Dual Sites for SEC11 on the SNARE SYP121 Implicate a Binding Exchange during Secretory Traffic.

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 F ⁹ xRF motif that is shared with the K ⁺ channels. This second, previously unrecognized motif is centered on residues R ²⁰ R ²¹ of SYP121 and is essential for SEC11 interaction with SYP121. Mutation of the R ²⁰ R ²¹ motif blocked vesicle traffic without uncoupling the effects of SYP121 on solute and K ⁺ uptake associated with the F ⁹ xRF motif; the mutation also mimicked the effects on traffic block observed on coexpression of the dominant-negative SEC11 ^Δ149 fragment. We conclude that the R ²⁰ R ²¹ 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.
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