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. 2018 Jul 12:9:991.
doi: 10.3389/fpls.2018.00991. eCollection 2018.

Mapping of Plasma Membrane Proteins Interacting With Arabidopsis thaliana Flotillin 2

Petra Junková  1 , Michal Daněk  2   3 , Daniela Kocourková  2 , Jitka Brouzdová  2 , Kristýna Kroumanová  2 , Enric Zelazny  4 , Martin Janda  1   2 , Radovan Hynek  1 , Jan Martinec  2 , Olga Valentová  1
Affiliations

Mapping of Plasma Membrane Proteins Interacting With Arabidopsis thaliana Flotillin 2

Petra Junková et al. Front Plant Sci. .

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 protein-like 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.

Keywords: Arabidopsis flotillin 2; immunopurification; intracellular trafficking; mass spectrometry; plant–pathogen interaction; protein–protein interactions; split-ubiquitin yeast system; water transport.

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Figures

FIGURE 1
FIGURE 1
Flot2-GFP localization at plasma membrane. (A) Confocal microscopy images showing Flot2-GFP localization at plasma membrane in epidermal cells of roots and cotyledons of Arabidopsis Flot2-GFP plants; (B) Confirmation of Flot2-GFP localization at plasma membrane in plasmolyzed root epidermal cells. The Flot2-GFP signal is detected at the plasma membrane of contracted protoplasts. Seedlings were treated with 0.8 M mannitol and subsequently stained with propidium iodide to mark cell walls.
FIGURE 2
FIGURE 2
Preparation and characterization of Flot2-GFP membrane fractions. (A) Flow chart of the preparation and analysis of membrane fractions; (B) Immunoblot analysis of Flot2-GFP content in cytosolic fraction (C), microsomal fraction (M), supernatant obtained after pelleting of cross-linked microsomal fraction (Sx), cross-linked microsomal fraction (Mx), plasma membrane fraction isolated from cross-linked microsomal fraction (PMx), 0.5 μg of total proteins were loaded into all lines; (C) Immunoblot analysis of Flot2-GFP content in whole cross-linked microsomal fraction (Mx), fraction dissolved by 0.5% (w/v) sodium deoxycholate (Mx-S) and fraction undissolved by 0.5% (w/v) sodium deoxycholate (Mx-P), 10 μg of total proteins were loaded into all lines.
FIGURE 3
FIGURE 3
Cluster analysis of GO annotation terms of proteins enriched by IP of microsomal and plasma membrane fraction. The DAVID Bioinformatics Resources functional annotation clustering algorithm was used to cluster the most significant terms in the GO Cellular Component and GO Biological Process annotations. The terms are ordered from least frequent (top) to most frequent (bottom). Black bars indicate terms enriched with p < 0.01, gray bars indicate terms enriched with p ≥ 0.01.
FIGURE 4
FIGURE 4
SUS test. Yeast strain THY.AP4 co-expressing Flot2-Cub-PLV and Nub fused with investigated possible interactors were plated in decimal dilution (OD600 = 1.00 or 0.10 or 0.01) onto selective media (without Ade, His, Leu, Trp) which was supplemented with 50, 250, or 500 μM Met in order to obtain a different expression level of the bait. Yeast growth rate was gradually repressed with an increasing concentration of Met. Only plates with 500 μM Met are presented. Plates were incubated for 48 h at 28°C. NubWT and NubG as prey were used as positive and negative controls, respectively. Similar growth performance was observed for each interaction with at least three biological replicates.
FIGURE 5
FIGURE 5
Graphical representation of Flot2 interaction network. Interactors experimentally determined in our study are displayed in red boxes. Solid red lines represent direct binding with Flot2 verified by SUS, while dashed red lines depict putative indirect interactions with Flot2 with negative SUS results. Blue boxes and lines represent direct (solid lines) or indirect (dashed lines) interactions published in literature and mentioned in discussion. Pair–wise interactions confirmed by yeast two hybrid or split ubiquitin assay, bimolecular fluorescence complementation or FRET/FLIM measurement are assumed as direct. Proteins found in co-IP assays and determined by mass spectrometry or revealed in tagged-protein pull-down assays, which were neither tested nor confirmed to be direct (by the methods listed above), were considered putative indirect interactors. Black boxes and lines represent most common direct interactions retrieved from Associomics database, which are common to several proteins detected in our study as well as Flot2. The implication of interactors in important biological processes is distinguished by colored fields.
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