Taillon B E, Adler S A, Suhan J P, Jarvik J W
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.
J Cell Biol. 1992 Dec;119(6):1613-24. doi: 10.1083/jcb.119.6.1613.
Centrin, a 20-kD phosphoprotein with four calcium-binding EF-hands, is present in the centrosome/basal body apparatus of the green alga Chlamydomonas reinhardtii in three distinct locations: the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions. In each location, centrin is found in fibrous structures that display calcium-mediated contraction. The mutant vfl2 has structural defects at all of these locations and is defective for basal body localization and/or segregation. We show that the vfl2 mutation is a G-to-A transition in the centrin structural gene which converts a glutamic acid to a lysine at position 101, the first amino acid of the E-helix of the protein's third EF-hand. This proves that centrin is required to construct the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions, and it demonstrates the importance of amino acid 101 to normal centrin function. Based on immunofluorescence analysis using anti-centrin antibodies, it appears that vfl2 centrin is capable of binding to the basal body but is incapable of polymerizing into filamentous structures. 19 phenotypic revertants of vfl2 were isolated, and 10 of them, each of which had undergone further mutation at codon 101, were examined in detail. At the DNA level, 1 of the 10 was wild type, and the other 9 were pseudorevertants encoding centrins with the amino acids asparagine, threonine, methionine, or isoleucine at position 101. No ultrastructure defects were apparent in the revertants with asparagine or threonine at position 101, but in those with methionine or isoleucine at position 101, the distal striated fibers were found to be incomplete, indicating that different amino acid substitutions at position 101 can differentially affect the assembly of the three distinct centrin-containing fibrous structures associated with the Chlamydomonas centrosome.
中心蛋白是一种具有四个钙结合EF手结构域的20kD磷蛋白，存在于莱茵衣藻的中心体/基体装置中的三个不同位置：核-基体连接体、远端横纹纤维和鞭毛过渡区。在每个位置，中心蛋白都存在于显示钙介导收缩的纤维结构中。突变体vfl2在所有这些位置都有结构缺陷，并且在基体定位和/或分离方面存在缺陷。我们表明，vfl2突变是中心蛋白结构基因中的一个G到A的转变，它在第101位将谷氨酸转化为赖氨酸，该位置是蛋白质第三个EF手结构域E螺旋的第一个氨基酸。这证明了中心蛋白是构建核-基体连接体、远端横纹纤维和鞭毛过渡区所必需的，并且它证明了第101位氨基酸对正常中心蛋白功能的重要性。基于使用抗中心蛋白抗体的免疫荧光分析，vfl2中心蛋白似乎能够与基体结合，但不能聚合成丝状结构。分离出了19个vfl2的表型回复突变体，并对其中10个在密码子101处发生进一步突变的突变体进行了详细检查。在DNA水平上，10个中有1个是野生型，其他9个是编码在第101位具有天冬酰胺、苏氨酸、甲硫氨酸或异亮氨酸的中心蛋白的假回复突变体。在第101位为天冬酰胺或苏氨酸的回复突变体中没有明显的超微结构缺陷，但在第101位为甲硫氨酸或异亮氨酸的回复突变体中，发现远端横纹纤维不完整，这表明第101位不同的氨基酸取代可以不同程度地影响与衣藻中心体相关的三种不同的含中心蛋白纤维结构的组装。
