Single Point Mutations in the Small Cytoplasmic Loop of ACA8, a Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana, Generate Partially Deregulated Pumps
Single Point Mutations in the Small Cytoplasmic Loop of ACA8, a Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana, Generate Partially Deregulated Pumps
复制标题
DOI：
10.1074/jbc.m109.006148
复制
发表时间：
2009-11-06
影响因子：
4.8
通讯作者：
De Michelis, Maria Ida
中科院分区：
文献类型：
--
作者：
Fusca, Tiziana;Bonza, Maria Cristina;De Michelis, Maria Ida
ACA8是一种2B型Ca2+- atp酶，具有调节N端，其自身抑制作用可通过钙调蛋白（CaM）或酸性磷脂的结合来抑制。aca8n末端能够与连接跨膜结构域2和3的小细胞质环区域相互作用。为了确定这种相互作用在自抑制中的作用，我们分析了ACA8 Glu（252）到Asn（345）序列突变产生的单点突变体。六种测试酸性残基（Glu（252）、Asp（273）、Asp（291）、Asp（303）、Glu（302）或Asp(332)）中任何一种的Ala突变都会使酶对CaM的依赖性降低。这些结果强调了ACA8对小细胞质环表面负电荷的自抑制的相关性。这些突变体中最不受调控的是D291A ACA8，它被受控蛋白水解或酸性磷脂激活的能力较弱；D291A突变体对CaM的亲和力明显高于野生型ACA8。此外，它的表型比D291NACA8强，这表明该残基更直接地参与了自抑制机制。在其他产生的突变体（I284A, N286A, P289A, P322A， V344A和N345A）中，只有P322A ACA8对CaM的依赖性低于野生型。本研究报告的结果提供了第一个证据，证明2B型Ca2+- atp酶的小细胞质环在实现自动抑制状态中起作用。
ACA8 is a type 2B Ca2+-ATPase having a regulatory N terminus whose auto-inhibitory action can be suppressed by binding of calmodulin (CaM) or of acidic phospholipids. ACA8 N terminus is able to interact with a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To determine the role of this interaction in auto-inhibition we analyzed single point mutants produced by mutagenesis of ACA8 Glu(252) to Asn(345) sequence. Mutation to Ala of any of six tested acidic residues (Glu(252), Asp(273), Asp(291), Asp(303), Glu(302), or Asp(332)) renders an enzyme that is less dependent on CaM for activity. These results highlight the relevance in ACA8 auto-inhibition of a negative charge of the surface area of the small cytoplasmic loop. The most deregulated of these mutants is D291A ACA8, which is less activated by controlled proteolysis or by acidic phospholipids; the D291A mutant has an apparent affinity for CaM higher than wildtype ACA8. Moreover, its phenotype is stronger than that of D291NACA8, suggesting a more direct involvement of this residue in the mechanism of auto-inhibition. Among the other produced mutants (I284A, N286A, P289A, P322A, V344A, and N345A), only P322A ACA8 is less dependent on CaM for activity than the wild type. The results reported in this study provide the first evidence that the small cytoplasmic loop of a type 2B Ca2+-ATPase plays a role in the attainment of the auto-inhibited state.