Interactions between SNAREs, plasma membrane aquaporins and the potassium channel KC1

Plasma membrane intrinsic proteins (PIPs) are channels facilitating the passive diffusion of water and small solutes. PIP trafficking occurs through physical interaction with SNARE proteins including the syntaxin SYP121, which is a plasma membrane Qa-SNARE involved in membrane fusion. We have previously shown that Arabidopsis SYP121 physically interacts with PIP2;7 to regulate its trafficking to the plasma membrane. As this syntaxin also regulates the trafficking and activity of the potassium channels, including KC1, we hypothesized that SYP121 is coordinating the trafficking and activity of both potassium channels and aquaporins to optimize the cell osmoregulation. To better understand the mechanism of interaction, we aimed at identifying the interaction motifs in SYP121 and PIP2;7 using ratiometric bimolecular fluorescence complementation assays in N. benthamania and the split-ubiquitin system in yeast. Mutation of the SYP121 motif required for its interaction with KC1 did not prevent the interaction between SYP121 and PIP2;7, indicating that the SYP121 motifs involved in the interactions with KC1 and PIP2;7 are different. As for other Qa-SNAREs, SYP121 can be divided into four regions from the N- to the C-terminus: N, H, Q and C. Sequential deletions of these regions revealed that the C region, containing the transmembrane domain, as well as the H and Q regions, containing the Habc and Qa-SNARE domains, interact separately with PIP2;7. Neither the linker between the Habc and the Qa-SNARE domains nor the H or the Q region alone could interact with PIP2;7, indicating that the motif depends on the conformation taken by the HQ region. Regarding the PIP2;7 motif(s), deletion of the cytosolic N- and/or C- terminus led to a significant decrease in the interaction with SYP121. Shorter deletions revealed that the amino acid residues 18 to 26 of the N-terminus are involved in the interaction. As deletions can affect protein conformation, we decided to identify a PIP protein that does not interact with PIP2;7 to perform swapping between the two isoforms. PIP2;6 was identified as non-interacting homolog of PIP2;7. Domain swapping between PIP2;6 and PIP2;7 showed that the N- and C-termini of PIP2;7 in PIP2;6 were not sufficient to create an interaction with SYP121, and that the PIP2;7 N-terminal part up to the loop C was required to restore the full interaction signal. These results suggest that, as it is the case for SYP121, the motif(s) of interaction in PIP2;7 depend on the protein conformation. We did not succeed to demonstrate the presence of a tripartite complex between PIP2;7, SYP121 and KC1 using the yeast split-ubiquitin bridge system and pull-down assays. Altogether, the data obtained in this work indicates that the interaction between SYP121 and PIP2;7 depends on the conformation of both proteins. The possible physiological role of this interaction is discussed in the manuscript.(SC - Sciences) -- UCL, 201