ArabidopsisSec1/Munc18 Protein SEC11 Is a Competitive and Dynamic Modulator of SNARE Binding and SYP121-Dependent Vesicle Traffic  

Abstract

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual "handshaking" mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane

“…The cytosolic fragments of the Qa-SNAREs SYP121 DC (=SYP121-Sp2) and SYP122 DC (=SYP122-Sp2) were shown previously to block secretion at a stage late in transit to the plasma membrane in tobacco (Nicotiana tabacum) and Arabidopsis (Geelen et al, 2002;Sutter et al, 2006;Tyrrell et al, 2007;Besserer et al, 2012;Karnik et al, 2013b). Secretory block was proposed ( Figure 1A) to arise by the titration of cognate SNARE partners, thus accounting for the lack of selectivity between the cytosolic fragments (Tyrrell et al, 2007), but this interpretation was not addressed directly using the complementary mutant backgrounds.…”

“…Cocultivation with Agrobacterium tumefaciens normally gives a strong transformation of the root epidermis (Honsbein et al, 2009;Grefen et al, 2010b;Karnik et al, 2013b). Therefore, we quantified secYFP traffic by confocal microscopy of seedling roots, using standardized excitation and emission settings, and analyzed 3D projections for YFP and GFP fluorescence after subtracting background fluorescence signals recorded from untransformed seedlings.…”

“…Secretory block was proposed ( Figure 1A) to arise by the titration of cognate SNARE partners, thus accounting for the lack of selectivity between the cytosolic fragments (Tyrrell et al, 2007), but this interpretation was not addressed directly using the complementary mutant backgrounds. We expressed the fluorescent secretory marker secreted yellow fluorescent protein (secYFP) and the endoplasmic reticulum marker GFP-HDEL with and without SYP121 DC ( Figure 1B) and SYP122 DC using the tetracistronic vector pTecG-2in1-CC ( Figure 1C) (Karnik et al, 2013b) to transiently transform Arabidopsis Columbia-0 (wild type), syp121-1 (syp121), and syp122-1 (syp122) mutant seedlings Honsbein et al, 2009;Grefen et al, 2010b). secYFP is exported from the cell and normally yields little or no fluorescence once in the apoplast.…”

“…Both SYP121 and SYP122 are expressed throughout the development of the plant and in most, if not all, tissues (Uemura et al, 2004;Enami et al, 2009). The Qa-SNAREs also share the cognate partner proteins SNAP33, VAMP721, and VAMP722 Rehman et al, 2008;Karnik et al, 2013b), indicating substantial mechanistic overlaps in driving vesicle fusion. Nonetheless, a number of functional differences between SYP121 and SYP122 have surfaced.…”

“…Nonetheless, SEC11 is expressed throughout the vegetative plant and not just during cell division. Karnik et al (2013b) demonstrated that SEC11 will bind with SYP121 and regulate its activity at the plasma membrane. Significantly, SEC11 binding to the N terminus of SYP121 affected binary interaction with the Qa-SNARE but not its assembly in the SNARE core complex.…”