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

我们从丝状真菌构巢曲霉中克隆了5个几丁质合成酶基因，并对其功能进行了研究。在本项目中，我们构建了产生功能性表位标记的几丁质酶的菌株，以阐明其基因产物的定位。HA表位标记的ChsA（HA-ChsA）主要定位于菌丝形成隔，GFP或FLAG表位标记的ChsB（GFP-ChsB或FLAG-ChsB）和FLAG表位标记的ChsC（FLAG-ChsC）主要定位于菌丝形成隔和菌丝顶端。我们接下来构建了产生HA-ChsA和FLAG-ChsC的菌株，并研究了它们的共定位。HA-ChsA和FLAG-ChsC的定位有部分重叠。针对表达HA-ChsA、FLAG-ChsC和FLAG-ChsB的菌株的细胞裂解物的蛋白质印迹分析显示，它们中的每一个在细胞中以几个条带存在，表明它们经历了转录后修饰。用磷酸酶处理后，发现FLAG-ChsB有一部分被磷酸化， ...更多信息 D.我们还构建了chsA chsC chsD和chsA chsC csmA三重缺失突变体，并表明csmA在chsA chsC双突变体的生长中起着关键作用。CsmA-HA定位于菌丝顶端，形成深褐色。我们发现，肌球蛋白马达样结构域的CsmA能够结合肌动蛋白在体内和体外，这种结合是必不可少的正确定位和功能的CsmA。我们从A. nidulans，并对其功能进行了表征。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。我们发现csmA和csmB的缺失是合成致死的。我们接下来构建了产生CsmA-HA和CsmB两者的菌株，所述CsmA-HA和CsmB在其C末端标记有三个拷贝的FLAG表位，并研究了它们的共定位。它们的定位部分重叠。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