Potential role of annexin AnnAt1 from Arabidopsis thaliana in pH-mediated cellular response to environmental stimuli
Potential role of annexin AnnAt1 from Arabidopsis thaliana in pH-mediated cellular response to environmental stimuli
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DOI：
10.1093/pcp/pcm046
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发表时间：
2007-06-01
影响因子：
4.9
通讯作者：
Pikula, Slawomir
中科院分区：
文献类型：
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作者：
Gorecka, Karolina M.;Thouverey, Cyrit;Pikula, Slawomir
植物膜联蛋白、钙离子和膜结合蛋白可能参与了细胞对细胞质酸化引起的胁迫的反应。为了了解膜联蛋白如何促进细胞离子动态平衡，我们研究了pH诱导的拟南芥重组膜联蛋白Annat1的结构和功能的变化。当pH从7.0降到5.8时，AnnAt1在人工脂膜上形成离子通道的时间从3.5h缩短到15-20min，单位电导从32ps增加到63ps。伴随着这些变化的是Annat1疏水性的增加，疏水性预测表明，与Annat1结合的2-(对甲苯基)萘-6-磺酸(TNS)的荧光增加，以及荧光共振能量从Annat1色氨酸残基转移到TNS。在酸性pH条件下，AnnAt1伴随的脂质分配导致其对蛋白水解性消化具有部分保护作用。圆二色谱和红外光谱测定的AnnAt1的二级结构也受pH从7.2降到5.2的影响。这些变化的特点是Beta-Sheet含量增加。α-螺旋结构的代价，并伴随着通过蔗糖梯度超速离心法探测的Annat1低聚物的可逆形成。当pH从5.2进一步降低到4.5或更低时，会形成不可逆的聚集体，导致AnnAt1离子电导的损失。我们的发现表明，AnnAt1可以在pH为7到5的范围内感知pH环境的变化，并通过离子通道电导、疏水性、蛋白质的二级结构和低聚物的形成来响应。进一步酸化不可逆转地灭活Annat1。提示Annat1的pH敏感离子通道活性可能在细胞内离子动态平衡中起作用。
Plant annexins, Ca2+ and membrane-binding proteins, are probably implicated in the cellular response to stress resulting from acidification of cytosol. To understand how annexins can contribute to cellular ion homeostasis, we investigated the pH-induced changes in the structure and function of recombinant annexin AnnAt1 from Arabidopsis thaliana. The decrease of pH from 7.0 to 5.8 reduced the time of the formation of ion channels by AnnAt1 in artificial lipid membranes from 3.5h to 15-20min and increased their unitary conductance from 32 to 63 pS. These changes were accompanied by an increase in AnnAt1 hydrophobicity as revealed by hydrophobicity predictions, by an increase in fluorescence of 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) bound to AnnAt1 and fluorescence resonance energy transfer from AnnAt1 tryptophan residues to TNS. Concomitant lipid partition of AnnAt1 at acidic pH resulted in its partial protection from proteolytic digestion. Secondary structures of AnnAt1 determined by circular dichroism and infrared spectroscopy were also affected by lowering the pH from 7.2 to 5.2. These changes were characterized by an increase in beta-sheet content at. the expense of alpha-helical structures, and were accompanied by reversible formation of AnnAt1 oligomers as probed by ultracentrifugation in a sucrose gradient. A further decrease of pH from 5.2 to 4.5 or lower led to the formation of irreversible aggregates and loss of AnnAt1 ionic conductance. Our findings suggest that AnnAt1 can sense changes of the pH milieu over the pH range from 7 to 5 and respond by changes in ion channel conductance, hydrophobicity, secondary structure of the protein and formation of oligomers. Further acidification irreversibly inactivated AnnAt1. We suggest that the pH-sensitive ion channel activity of AnnAt1 may play a role in intracellular ion homeostasis.