体，表明CSMA对CHSA-CHSC双变异体的生长起着至关重要的作用(S)。CSMA-HA定位于菌丝顶端，形成深褐色。我们发现CSMA的肌球蛋白马达样结构域能够在体内和体外与肌动蛋白结合，这种结合对于CSMA的正确定位和功能是必不可少的。我们从根瘤拟青霉中分离到一个类似于CSMA的基因CSMB，并对其功能进行了研究。CSMB缺失突变体的生长和形态缺陷与CSMA缺失突变体部分相似。我们证明了CSMA和CSMB的缺失是综合致死的。接下来，我们构建了同时产生CSMA-HA和CSMB的菌株，并在其C端标记了三个标记表位的副本，并研究了它们的共定位。它们的定位部分重叠。较少

项目成果

The class V chitin synthase gene csmA is crucial for the growth of the chsA chsC double mutant in Aspergillus nidulans.

V 类几丁质合酶基因 csmA 对于构巢曲霉中 chsA chsC 双突变体的生长至关重要。

• 发表时间: 2005
• 期刊: Biosci.Biotechnol.Biochem. 69
• 作者: 山田絵美;一宮維幸;太田明徳;堀内裕之
• 通讯作者: 堀内裕之

CsmA, a class V chitin synthase with a myosin motor-like domain, is localized through direct interaction with the actin cytoskeleton in Aspergillus nidulans

• DOI: 10.1091/mbc.e04-09-0761
• 发表时间: 2005-04-01
• 期刊: MOLECULAR BIOLOGY OF THE CELL
• 影响因子: 3.3
• 作者: Takeshita, N;Ohta, A;Horiuchi, H
• 通讯作者: Horiuchi, H