Abstract
SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitate vesicle traffic through their assembly in a heteromeric complex that drives membrane fusion. Much of vesicle traffic at the Arabidopsis (Arabidopsis thaliana) plasma membrane is subject to the Sec1/Munc18 protein SEC11, which, along with plasma membrane K+ channels, selectively binds with the SNARE SYP121 to regulate its assembly in complex. How SEC11 binding is coordinated with the K+ channels is poorly understood, as both SEC11 and the channels are thought to compete for the same SNARE binding site. Here, we identify a second binding motif within the N terminus of SYP121 and demonstrate that this motif affects SEC11 binding independently of the F9xRF motif that is shared with the K+ channels. This second, previously unrecognized motif is centered on residues R20R21 of SYP121 and is essential for SEC11 interaction with SYP121. Mutation of the R20R21 motif blocked vesicle traffic without uncoupling the effects of SYP121 on solute and K+ uptake associated with the F9xRF motif; the mutation also mimicked the effects on traffic block observed on coexpression of the dominant-negative SEC11Δ149 fragment. We conclude that the R20R21 motif represents a secondary site of interaction for the Sec1/Munc18 protein during the transition of SYP121 from the occluded to the open conformation that leads to SNARE complex assembly.
Highlights
SNARE proteins facilitate vesicle traffic through their assembly in a heteromeric complex that drives membrane fusion
Growth was weaker with SYP122 as the prey, consistent with the preferential binding and action of SEC11 with SYP121 (Karnik et al, 2013b, 2015)
We used the mCherry fluorescence excited at 552 nm as a reference for comparisons. mCherry fluorescence excited by 488-nm light showed strong and highly significant signals when SEC11 was coexpressed with the SYP121 construct and with chimeras
Summary
SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitate vesicle traffic through their assembly in a heteromeric complex that drives membrane fusion. Much of vesicle traffic at the Arabidopsis (Arabidopsis thaliana) plasma membrane is subject to the Sec1/Munc protein SEC11, which, along with plasma membrane K+ channels, selectively binds with the SNARE SYP121 to regulate its assembly in complex. Like other SM proteins, SEC11 incorporates a minor cleft that interacts with the N terminus of SYP121 and is important for the conformational changes that lead to SNARE complex assembly (Karnik et al, 2013b, 2015). Identifying the additional motif(s) of SYP121 for binding with the SEC11 minor cleft will likely clarify the mechanics of SEC11 and the K+ channels within the SNARE cycle of SYP121. We identify a second, N-terminal SEC11-binding motif associated with residues R20R21 of SYP121 We show that this motif is essential but distinguishable in its SNARE-binding characteristics from the F9xRF motif of SYP121. We demonstrate that the R20R21 motif is important for the selectivity of SEC11 for SYP121 in secretory traffic and for the link between vesicle traffic and osmotic solute uptake during plant growth
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