Expression and characterization of Cys374 mutated human β‐actin in two different mammalian cell lines: impaired microfilament organization and stability

FEBS Letters

Moustakas, A²  Kardassis, D²  Stournaras, C²  Tsapara, A²  Gravanis, A¹ 

[1] Department of Pharmacology, Division of Basic Sciences, School of Medicine, University of Crete, GR-71110 Heraklion, Greece;Department of Biochemistry, Division of Basic Sciences, School of Medicine, University of Crete, GR-71110 Heraklion, Greece

关键词: β-Actin-Cys374 mutant;    Flag-tagged actin;    Microfilament organization;   

DOI  :  10.1016/S0014-5793(99)00848-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Previous studies have demonstrated that addition of glutathione at the penultimate Cys³⁷⁴ residue of actin results in filaments with diminished mechanical stability. In the present work substitutions introducing a negatively charged (Asp and Glu) or a neutral (Ala) amino acid at position 374 of the human β-actin and tagged at the N-terminus with the flag epitope were studied by transient transfections into Ishikawa human endometrial and opossum kidney cells. Immunofluorescence revealed that microfilaments which incorporated negatively charged mutants were partially to severely disorganized when compared to the almost well-formed actin-Ala³⁷⁴ filaments or the wild type actin filaments. Furthermore, microfilaments containing either negatively charged mutant were more sensitive to the destabilizing action of cytochalasin B. In addition, Triton fractionation revealed a considerable reduction of flag-actin content in the Triton insoluble fraction for cells expressing Asp³⁷⁴ or Glu³⁷⁴ mutant compared to wild type actin. These results demonstrate that negatively charged amino acid residues at the exposed C-terminal tail strongly affect actin microfilament organization and dynamics in vivo.

【 授权许可】

Unknown