Servicenavigation

Detailseite

Zurück

Projekt Druckansicht

R-SNARE Kaliumkanal Interaktionen in der Koordinierung von Vesikel- und Ionentransport

Fachliche Zuordnung Biochemie und Biophysik der Pflanzen
Genetik und Genomik der Pflanzen
Zell- und Entwicklungsbiologie der Pflanzen
Förderung Förderung von 2012 bis 2022
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 226667644
 
Erstellungsjahr 2019

Zusammenfassung der Projektergebnisse

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.

Projektbezogene Publikationen (Auswahl)

 
 

Zusatzinformationen

© 2025 DFG Kontakt / Impressum / Datenschutz

Textvergrößerung und Kontrastanpassung