Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain
Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain
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DOI：
10.1074/jbc.m002047200
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发表时间：
2000-09-29
影响因子：
4.8
通讯作者：
Harper, JF
中科院分区：
文献类型：
--
作者：
Curran, AC;Hwang, I;Harper, JF
Ca 2+泵的调节对于控制所有真核生物细胞质和细胞器中的[Ca 2 +]是重要的。在这里，我们报告了一种遗传策略，以确定一种新的钙调素激活的钙泵与N-末端调控结构域（异构体ACA 2从拟南芥）的自抑制功能的残基。突变泵组成的活动进行了鉴定，通过互补的酵母（K616）缺乏两个Ca 2+泵。发现15个突变破坏了位于Lys（23）和Arg（54）之间的N-末端自身抑制剂片段。发现三个突变（E167 K、D219 N和E341 R）与连接ATP酶催化结构域（头部）的茎部和跨膜结构域相关。酶分析表明，茎突变导致钙调素独立的活动，与V-max K-mATP，和K（mCa）2+类似的泵，其中N-末端自身抑制剂已被删除。在Asp（219）（D219 E）处的高度保守取代仍然产生失调的泵，表明茎中的自抑制结构对扰动高度敏感。在来自酵母和植物的质膜H+-ATP酶中，类似定位的突变导致了过度活跃的泵。总之，这些结果表明，茎的结构特征是在调节不同的P-型ATP酶的一般重要性。
The regulation of Ca2+-pumps is important for controlling [Ca2+] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca2+-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca2+-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341R) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V-max K-mATP, and K(mCa)2+ similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H+-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.