VAMP721 Conformations Unmask an Extended Motif for K⁺ Channel Binding and Gating Control

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play a major role in membrane fusion and contribute to cell expansion, signaling, and polar growth in plants. The SNARE SYP121 of Arabidopsis thaliana that facilitates vesicle fusion at the plasma membrane also binds with, and regulates, K + channels already present at the plasma membrane to affect K + uptake and K + -dependent growth. Here, we report that its cognate partner VAMP721, which assembles with SYP121 to drive membrane fusion, binds to the KAT1 K + channel via two sites on the protein, only one of which contributes to channelgating control. Binding to the VAMP721 SNARE domain suppressed channel gating. By contrast, interaction with the aminoterminal longin domain conferred specificity on VAMP721 binding without influencing gating. Channel binding was defined by a linear motif within the longin domain. The SNARE domain is thought to wrap around this structure when not assembled with SYP121 in the SNARE complex. Fluorescence lifetime analysis showed that mutations within this motif, which suppressed channel binding and its effects on gating, also altered the conformational displacement between the VAMP721 SNARE and longin domains. The presence of these two channel-binding sites on VAMP721, one also required for SNARE complex assembly, implies a well-defined sequence of events coordinating K + uptake and the final stages of vesicle traffic. It suggests that binding begins with VAMP721, and subsequently with SYP121, thereby coordinating K + channel gating during SNARE assembly and vesicle fusion. Thus, our findings also are consistent with the idea that the K + channels are nucleation points for SNARE complex assembly.

SNAREs SYP121 and SYP122 Mediate the Secretion of Distinct Cargo Subsets

“…To test the findings of our proteomic analysis, we analyzed a subset of cargo proteins to determine if their secretion was mediated by SYP121 and SYP122. We performed secretion assays in Arabidopsis roots after transient transformation to express the fluorescently tagged cargo proteins (Honsbein et al, 2009;Grefen et al, 2010b;Zhang et al, 2017). We tested Protein Disulfide Isomerase-Like1 (PDIL1; AT1G21750), GDSL (AT1G28660), and Pectin Methylesterase Inhibitor Protein (PMEI; AT2G26440) as potential candidates associated with traffic mediated by SYP121 and BETA-GLUCOSIDASE34 (BGLU34; AT1G47600) as mediated by SYP122.…”

Vesicle transport in plants: A revised phylogeny of SNARE proteins

“…This specificity of t-SNARE and v-SNARE complex formation ensures that vesicles are targeted to the correct compartment and induce membrane fusion [61]. For example, SYP111 takes part in membrane fusion events forming the cell plate and the transport of secretory vesicles at the plasma membrane [34,[62][63][64]. SYP122 plays a role in cell wall deposition and in tethering of donor and target membrane [65].…”

“…Xue et al [26] found that VAMP711 regulates ABA-mediated inhibition of PM H+-ATPase activity and drought stress response by regulating stoma closure. Zhang et al [19,64] found that VAMP721 and VAMP722 interact with the same K+ channels and that this interaction suppresses channel activity, and VAMP721 assembles with SYP121 to coordinate K+ channel gating during SNARE assembly and vesicle fusion. Kim et al [102] found that CALRETICULIN 1 (CRT1) and CRT2 are critical components in the accumulation of VAMP721 and VAMP722 during ER stress responses.…”

K+ Channel-SEC11 Binding Exchange Regulates SNARE Assembly for Secretory Traffic

“…We used the mating-based split-ubiquitin system (mbSUS) assay, previously employed successfully to identify K 1 channel binding motifs (Honsbein et al, 2009;Grefen et al, 2010;Karnik et al, 2015;Zhang et al, 2015Zhang et al, , 2017Horaruang and Zhang, 2017), to assess channel binding with the cognate SNAREs and identify the relevant binding motifs. The mbSUS assay takes advantage of protein fusions with the N-and C-terminal halves (Nub-X and Y-Cub) of ubiquitin.…”

“…The cognate R-SNAREs VAMP721 and VAMP722 also bind with these K 1 channels, but in contrast with SYP121, R-SNARE binding suppresses channel gating (Zhang et al, 2015). The R-SNAREs incorporate, within their regulatory (so-called longin) domain, a linear binding motif for the channels of GHTFNYLVExGxxY centered around Tyr-57 (Zhang et al, 2015(Zhang et al, , 2017. At present, we do not know the complementary motif on the K 1 channels.…”