Cytoskeletal Components Define Protein Location to Membrane Microdomains*

Abstract

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases. Molecular & Cellular Proteomics 14: 10.1074/ mcp.M114.046904, 2493-2509, 2015.Living cells need borders and molecular compartments for biochemical reactions and storage of metabolites. The plasma membrane therefore is a prerequisite for the evolution of different life forms. It consists of a phospholipid bilayer into which proteins and special lipid species such as sterols, sphingolipids, and glycolipids are inserted. The first complex model of plasma membrane was proposed in 1972 by Jonathan Singer and Garth Nicolson (1), replacing the concept of the plasma membrane as a strict protein-lipid-protein sandwich that was generally accepted until then. In Singer and Nicolson's model, the cell membrane is a two-dimensionally oriented viscous solution in which the membrane constituents are orientated in the most thermodynamically favorable manner, hiding hydrophobic hydrocarbon chains inside the lipid bilayer and exposing polar and ionic groups to the aqueous phase. This fluid mosaic model also implied that membrane proteins as well as lipid components are distributed in a homogeneous lipid bilayer at long range, but they can form specific aggregates and phases at short range, which were also termed "lipid rafts" or membrane microdomains.Over the past 30 years, it has become evident that the plasma membrane is not such a homogeneous structure as it was initially proposed. We now know that the lipid bilayer is asymmetric (2) and that the free diffusion of membrane proteins is restricted by their interactions with intracellular and extracellular components (3). More recently, Simons and Ikonen suggested that large ordered phases, enriched with cholesterol and sphin...

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics

“…As a result, polar auxin transport is drastically reduced in abcb1 abcb19 and twd1, leading to widely overlapping phenotypes, including dwarfism, disoriented growth, and helical rotation (twisting) of epidermal layers (Geisler et al, 2003;Wu et al, 2010;Wang et al, 2013). Epidermal twisting in twd1/fkbp42 is in contrast to mutations of tubulin subunits, such as the rice (Oryza sativa) mutant twisted dwarf1 (Szymanski et al, 2015), nonhanded. The chaperone function of TWD1/FKBP42 is in functional analogy with the closest mammalian ortholog, FKBP38, shown to chaperone ABCC7/CFTR to the PM (Banasavadi-Siddegowda et al, 2011), but underlying mechanisms are not clear.…”

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

“…Szymanski et al (2015) recently demonstrated that actin filaments are related to the formation and dynamics of microdomains and that microtubules regulate the amounts of microdomain proteins by observation of microdomain marker proteins such as Remorin1.2 during deletion of actin filaments or microtubules. Therefore, the proper organization of PM microdomains would need not only lipids such as sphingolipids but also proteins, especially cytoskeleton.…”

Mobility of Group 1b Remorins in Membrane Nanodomains Differs Significantly between Mesophyll Cells and Protoplasts

“…In addition there is only an estimated one third of Arabidopsis plasma membrane proteins reported to exhibit strong sterol-dependencies [5] a basic requirement of proteins for possible raft-associations [11]. In plants associations of plasma membrane proteins or whole protein assemblies with stabilizing structures could therefore overcome decreased sterol levels by, for example, interactions or stabilization with the cytoskeleton [7]. There were less cytoskeletal proteins found in sterol-rich membrane domains in plants compared to those found in animal plasma membrane preparations; at the same time plant cell wall proteins made up the biggest functional group of proteins identified as responding to methyl-ß-cyclodextrin [5].…”

“…Subsequently, it was demonstrated that almost all of the 16 remorin proteins known in Arabidopsis thaliana localize to (different) membrane microdomain platforms [2]. Concerning physiological roles of sterol-and sphingolipid-enriched compartments it was demonstrated in Arabidopsis that regulatory protein-protein interactions occur therein, as shown for the CPK21-mediated activation of the ion channel SLAH3 [7]. It is now established that sterols [8] and sterol-protein interactions play a role in the compartmentalization of plasma membrane proteins in plants and that these interactions play regulatory roles [9].…”