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

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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