Protein phosphatases PP1 and PP2A are located in distinct positions in the Chlamydomonas flagellar axoneme.
Protein phosphatases PP1 and PP2A are located in distinct positions in the Chlamydomonas flagellar axoneme.
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发表时间：
2000
影响因子：
4
通讯作者：
Pinfen Yang;L. Fox;R. Colbran;W. Sale
中科院分区：
文献类型：
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作者：
Pinfen Yang;L. Fox;R. Colbran;W. Sale
我们推测，微囊藻毒素敏感的蛋白磷酸酶的衣原体鞭毛轴丝，定位调节内臂动力蛋白活性的组成部分。为了验证这一点，我们采用了直接的生物化学方法。微囊藻毒素-琼脂糖亲和纯化揭示了一个突出的35-kDa的轴丝蛋白，预测是1型蛋白磷酸酶（PP 1c）的催化亚基。我们克隆了衣原体PP 1c并制备了特异性多克隆肽抗体。基于蛋白质印迹分析，35-kDa PP 1c锚定在轴丝中。此外，突变株的鞭毛和轴丝的分析表明，PP 1C主要是，但不限于，锚定在中央对装置，与C1微管。因此，PP 1是控制鞭毛运动的中心对机制的一部分。另外两个轴丝蛋白的62和37 kDa的分离也使用微囊藻毒素-琼脂糖亲和。基于直接肽序列和蛋白质印迹，这些蛋白质是2A型蛋白磷酸酶（PP 2A）的A-和C-亚基。轴丝PP 2A不是先前确定的中心对装置、外臂动力蛋白、内臂动力蛋白、动力蛋白调节复合物或径向辐条的组成部分之一。我们假设PP 2A是锚定的双微管，可能在位置直接控制内臂动力蛋白的活动。
We postulated that microcystin-sensitive protein phosphatases are integral components of the Chlamydomonas flagellar axoneme, positioned to regulate inner arm dynein activity. To test this, we took a direct biochemical approach. Microcystin-Sepharose affinity purification revealed a prominent 35-kDa axonemal protein, predicted to be the catalytic subunit of type-1 protein phosphatase (PP1c). We cloned the Chlamydomonas PP1c and produced specific polyclonal peptide antibodies. Based on western blot analysis, the 35-kDa PP1c is anchored in the axoneme. Moreover, analysis of flagella and axonemes from mutant strains revealed that PP1c is primarily, but not exclusively, anchored in the central pair apparatus, associated with the C1 microtubule. Thus, PP1 is part of the central pair mechanism that controls flagellar motility. Two additional axonemal proteins of 62 and 37 kDa were also isolated using microcystin-Sepharose affinity. Based on direct peptide sequence and western blots, these proteins are the A- and C-subunits of type 2A protein phosphatase (PP2A). The axonemal PP2A is not one of the previously identified components of the central pair apparatus, outer arm dynein, inner arm dynein, dynein regulatory complex or the radial spokes. We postulate PP2A is anchored on the doublet microtubules, possibly in position to directly control inner arm dynein activity.