R-SNARE K-channel interactions in coordinating vesicle trafficking and ion transport
Plant Genetics and Genomics
Plant Cell and Developmental Biology
Final Report Abstract
Previously, we had established a novel, functional role for SNAREs in coordinating traffic and transport. Using a number of different protein-protein interaction techniques we demonstrated a physical interaction of the Qa-SNARE SYP121 with the K+-channel subunit KC1. Voltage-clamp experiments in Xenopus oocytes revealed that coexpression with SYP121 altered the gating behaviour of the AKT1-KC1 heterotetramer. Under K+-limiting conditions the Arabidopsis syp121 line shows reduced growth phenocopying akt1 and kc1. In the course of these analyses, we have also identified specific interactions of K+-channels with the VAMP72-subfamily of R-SNAREs some of which are cognate binding partners of SYP121 and which formed the basis for this Emmy Noether fellowship. As part of this project we found VAMP721 - the cognate SNARE partner of SYP121 - counterbalancing the effect of the latter that we had previously discovered, namely the enhancement of K+-uptake. Conversely, overexpression of VAMP721 leads to a reduced ‘open-probability’ of the K+-channels KC1 and KAT1 in oocyte experiments and a decrease in K+-uptake under limiting conditions in stably transformed Arabidopsis plants. A single amino-acid residue in the Longin domain of the VAMP (Tyr57) seems responsible for this effect. The Longin domain of R-SNAREs is thought to shield the R-SNARE motif prior interaction with cognate SNARE partners. However, other studies also pointed towards a potential role in determining subcellular localisation. We found that mutation of this tyrosine residue leads to a loss in SNARE-SNARE interaction. Consequently, the VAMP721Y57D mutant cannot complement the seedling-lethal phenotype of homozygous vamp721vamp722 lines. We also observed that expression of the mutated SNARE under its native promoter was reduced significantly in Arabidopsis. This could protect the cells from a cytotoxic effect, as overexpression of VAMP721Y57D leads to seedling lethality, the mechanism of which we are currently investigating. In a project that directly arose from our work on the R-SNAREs, we identified the orthologues of the Guided Entry of Tail-anchored (TA) proteins (GET) pathway. Arabidopsis has three paralogues of the cytosolic chaperone GET3, which in yeast binds nascent tail-anchored (TA) proteins (such as SNAREs) from the ribosome, and one candidate for the ER-membrane localised GET1, which is both, docking station for GET3 and ‘insertase’ of the TA protein. Work in the mammalian/yeast field has focussed on structural and biochemical analyses, which corroborated the general ability of GET pathway components to facilitate ER insertion of TA proteins. Most of this work, however, was executed in vitro or unicellular model organisms as the lethality of pathway knockouts in animals impaired studies on an organismic level. As global analyses in animal systems are impaired, Arabidopsis is indeed the first multicellular model to be analysed for universal consequences of loss of GET function. As we found specific interaction with VAMP721 but not its mutant version, we followed up on our analysis of GET orthologues in Arabidopsis. We have since identified the key players of the pathway in planta, and found a novel stunted-growth phenotype in root hairs of Atget lines. This effect seems to stem from reduced abundance of a specific tail-anchored SNARE protein, which is necessary for the fast polar growth of root hairs. Noteworthy, AtGET3a overexpression in the absence of its membrane receptor gives rise to a drastic phenotype (lethal) and implies an intricate yet poorly understood integration of the GET pathway into cellular homeostasis. Our findings substantially extend current observations in the field and will hence provide important impulses to solving the problem of how the GET pathway is integrated into the cellular proteostasis network.
Publications
- (2014) Voltage-sensor transitions of the inward-rectifying K+ channel KAT1 indicate a latching mechanism biased by hydration within the voltage sensor. Plant Physiology 166:960-75
Lefoulon C, Karnik R, Honsbein A, Gutla PV, Grefen C, Riedelsberger J, Poblete T, Dreyer I, Gonzalez W, Blatt MR
(See online at https://doi.org/10.1104/pp.114.244319) - (2015) A vesicletrafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion. Nature Plants 1:15108
Grefen C, Karnik R, Larson E, Lefoulon C, Wang YZ, Waghmare S, Zhang B, Hills A, Blatt MR
(See online at https://doi.org/10.1038/NPLANTS.2015.108) - (2015) Binary 2in1 vectors improve in planta (co- ) localisation and dynamic protein interaction studies. Plant Physiology 168:776-787
Hecker A, Wallmeroth N, Peter S, Blatt MR, Harter K, Grefen C
(See online at https://doi.org/10.1104/pp.15.00533) - (2015) Binding of SEC11 indicates its role in SNARE recycling after vesicle fusion and identifies two pathways for vesicular traffic to the plasma membrane. The Plant Cell 27:675-694
Karnik R, Zhang B, Waghmare S, Aderhold C, Grefen C, Blatt MR
(See online at https://doi.org/10.1105/tpc.114.134429) - (2015) The R-SNARE VAMP721 Interacts with KAT1 and KC1 K+ Channels to Moderate K+ Current at the Plasma Membrane. The Plant Cell 27:1697-717
Zhang B, Karnik R, Wang Y, Wallmeroth N, Blatt MR, Grefen C
(See online at https://doi.org/10.1105/tpc.15.00305) - (2016) Techniques for the Analysis of Protein-Protein Interactions in Vivo. Plant Physiology 171:727–758
Xing S, Wallmeroth N, Berendzen KW, Grefen C
(See online at https://doi.org/10.1104/pp.16.00470) - (2017) Loss of GET pathway orthologues in Arabidopsis thaliana causes root hair growth defects and affects SNARE abundance. Proc Natl Acad Sci USA 114:E1544–E1553
Xing S, Mehlhorn DG, Wallmeroth N, Asseck LY, Kar R, Voss A, Denninger P, Schmidt VAF, Schwarzländer M, Stierhof YD, Grossmann G, Grefen C
(See online at https://doi.org/10.1073/pnas.1619525114) - (2018) 2in1 Vectors Improve In Planta BiFC and FRET Analyses. Methods in Molecular Biology 1691:139-158
Mehlhorn DG, Wallmeroth N, Berendzen KW, Grefen C
(See online at https://doi.org/10.1007/978-1-4939-7389-7_11) - (2018) ER Membrane Protein Interactions Using the Split-Ubiquitin System (SUS). Methods in Molecular Biology 1691:191-203
Asseck LY, Wallmeroth N, Grefen C
(See online at https://doi.org/10.1007/978-1-4939-7389-7_15) - (2018) The GET pathway can increase the risk of mitochondrial outer membrane proteins to be mistargeted to the ER. Journal of Cell Science 131: jcs211110
Vitali DG, Sinzel M, Bulthuis EP, Kolb A, Zabel S, Mehlhorn DG, Costa BF, Farkas A, Clancy A, Schuldiner M, Grefen C, Schwappach B, Borgese N, Rapaport D
(See online at https://doi.org/10.1242/jcs.211110)
