Mapping of plasma membrane proteins interacting with Arabidopsis thaliana Flotillin 2

Junková P., Daněk M., Kocourková D., Brouzdová J., Kroumanová K., Zelazny E., Janda M., Hynek R., Martinec J., Valentová O.

Keywords: Arabidopsis flotillin 2, protein–protein interactions, immunopurification, mass spectrometry, splitubiquitin yeast system, plant–pathogen interaction, water transport, intracellular trafficking
Abstract: Arabidopsis flotillin 2 (At5g25260) belongs to the group of plant flotillins, which are not well characterized. In contrast, metazoan flotillins are well known as plasma membrane proteins associated with membrane microdomains that act as a signaling hub. The similarity of plant and metazoan flotillins, whose functions most likely consist of affecting other proteins via protein–protein interactions, determines the necessity of detecting their interacting partners in plants. Nevertheless, identifying the proteins that form complexes on the plasma membrane is a challenging task due to their low abundance and hydrophobic character. Here we present an approach for mapping Arabidopsis thaliana flotillin 2 plasma membrane interactors, based on the immunoaffinity purification of crosslinked and enriched plasma membrane proteins with mass spectrometry detection. Using this approach, 61 proteins were enriched in the AtFlot-GFP plasma membrane fraction, and 19 of them were proposed to be flotillin 2 interaction partners. Among our proposed partners of Flot2, proteins playing a role in the plant response to various biotic and abiotic stresses were detected. Additionally, the use of the split-ubiquitin yeast system helped us to confirm that plasma-membrane ATPase 1, early-responsive to dehydration stress protein 4, syntaxin-71, harpin-induced proteinlike 3, hypersensitive-induced response protein 2 and two aquaporin isoforms interact with flotillin 2 directly. Based on the results of our study and the reported properties of Flot2 interactors, we propose that Flot2 complexes may be involved in plant–pathogen interactions, water transport and intracellular trafficking.
DOI: 10.3389/fpls.2018.00991
IEB authors: Jitka Brouzdová, Michal Daněk, Martin Janda, Daniela Kocourková, Kristýna Kroumanová, Jan Martinec