Wang Hao, Zhang Liyuan, Tao Yuan, Wang Zuodong, Shen Dan, Dong Hansong
Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China.
Department of Plant Pathology, Shandong Agricultural University, Taian, China.
Front Plant Sci. 2020 Jan 10;10:1671. doi: 10.3389/fpls.2019.01671. eCollection 2019.
In plants, plasma membrane intrinsic protein (PIP) PIP1s and PIP2s mediate the transport of disparate substrates across plasma membranes (PMs), with a prerequisite that the proteins correctly localize to the PMs. While PIP2s can take correct localization by themselves in plant cells, PIP1s cannot unless aided by a specific PIP2. Here, we analyzed the localization of the aquaporins, AtPIP1s, AtPIP2;4, and their mutants in yeast, oocytes, and protoplasts of . Most of AtPIP2;4 localized in the PM when expressed alone, whereas AtPIP1;1 failed to realize it in yeast and oocytes. Switch of the transmembrane helix 2 (TM2) or TM3 from AtPIP1;1 to AtPIP2;4 disabled the latter's PM targeting activity. Surprisingly, a replacement of TM2 and TM3 of AtPIP1;1 with those of AtPIP2;4 created a PM-localized AtPIP1;1 mutant, 1;1Δ(TM2+TM3)/2;4(TM2+TM3), which could act as a water and hydrogen peroxide channel just like AtPIP2;4. A localization and function analysis on mutants of AtPIP1;2, AtPIP1;3, AtPIP1;4, and AtPIP1;5, with the same replaced TM2 and TM3 from AtPIP2;4, showed that these AtPIP1 variants could also localize in the PM spontaneously, thus playing an inherent role in transporting solutes. Sequential and structural analysis suggested that a hydrophilic residue and a defective LxxxA motif are modulators of PM localization of AtPIP1s. These results indicate that TM2 and TM3 are necessary and, more importantly, sufficient in AtPIP2 for its PM localization.
在植物中，质膜内在蛋白（PIP）中的PIP1s和PIP2s介导不同底物跨质膜（PM）的运输，前提是这些蛋白质能正确定位于质膜。虽然PIP2s能在植物细胞中自行正确定位，但PIP1s却不能，除非有特定的PIP2协助。在此，我们分析了水通道蛋白AtPIP1s、AtPIP2;4及其突变体在酵母、卵母细胞和拟南芥原生质体中的定位。单独表达时，大多数AtPIP2;4定位于质膜，而AtPIP1;1在酵母和卵母细胞中未能实现这一定位。将AtPIP1;1的跨膜螺旋2（TM2）或TM3替换为AtPIP2;4的相应结构域后，会使后者丧失质膜靶向活性。令人惊讶的是，将AtPIP1;1的TM2和TM3替换为AtPIP2;4的TM2和TM3后，产生了一个定位于质膜的AtPIP1;1突变体，即1;1Δ(TM2 + TM3)/2;4(TM2 + TM3)，它能像AtPIP2;4一样作为水和过氧化氢通道发挥作用。对AtPIP1;2、AtPIP1;3、AtPIP1;4和AtPIP1;5的突变体进行定位和功能分析，这些突变体的TM2和TM3同样被AtPIP2;4的相应结构域替换，结果表明这些AtPIP1变体也能自发定位于质膜，从而在溶质运输中发挥内在作用。序列和结构分析表明，一个亲水残基和一个有缺陷的LxxxA基序是AtPIP1s质膜定位的调节因子。这些结果表明，TM2和TM3对于AtPIP2定位于质膜是必要的，更重要的是，也是充分的。
