Plasma membrane intrinsic proteins (PIPs) are channels facilitating the passive diffusion of water and small solutes. Arabidopsis PIP2;7 trafficking occurs through physical interaction with SNARE proteins including the syntaxin SYP121, a plasma membrane Qa-SNARE involved in membrane fusion. To better understand the interaction mechanism, we aimed at identifying the interaction motifs in SYP121 and PIP2;7 using ratiometric bimolecular fluorescence complementation assays in Nicotiana benthamiana. SYP121 consists of four regions, N, H, Q, and C, and sequential deletions revealed that the C region, containing the transmembrane domain, as well as the H and Q regions, containing the Habc and Qa-SNARE functional domains, interact with PIP2;7. Neither the linker between the Habc and the Qa-SNARE domains nor the H or Q regions alone could fully restore the interaction with PIP2;7, suggesting that the interacting motif depends on the conformation taken by the HQ region. When investigating the interacting motif(s) in PIP2;7, we observed that deletion of the cytosolic N- and/or C- terminus led to a significant decrease in the interaction with SYP121. Shorter deletions revealed that at the N-terminal amino acid residues 18–26 were involved in the interaction. Domain swapping experiments between PIP2;7 and PIP2;6, a PIP isoform that does not interact with SYP121, showed that PIP2;7 N-terminal part up to the loop C was required to restore the full interaction signal, suggesting that, as it is the case for SYP121, the interaction motif(s) in PIP2;7 depend on the protein conformation. Finally, we also showed that PIP2;7 physically interacted with other Arabidopsis SYP1s and SYP121 orthologs.

质膜内在蛋白（PIP）是促进水和小溶质被动扩散的通道。拟南芥PIP2; 7的运输是通过与SNARE蛋白质的物理相互作用而发生的，包括SYP121语法，这是一种参与膜融合的质膜Qa-SNARE。为了更好地了解相互作用机制，我们旨在通过比例双分子荧光互补测定法确定SYP121和PIP2; 7中的相互作用基序。烟草。SYP121由N，H，Q和C四个区域组成，顺序删除显示，含有跨膜结构域的C区以及含有Habc和Qa-SNARE功能域的H和Q区与PIP2; 7。Habc和Qa-SNARE域之间的连接子以及单独的H或Q区都不能完全恢复与PIP2的相互作用； 7，这表明相互作用的基序取决于HQ区所采取的构象。当研究PIP2; 7中的相互作用基序时，我们观察到胞质N-和/或C-末端的缺失导致与SYP121的相互作用显着降低。较短的删除表明，在N末端氨基酸残基18-26参与了相互作用。PIP2; 7和PIP2; 6之间的域交换实验，不与SYP121相互作用的PIP同工型表明，要恢复完整的相互作用信号，需要至环C的PIP2; 7 N末端部分才能恢复完整的相互作用信号，这表明，与SYP121一样，相互作用基序PIP2; 7中的蛋白依赖于蛋白质构象。最后，我们还表明，PIP2; 7与其他拟南芥SYP1和SYP121直系同源物发生了物理相互作用。