A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain
A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain
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DOI：
10.1074/jbc.273.2.1099
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发表时间：
1998-01-09
影响因子：
4.8
通讯作者：
Sze, H
中科院分区：
文献类型：
--
作者：
Harper, JF;Hong, BM;Sze, H
为了研究参与调节细胞内Ca2+的转运体，我们从模式植物拟南芥中分离出编码Ca2+-ATPase的全长cDNA，并将其命名为ACA2（拟南芥Ca2+-ATPase， isoform 2）。ACA2p最类似于“质膜型”Ca2+- atp酶，但更小（110 kDa），包含独特的n端结构域，并且缺少长c端钙调素结合调节结构域。此外，正如微粒体膜的水-两相分离所示，ACA2p定位于膜系统而不是质膜。ACA2p在酵母中以全长蛋白（ACA2-1p）和n端截断突变体（ACA2-2p； δ残基2-80）的形式表达。只有截断突变体恢复了内源性Ca2+泵（PMR1和PMC1）缺陷的酵母突变体在Ca2+缺失的培养基上的生长。尽管全长蛋白的基础Ca2+- atp酶活性较低，但它被钙调素刺激了5倍（在30 nM左右激活50%）。在融合蛋白上的钙调蛋白凝胶覆盖表明，在n端结构域的前36个残基中鉴定出了钙调蛋白结合序列。因此，ACA2编码了一种新的钙调蛋白调节的Ca2+- atp酶，其独特的n端调节结构域和非质膜定位。
To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1, Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin, A calmodulin binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins, Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.