Tonoplast of Beta vulgaris L. contains detergent-resistant membrane microdomains

Abstract

The experiments conducted on tonoplast of Beta vulgaris L. roots were performed to identify detergent-resistant lipid-protein microdomains (DRMs, interpreted as lipid rafts).The presence of DRMs can be found when dynamic clustering of sphingolipids, sterols, saturated fatty acids is registered, and the insolubility of these microdomains in nonionic detergents at low temperatures is proven. The elucidation of tonoplast microdomains has been based on results obtained with the aid of high-speed centrifuging in the sucrose gradient. The experiments have shown that tonoplast microdomains are rich in sphingolipids, free sterols and saturated fatty acids (such a lipid content is also typical of lipid-protein microdomains of other membranes), while only few phospholipids are present in tonoplast microdomains. The presence of microdomains has been confirmed by fluorescence and confocal microscopy using filipin and Laurdan as fluorescent probes. The experiments with Laurdan have shown that tonoplast microdomains are characterized by a high order compared to characteristics of the rest of the tonoplast. Thus, the presence of detergent-resistant lipid-protein microdomains in the tonoplast has been demonstrated.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…Recently, similar sphingolipid desaturases have been characterized in the diatom T. pseudonana (Michaelson et al, 2013;Tonon et al, 2005). In yeast, and in Arabidopsis, regions within the vacuolar bilayer, enriched in sphingolipids and sterols (Chung et al, 2003;Finnigan et al, 2011), have been identified as essential to the function of vacuolar-ATPases (Ozolina et al, 2013;Yoshida et al, 2013). Vacuolar-ATPases are the proton pumps in plants (reviewed in Martinoia et al, 2007) that provide energy for NO 3 2 transport into the vacuole via NO 3 2 ClC transporters identified in this study.…”

“…Vacuolar membrane lipids are poorly characterized in diatoms, but those that make up the vacuolar membranes in plants that store NO 3 2 provide a starting point for consideration of the composition of P. tricornutum vacuolar membranes. In vascular plants, the majority of vacuolar membrane lipids are phospholipids PE and PC, sterols, and sphingolipids, with sterols and sphingolipids comprising 30% and 10 to 20%, respectively (Ozolina et al, 2013;Yoshida and Uemura, 1986;reviewed in Zhang et al, 2015). Within the lipid cluster subset, we highlighted five genes encoding proteins with functions that are putatively well suited to vacuolar membrane lipid biosynthesis in P. tricornutum.…”

The Endoplasmic Reticulum Is the Main Membrane Source for Biogenesis of the Lytic Vacuole inArabidopsis

“…Alternatively, the presence of VHA-a2 and VHA-a3 in DRMs could simply be due to residual tonoplast membranes in the PMenriched samples. Indeed, the presence of DRMs in tonoplast preparations has recently been confirmed (Ozolina et al, 2012) and is in agreement with the fact that both sterols and sphingolipids, two classes of lipids typically enriched in DRMs, are present in amounts comparable to the PM (Yoshida and Uemura, 1986). Here, using the sterol binding probe filipin III, we have not only shown that sterols are indeed present in the tonoplast of Arabidopsis root tip cells but also that they still reach the tonoplast when ER export is blocked by BFA treatment in the sensitized GNL1-BFA S background.…”

Lipid-Protein Microinclusions in the Morphological Structures of Organelle Membranes Studied by Fluorescent Confocal Microscopy

“…The stimulus for our investigation was absence of plausible knowledge about the structure and the functions of lipid-protein microformations in cell organelles, as well as the interest to the unusual membrane microinclusions registered in our observations earlier [3]. In particular, the domain structure of vacuolar membranes has not been plausibly scanned and investigated before.…”

“…As obvious from earlier publications of several authors, the cell membrane is characterized by mobile lateral nonhomogenieties of its lipid content represented in the form of microinclusions [1] [2] [3]. These microinclusions may be studied in experiments presuming 1) visualization of the cell membranes with finding out examples of such microinclusions and 2) investigation of their characteristics.…”