The topology of plastid inner envelope potassium cation efflux antiporter KEA1 provides new insights into its regulatory features
The topology of plastid inner envelope potassium cation efflux antiporter KEA1 provides new insights into its regulatory features
复制标题
DOI：
10.1007/s11120-019-00700-2
复制
发表时间：
2020-07-01
影响因子：
3.7
通讯作者：
Kunz, Hans-Henning
中科院分区：
文献类型：
--
作者：
Boelter, Bettina;Mitterreiter, Melanie J.;Kunz, Hans-Henning
质体钾离子外排反转运蛋白（KEAs）对叶绿体功能、发育和光合作用具有重要意义。因此，了解它们在蛋白质水平上的调控是至关重要的。先前的研究主要集中在类囊体载体KEA3的c端K（+）转运和nad结合（KTN）结构域，但该结构域的定位尚不清楚。虽然所有三个质体KEA成员在其跨膜区和c端KTN结构域高度保守，但只有内膜KEA家族成员KEA1和KEA2携带长可溶性n端。有趣的是，这个区域的赖氨酸168被基质乙酰转移酶NSI乙酰化。如果内包膜KEAs存在奇数个跨膜结构域，正如所有三种质体KEA载体所建议的那样，那么调节结构域和蛋白质调控将发生在内包膜的相反两侧。因此，在这项研究中，我们着手研究内膜KEA蛋白的拓扑结构。利用新设计的KEA1包膜n端特异性抗体和表达c端KEA1- yfp融合蛋白的转基因拟南芥植物，我们发现KEA1的n端和c端调控区域都位于叶绿体基质中，而不是在膜间空间中。考虑到KEA1和KEA2之间的高度同源性，因此我们推断包膜KEAs必须由偶数个跨膜结构域组成。
The plastid potassium cation efflux antiporters (KEAs) are important for chloroplast function, development, and photosynthesis. To understand their regulation at the protein level is therefore of fundamental importance. Prior studies have focused on the regulatory K(+)transport and NAD-binding (KTN) domain in the C-terminus of the thylakoid carrier KEA3 but the localization of this domain remains unclear. While all three plastid KEA members are highly conserved in their transmembrane region and the C-terminal KTN domain, only the inner envelope KEA family members KEA1 and KEA2 carry a long soluble N-terminus. Interestingly, this region is acetylated at lysine 168 by the stromal acetyltransferase enzyme NSI. If an odd number of transmembrane domains existed for inner envelope KEAs, as it was suggested for all three plastid KEA carriers, regulatory domains and consequently protein regulation would occur on opposing sides of the inner envelope. In this study we therefore set out to investigate the topology of inner envelope KEA proteins. Using a newly designed antibody specific to the envelope KEA1 N-terminus and transgenic Arabidopsis plants expressing a C-terminal KEA1-YFP fusion protein, we show that both, the N-terminal and C-terminal, regulatory domains of KEA1 reside in the chloroplast stroma and not in the intermembrane space. Considering the high homology between KEA1 and KEA2, we therefore reason that envelope KEAs must consist of an even number of transmembrane domains.