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

从丝状真菌中克隆了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几丁质合成酶的菌株，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，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隔和尖端。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的菌株的细胞裂解物进行Western blot分析显示，它们在细胞中以多个条带的形式存在，表明它们经历了转录后修饰。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至关重要的作用。接下来，我们构建了表达带有9拷贝HA表位（CsmA-HA）标记的CsmA的菌株，而不是野生型CsmA。CsmA-HA定位于菌丝尖端，形成棕褐色。我们发现CsmA的肌凝蛋白运动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。本研究分离了一株csmA同源基因csmB，并对其功能进行了表征。csmB缺失突变体的生长和形态缺陷部分与csmA缺失突变体相似。我们发现csmA和csmB的缺失是合成致死的。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FLAG表位拷贝，并研究了它们的共定位。他们的定位部分重叠。少

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