Selective Mobility and Sensitivity to SNAREs Is Exhibited by theArabidopsisKAT1 K+ Channel at the Plasma Membrane

Abstract

Recent findings indicate that proteins in the SNARE superfamily are essential for cell signaling, in addition to facilitating vesicle traffic in plant cell homeostasis, growth, and development. We previously identified SNAREs SYP121/Syr1 from tobacco (Nicotiana tabacum) and the Arabidopsis thaliana homolog SYP121 associated with abscisic acid and drought stress. Disrupting tobacco SYP121 function by expressing a dominant-negative Sp2 fragment had severe effects on growth, development, and traffic to the plasma membrane, and it blocked K þ and Cl ÿ channel responses to abscisic acid in guard cells. These observations raise questions about SNARE control in exocytosis and endocytosis of ion channel proteins and their organization within the plane of the membrane. We have used a dual, in vivo tagging strategy with a photoactivatable green fluorescent protein and externally exposed hemagglutinin epitopes to monitor the distribution and trafficking dynamics of the KAT1 K þ channel transiently expressed in tobacco leaves. KAT1 is localized to the plasma membrane within positionally stable microdomains of ;0.5 mm in diameter; delivery of the K þ channel, but not of the PMA2 H þ -ATPase, to the plasma membrane is suppressed by Sp2 fragments of tobacco and Arabidopsis SYP121, and Sp2 expression leads to profound changes in KAT1 distribution and mobility within the plane of the plasma membrane. These results offer direct evidence for SNARE-mediated traffic of the K þ channel and a role in its distribution within subdomains of the plasma membrane, and they implicate a role for SNAREs in positional anchoring of the K þ channel protein.

Reconstitution of Abscisic Acid Signaling from the Receptor to DNA via bHLH Transcription Factors

“…Rapid events occur within seconds to promote stomatal closure through physiological cellular responses, including Ca 2+ elevation and regulation of ion channels and pump activities by protein phosphorylation and dephosphorylation . Slow events are thought to inhibit stomatal reopening in the late periods after the increase in ABA concentration and are mediated by gene expression, ion channel modifications, and intracellular relocation of the transporter proteins (Sutter et al, 2006(Sutter et al, , 2007Eisenach, et al, 2012). AKS1 dissociation from the KAT1 promoter reconstituted in this study most likely mediates late events through a decrease in the population of KAT1 proteins in the plasma membrane in coordination with KAT1 endocytosis in response to ABA (Sutter et al, 2007).…”

Roles of ion channels and transporters in guard cell signal transduction

“…post-transcriptional level. Additional regulation of K + channels by trafficking and relocalization may also be an important determinant [41]. Recent elegant studies with transiently expressed GFP-tagged KAT1 and dominant negative SNARE fragments show that KAT1 is localized at the PM in punctuate structures and that syntaxins regulate the trafficking and clustering of these channels [41].…”

Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

“…It seems possible, then, that the Golgi apparatus in plants moves with, and not over, the surface of the ER, a possibility that supports the idea of continuity between Golgi bodies and discrete ER export sites on the ER membrane (daSilva et al, 2004; Hanton et al, 2007a). Another version of PA-GFP that is available for plant endomembrane studies was generated by introducing a T207H site mutation in the plant-codon optimized mGFP5 (Sutter et al, 2006). This PA-GFP variant was used to tag the Arabidopsis (Arabidopsis thaliana) KAT1 K 1 channel to monitor its distribution and trafficking dynamics in tobacco leaves (Sutter et al, 2006).…”

“…Another version of PA-GFP that is available for plant endomembrane studies was generated by introducing a T207H site mutation in the plant-codon optimized mGFP5 (Sutter et al, 2006). This PA-GFP variant was used to tag the Arabidopsis (Arabidopsis thaliana) KAT1 K 1 channel to monitor its distribution and trafficking dynamics in tobacco leaves (Sutter et al, 2006). As mGFP5 may be used in coexpression analyses with YFP with a good spectral separation using the appropriate microscope settings (Brandizzi et al, 2002a, it would be interesting to determine whether the spectral properties of the photoactivated PA-GFP derived from mGFP5 could be used in combination with YFP for colocalization studies.…”