Mapping of Plasma Membrane Proteins Interacting With Arabidopsis thaliana Flotillin 2

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.

Cell wall contributes to the stability of plasma membrane nanodomain organization of Arabidopsis thaliana FLOTILLIN2 and HYPERSENSITIVE INDUCED REACTION1 proteins

“…The interaction of At FLOTs (or possibly also At HIRs) with the CW constituents is probably indirect, that is via other proteins, supposedly with the ones containing extracellular domains. In our previous study, we found several transmembrane proteins, such as receptor‐like kinases, aquaporins, ATPases or other transporters in the interactome of At FLOT2 (Junková et al , ). At FLOT2 and At FLOT3 also interact with At TBL36 (associomics.org), a homologue of transmembrane trichome birefringence protein which is necessary for proper cellulose composition of the CW (Bischoff et al , ).…”

“…Similar effect was observed in mammalian homologues of flotillins in which hetero‐oligomerization is important for endocytosis provided by flotillin nanodomains (Neumann‐Giesen et al , ; Solis et al , ; Babuke and Tikkanen, ). Recently, we have revealed several PM proteins constituting the interactome of At FLOT2 (Junková et al , ). In addition, At HIRs were found to be a part of PM‐associated complexes, which also contained phospholipase Dδ, cytoskeletal proteins, heat‐shock proteins, clathrin chains, transporters or kinases (Ho et al , ; Qi and Katagiri, ).…”

Flotillin membrane domains in cancer

“…In various plant genomes, flotillin homologs were identified. For instance, in Arabidopsis thaliana, there are three homologs that are detected in plasma membrane DRM [40].…”

Ubiquitin recognition in endocytic trafficking – with or without ESCRT-0

“…In mammals, among others, endophilins, caveolins, and flotillins are important in clathrin-independent endocytosis events [reviewed in (Ferreira and Boucrot, 2018)]. There are three homologues of flotillin in Arabidopsis that localise on the plasma membrane; however, none of them has been well characterised to date (Junkova et al, 2018). Homologues of caveolin do not exist in plants and, as discussed before, a true functional orthologue of endophilins has not been identified yet, though SH3Ps could represent possible candidates.…”