Recognition of lipid-protein rafts in vacuolar membrane

Abstract

120 The well known Singer–Nicholson model, according to which proteins “float” in a homogeneous lipid sea, has undergone significant changes. Today it is generally accepted that cell membranes are highly heterogeneous: they contain various lipid–protein structures that are involved in many metabolic pro cesses occurring in living cells [1]. According to the model of lipid–protein microdomains (rafts), which is currently most widespread, areas enriched in gly cosphingolipids, sterols, and lipids with saturated fatty acids occur around certain proteins; lipids in these areas are characterized by a denser packing of mole cules compared to the surrounding areas of mem branes [2]. The interest to rafts is determined by the increasing number of experimental data that confirm their involvement in vital cellular processes, such as intrac ellular signaling, endocytosis, apoptosis, sorting of proteins, as well as internalization of toxins, bacteria, viruses, etc. [3, 4]. In addition to specific lipid compo sition, these membrane domains are characterized by specific protein profiles. In plant plasma membrane rafts, P and V type ATPases, protein kinases, mono meric and heteromeric G proteins, 14 3 3 family proteins, aquaporins, etc. were identified [5]. Rafts have been found in the plasma membrane and in endoplasmic reticulum and Golgi membranes. Mitochondrial membranes do not contain rafts [6]. Data on the lipid composition of tonoplasts [7] suggest that plant vacuolar membranes may also contain pro tein–lipid rafts. In this study, we found lipid protein domains (rafts) in the vacuolar membrane. Experiments were performed with isolated vacu oles and membrane vesicles of dormant storage roots of the red beet (Beta vulgaris L.) variety Bordeaux. Vacuoles and tonoplast vesicles from the storage root tissue were isolated and purified as described in [8]. Sterol enriched microdomains (rafts) were detected by fluorescent microscopy using the sterol binding antibiotic filipin [9]. To ensure the binding of filipin to the vacuolar membrane, a suspension of isolated vac uoles in the isolation medium (0.8 M KCl, 20 mM EDTA, and 50 mM Na2HPO4 (pH 8.0)) was supple mented with filipin (0.2% solution in DMSO) to a final concentration of 5 μM. The suspension was incu bated at 20 ± 2°С for 30 min, washed in the same buffer, and analyzed under an Axio observer Z1 fluo rescence microscope (Carl Zeiss, Germany) equipped with an Axio Cam MRm monochrome digital camera and Axio Vision Rel.4.8 software package for captur Recognition of Lipid–Protein Rafts in Vacuolar Membrane