Ueno Hironori, Gonda Kohsuke, Takeda Tetsuya, Numata Osamu
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
Cell Motil Cytoskeleton. 2003 May;55(1):51-60. doi: 10.1002/cm.10111.
Calmodulin (CaM) is known to be a ciliary component. However, the function of CaM in cilia or flagella has not been well understood. Immunoelectron microscopy using anti-CaM antibody showed that CaM was localized on the axonemal microtubules (MTs) and matrix of Tetrahymena cilia. To investigate the signal transduction of Ca(2+)/CaM in cilia, we performed Ca(2+)/CaM-affinity column chromatography in the membrane and matrix fraction. Elongation factor-1alpha (EF-1alpha) was identified as a Ca(2+)/CaM-binding protein in cilia. EF-1alpha is a highly conserved protein and functions in protein translation. In addition, EF-1alpha has been reported to interact with MTs and F-actin in several organisms. Immunoelectron microscopy showed that EF-1alpha was localized on the axonemal MTs. However, in immunoblot analysis, EF-1alpha was mainly extracted in the membrane and matrix fraction from the axonemal MTs by 1% Triton X-100 extraction. These results suggest that interaction between EF-1alpha and axonemal MTs is weak and sensitive to treatment with 1% Triton X-100 and that EF-1alpha mediates between axonemal MTs and CaM in the presence of Ca(2+). Moreover, EF-1alpha was also localized in cilia of Paramecium, suggesting that EF-1alpha functions as a target protein of Ca(2+)/CaM in ciliate cilia.
钙调蛋白（CaM）是一种已知的纤毛成分。然而，CaM在纤毛或鞭毛中的功能尚未得到很好的理解。使用抗CaM抗体的免疫电子显微镜显示，CaM定位于四膜虫纤毛的轴丝微管（MTs）和基质上。为了研究Ca(2+)/CaM在纤毛中的信号转导，我们在膜和基质部分进行了Ca(2+)/CaM亲和柱层析。延伸因子-1α（EF-1α）被鉴定为纤毛中的一种Ca(2+)/CaM结合蛋白。EF-1α是一种高度保守的蛋白质，在蛋白质翻译中发挥作用。此外，据报道EF-1α在几种生物体中与MTs和F-肌动蛋白相互作用。免疫电子显微镜显示EF-1α定位于轴丝MTs上。然而，在免疫印迹分析中，通过1% Triton X-100提取，EF-1α主要从轴丝MTs的膜和基质部分中提取出来。这些结果表明，EF-1α与轴丝MTs之间的相互作用较弱，且对1% Triton X-100处理敏感，并且在Ca(2+)存在的情况下，EF-1α在轴丝MTs和CaM之间起介导作用。此外，EF-1α也定位于草履虫的纤毛中，这表明EF-1α在纤毛虫纤毛中作为Ca(2+)/CaM的靶蛋白发挥作用。
