A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K + channel subunit that assembles with different inward-rectifying K + channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K + channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K + channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inwardrectifying K + current in Arabidopsis root epidermal protoplasts as well as K + acquisition and growth in seedlings when channel-mediated K + uptake was limiting. That SYP121 should be important for gating of a K + channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE-ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K + uptake coordinately with membrane expansion for cell growth.

Iron acquisition in Arabidopsis depends mainly on AtIRT1, a Fe 2؉ transporter in the plasma membrane of root cells. However, substrate specificity of AtIRT1 is low, leading to an excess accumulation of other transition metals in iron-deficient plants.In the present study we describe AtIREG2 as a nickel transporter at the vacuolar membrane that counterbalances the low substrate specificity of AtIRT1 and possibly other iron transport systems in iron-deficient root cells. showed an increased tolerance to elevated concentrations of nickel, whereas T-DNA insertion lines lacking AtIREG2 expression were more sensitive to nickel, particularly under iron deficiency, and accumulated less nickel in roots. We therefore propose a role of AtIREG2 in vacuolar loading of nickel under iron deficiency and thus identify it as a novel component in the iron deficiency stress response.Iron deficiency in plants is visually expressed as chlorosis, first appearing in the youngest developing leaves and accompanied by a reduction in growth rate, dry matter production, and in most cases by a decrease of iron concentration (1). At the same time, root uptake capacities and leaf concentrations of other divalent metal cations increase (2, 3). In soils or nutrient solutions with unbalanced microelement supply, iron deficiency can then promote the toxicity of other transition metals (4, 5). Such toxicity seems also to be the case with nickel, supported by the observation that phytotoxicity of nickel decreased with increasing iron:nickel ratios in the leaf tissue (6, 7). Thus, an increased sensitivity to heavy metals under iron deficiency might represent a secondary, growth-limiting factor besides the lack of iron itself.Molecular studies in yeast showed that an iron deficiencyinduced accumulation of transition metals other than iron was explained by a higher activity of non-selective low affinity iron transport. Deletion of FET3, an essential component for high affinity iron uptake in yeast, leads to a constitutive iron-deficient phenotype and a concomitant up-regulation of FET4, which encodes a low affinity Fe(II) transporter with poor substrate selectivity (8). As a consequence, sensitivity to elevated concentrations of the transition metals cobalt, copper, zinc, and manganese was higher in fet3 mutants than in the corresponding wild type, consistent with increased metal accumulation (8).In Arabidopsis, iron-dependent overaccumulation of divalent metal cations was found to be mediated by the Fe(II) transporter AtIRT1, which in fact transports a broad range of transition metals (9). Atirt1 T-DNA insertion lines no longer accumulated manganese, zinc, and cobalt under iron deficiency and even showed an increased tolerance to toxic levels of cadmium (10). Thus, accumulation of certain transition metals in iron-deficient Arabidopsis plants directly depends on AtIRT1 and appears as an unavoidable side effect of iron deficiencyinduced iron acquisition.In the search for genes that might be involved in metal transport in Arabidopsis roots, homology to ...

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.

The SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/ PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane, and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K + channel subunit KC1 and forms a tripartite complex with a second K + channel subunit, AKT1, to control channel gating and K + transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for proteinprotein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this motif is the minimal requirement for SNARE-dependent alterations in K + channel gating when heterologously expressed, and that rescue of KC1-associated K + current of the root epidermis in syp121 mutant Arabidopsis plants depends on expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously unidentified motif required for SNARE-ion channel interactions and lead us to suggest a mechanistic framework for understanding the coordination of vesicle traffic with transmembrane ion transport.

Multidomain scaffolding proteins organize the molecular machinery of neurotransmitter vesicle dynamics during synaptogenesis and synaptic activity. We find that domains of five active zone proteins converge on an interaction node that centers on the N-terminal region of Munc13-1 and includes the zinc-finger domain of Rim1, the C-terminal region of Bassoon, a segment of CAST1/ELKS2, and the third coiled-coil domain (CC3) of either Aczonin/Piccolo or Bassoon. This multidomain complex may constitute a center for the physical and functional integration of the protein machinery at the active zone. An additional connection between Aczonin and Bassoon is mediated by the second coiled-coil domain of Aczonin. Recombinant Aczonin-CC3, expressed in cultured neurons as a green fluorescent protein fusion protein, is targeted to synapses and suppresses vesicle turnover, suggesting involvements in synaptic assembly as well as activity. Our findings show that Aczonin, Bassoon, CAST1, Munc13, and Rim are closely and multiply interconnected, they indicate that Aczonin-CC3 can actively participate in neurotransmitter vesicle dynamics, and they highlight the N-terminal region of Munc13-1 as a hub of protein interactions by adding three new binding partners to its mechanistic potential in the control of synaptic vesicle priming.