Plasma Membranes Are Subcompartmentalized into a Plethora of Coexisting and Diverse Microdomains in Arabidopsis and Nicotiana benthamiana

Eukaryotic plasma membranes are highly compartmentalized structures. So far, only a few individual proteins that function in a wide range of cellular processes have been shown to segregate into microdomains. However, the biological roles of most microdomain-associated proteins are unknown. Here, we investigated the heterogeneity of distinct microdomains and the complexity of their coexistence. This diversity was determined in living cells of intact multicellular tissues using 20 different marker proteins from Arabidopsis thaliana, mostly belonging to the Remorin protein family. These proteins associate with microdomains at the cytosolic leaflet of the plasma membrane. We characterized these membrane domains and determined their lateral dynamics by extensive quantitative image analysis. Systematic colocalization experiments with an extended subset of marker proteins tested in 45 different combinations revealed the coexistence of highly distinct membrane domains on individual cell surfaces. These data provide valuable tools to study the lateral segregation of membrane proteins and their biological functions in living plant cells. They also demonstrate that widely used biochemical approaches such as detergent-resistant membranes cannot resolve this biological complexity of membrane compartmentalization in vivo.     

Species-sorting may explain an apparent minimal effect of immigration on freshwater bacterial community dynamics

Long distance atmospheric transport of bacterial cells is often implied as a driver of the apparent cosmopolitan distribution of bacterial taxa. Surprisingly, efforts to measure immigration in bacterial communities are rare. An 8-week time series of within-lake bacterial community composition and atmospheric deposition rates and composition were used to estimate the influence of immigration on bacterial community dynamics in two north temperate lakes. Characterization of bacterial community dynamics using automated ribosomal intergenic spacer analysis suggested moderate overlap in composition between the lakes and atmospherically deposited cells. However, taxa that appeared to be delivered by atmospheric deposition had a relatively minor influence on lake bacterial community dynamics. The weak influence of immigrating bacterial taxa suggests that a species-sorting concept best describes aquatic bacterial metacommunity dynamics.     

Ultrafine membrane compartments for molecular diffusion as revealed by single molecule techniques

Plasma membrane compartments, delimited by transmembrane proteins anchored to the membrane skeleton (anchored-protein picket model), would provide the membrane with fundamental mosaicism because they would affect the movement of practically all molecules incorporated in the cell membrane. Understanding such basic compartmentalized structures of the cell membrane is critical for further studies of a variety of membrane functions. Here, using both high temporal-resolution single particle tracking and single fluorescent molecule video imaging of an unsaturated phospholipid, DOPE, we found that plasma membrane compartments generally exist in various cell types, including CHO, HEPA-OVA, PtK2, FRSK, HEK293, HeLa, T24 (ECV304), and NRK cells. The compartment size varies from 30 to 230 nm, whereas the average hop rate of DOPE crossing the boundaries between two adjacent compartments ranges between 1 and 17 ms. The probability of passing a compartment barrier when DOPE is already at the boundary is also cell-type dependent, with an overall variation by a factor of approximately 7. These results strongly indicate the necessity for the paradigm shift of the concept on the plasma membrane: from the two-dimensional fluid continuum model to the compartmentalized membrane model in which its constituent molecules undergo hop diffusion over the compartments.     

The amino acid composition is different between the cytoplasmic and extracellular sides in membrane proteins.

The amino acid composition of transmembrane proteins was analyzed for their three separate portions: the transmembrane apolar, cytoplasmic and extracellular regions. The composition was different between cytoplasmic and extracellular peptides: alanine and arginine residues were preferentially sited on the cytoplasmic side, while the threonine and cysteine/cystine were preferentially sited on the extracellular side. The composition of cytoplasmic and extracellular peptides of membrane proteins corresponded to those of intracellular and extracellular types of soluble proteins, respectively. This difference in composition was independent of the peptide orientation against the membrane. Peptide chains could be correctly assigned as either cytoplasmic or extracellular, solely from an analysis of sequence composition. For single-spanning membrane proteins the predictive accuracy was 90%, whereas for multi-spanning proteins this was 85%.     

Characteristics affecting expression and solubilization of yeast membrane proteins

Biochemical and structural analysis of membrane proteins often critically depends on the ability to overexpress and solubilize them. To identify properties of eukaryotic membrane proteins that may be predictive of successful overexpression, we analyzed expression levels of the genomic complement of over 1000 predicted membrane proteins in a recently completed Saccharomyces cerevisiae protein expression library. We detected statistically significant positive and negative correlations between high membrane protein expression and protein properties such as size, overall hydrophobicity, number of transmembrane helices, and amino acid composition of transmembrane segments. Although expression levels of membrane and soluble proteins exhibited similar negative correlations with overall hydrophobicity, high-level membrane protein expression was positively correlated with the hydrophobicity of predicted transmembrane segments. To further characterize yeast membrane proteins as potential targets for structure determination, we tested the solubility of 122 of the highest expressed yeast membrane proteins in six commonly used detergents. Almost all the proteins tested could be solubilized using a small number of detergents. Solubility in some detergents depended on protein size, number of transmembrane segments, and hydrophobicity of predicted transmembrane segments. These results suggest that bioinformatic approaches may be capable of identifying membrane proteins that are most amenable to overexpression and detergent solubilization for structural and biochemical analyses. Bioinformatic approaches could also be used in the redesign of proteins that are not intrinsically well-adapted to such studies.     

