Molecular characterization of plasma membrane H+-ATPase in a carrot mutant cell line with enhanced citrate excretion
Molecular characterization of plasma membrane H+-ATPase in a carrot mutant cell line with enhanced citrate excretion
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DOI：
10.1111/j.1399-3054.2004.00399.x
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发表时间：
2004-10
影响因子：
6.4
通讯作者：
Takashi Ohno;S. Nakahira;Yuji Suzuki;T. Kani;T. Hara;H. Koyama
中科院分区：
文献类型：
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作者：
Takashi Ohno;S. Nakahira;Yuji Suzuki;T. Kani;T. Hara;H. Koyama
柠檬酸排泄增强的胡萝卜突变细胞系（IPG；不溶性磷酸盐生长者）的柠檬酸流出伴随着电等效的 H + 释放。此外，已知IPG细胞系中的质膜H + -ATP酶活性（EC 3.6.1.35）高于野生型细胞系。在本研究中，对 IPG 细胞系中质膜 H + -ATP 酶进行了分子表征，以检查该酶在增强柠檬酸盐排泄中的作用。克隆了编码胡萝卜质膜H + -ATP酶的四个II型（DcPA 1、2、4和5）和两个I型（DcPA 3和6）全长cDNA。 IPG 细胞系中最丰富的亚型 DcPA 1 的转录水平是野生型细胞的三倍。其他亚型的转录水平在细胞类型之间没有显示差异。通过蛋白质印迹分析，IPG细胞系中质膜H + -ATP酶蛋白水平是野生型细胞系的1.6倍。这些结果表明，IPG细胞系中质膜H + -ATP酶的较高活性是由于DcPA 1的上调所致。DcPA 1基因的反义抑制也导致转基因IPG细胞系中柠檬酸排泄减少，表明质膜H + -ATP酶的上调参与了柠檬酸排泄的增强。
The citrate efflux of a carrot mutant cell line (IPG; insoluble phosphate grower) with enhanced citrate excretion is concomitant with an electrically equivalent H + release. Furthermore, plasma membrane H + -ATPase activity (EC 3.6.1.35) in the IPG cell line is known to be greater than in the wild-type cell line. In the present study, molecular characterization of plasma membrane H + -ATPase in the IPG cell line was conducted to examine the involvement of this enzyme in enhanced citrate excretion. Four type-II (DcPA 1, 2, 4 and 5) and two type-I (DcPA 3 and 6) full-sized cDNAs encoding Daucus carota plasma membrane H + -ATPase were cloned. The transcript level of the most abundant isoform, DcPA 1, was three times higher in the IPG cell line than in the wild-type cells. The transcript levels of the other isoforms showed no differences between cell types. The plasma membrane H + -ATPase protein level in the IPG cell line was 1.6-fold that of the wild-type cell line when analysed by western blot analysis. These results indicate that the higher activity of plasma membrane H + -ATPase in the IPG cell line is due to up-regulation of DcPA 1. Antisense inhibition of the DcPA 1 gene also led to decreased citrate excretion from the transgenic IPG cell line, suggesting that the up-regulation of plasma membrane H + -ATPase is involved in enhanced citrate excretion.