Abstract
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.
Highlights
Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water and other small solutes across the plasma membrane
The N region (N-terminus) contains the FxRF motif required for SYP121 interaction with KC1 (Grefen et al, 2010), the H region contains the regulatory domain Habc, the Q region contains the Q-SNARE motif, and the C region (Cterminus) contains a transmembrane domains (TM) domain (Figure 1A)
Several genetic constructs encoding deletions starting from either the C- or the N-terminus of SYP121 were prepared to identify the region required for the interaction with PIP2;7, using rBiFC assays
Summary
Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water and other small solutes across the plasma membrane. SYP121, one of the nine SYP1s found in Arabidopsis, is involved in the fusion of vesicles with the plasma membrane, together with its cognate SNARE SynaptosomeAssociated Protein (SNAP) 33, a Qb+Qc-SNARE, and the R-SNAREs Vesicle-Associated Membrane Protein (VAMP) 721 and 722 (Kargul et al, 2001; Kwon et al, 2008; Kwaaitaal et al, 2010) It has the typical structure of Qa-SNAREs with, in front of the Habc, a 39-residue N-terminal region (Sanderfoot, 2007; Tyrrell et al, 2007; Grefen et al, 2010; Fujiwara et al, 2014). It is expressed in all tissues, albeit at lower levels (Enami et al, 2009)
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