Molecular convergence of bacterial and eukaryotic surface order

The conservation of fluidity is a theme common to all cell membranes. In this study, an analysis of lipid packing was conducted via C-laurdan spectroscopy of cell surface membranes prepared from representative species of Bacteria and Eukarya. We found that despite their radical differences in composition (namely the presence and absence of membrane-rigidifying sterol) the membrane order of all taxa converges on a remarkably similar level. To understand how this similarity is constructed, we reconstituted membranes with either bacterial or eukaryotic components. We found that transmembrane segments of proteins have an important role in buffering lipid-mediated packing. This buffering ensures that sterol-free and sterol-containing membranes exhibit similar barrier properties.     

Microbial mats and the early evolution of life

Microbial mats have descended from perhaps the oldest and most widespread biological communities known. Mats harbor microbes that are crucial for studies of bacterial phylogeny and physiology. They illustrate how several oxygen-sensitive biochemical processes have adapted to oxygen, and they show how life adapted to dry land long before the rise of plants. The search for the earliest grazing protists and metazoa in stromatolites is aided by observations of mats: in them, organic compounds characteristic of ancient photosynthetic protists can be identified. Recent mat studies suggest that the 13C/12C increase observed over geological time in stromatolitic organic matter was driven at least in part by a long-term decline in atmospheric carbon dioxide levels.     

Membrane lipids and transporter function

Transport proteins are essential for cells in allowing the exchange of substances between cells and their environment across the lipid bilayer forming a tight barrier. Membrane lipids modulate the function of transmembrane proteins such as transporters in two ways: Lipids are tightly and specifically bound to transport proteins and in addition they modulate from the bulk of the lipid bilayer the function of transport proteins. This overview summarizes currently available information at the ultrastructural level on lipids tightly bound to transport proteins and the impact of altered bulk membrane lipid composition. Human diseases leading to altered lipid homeostasis will lead to altered membrane lipid composition, which in turn affect the function of transporter proteins.     

An assessment of the impacts of molecular oxygen on the evolution of proteomes

Oxygen is not only one of life's essential elements but also a source of protein damage, mutagenesis, and ageing. Many proteome adaptations have been proposed to tackle such stresses and we assessed them using comparative genomics in a phylogenetic context. First, we find that aerobiosis is a trait with important phylogenetic inertia but that oxygen content in proteins is not. Instead, oxygen content is close to the expected values given the nucleotide composition. Accordingly, we find no evidence of oxygen being a scarce resource for protein synthesis even among anaerobes. Second, we searched for counterselection of amino acids more prone to oxidation among aerobes. Only cysteine follows the expected trend, whereas tryptophan follows the inverse one. When analyzing composition in the context of protein structures and residue accessibility, we find that all oxidable residues are avoided at the surface of proteins. Yet, there is no difference between aerobes and anaerobes in this respect, and the effect might be explained by the hydrophobicity of these residues. Third, we revisited the hypothesis that atmospheric enrichment in molecular oxygen led to the development of the communication capabilities of eukaryotes. With a larger data set and adequate controls, we confirm the trend of longer oxygen-rich outer domains in transmembrane proteins of eukaryotes. Yet, we find no significant association between oxygen concentration in the environment and this trait within prokaryotes, suggesting that this difference is clade specific and independent of oxygen availability. We find that genes involved in cellular responses to oxygen are much more frequent among aerobes, and we suggest that they erase most expected differences in terms of proteome composition between organisms facing high and low oxygen concentrations.     

Sphingolipid management by an orchestra of lipid transfer proteins

The various membranes in eukaryotic cells have unique lipid compositions. Despite important discoveries in lipid research over recent decades, the basic principles by which cells define their membrane compositions are essentially unknown. Cells must sense the concentration of each lipid, integrate such signals and regulate the activity of their metabolic enzymes and transport routes to dynamically meet their needs in terms of membrane composition. Sphingolipids constitute a lipid category that is essential for eukaryotic life and appears to be key to differences in lipid composition. Here we discuss recent findings that assign an important role to lipid transfer proteins in the regulation of sphingolipid metabolism, organization and function.     

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.     

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.     

Diacidic motifs influence the export of transmembrane proteins from the endoplasmic reticulum in plant cells

In yeast and mammals, amino acid motifs in the cytosolic tails of transmembrane domains play a role in protein trafficking by facilitating export from the endoplasmic reticulum (ER). However, little is known about ER export signals of membrane proteins in plants. Therefore, we investigated the role of diacidic motifs in the ER export of Golgi-localized membrane proteins. We show that diacidic motifs perform a significant function in the export of transmembrane proteins to the Golgi apparatus, as mutations of these signals impede the efficient anterograde transport of multispanning, type II, and type I proteins. Furthermore, we demonstrate that diacidic motifs instigate the export of proteins that reside in the ER due to the lengths of their transmembrane domains. However, not all of the diacidic motifs in the cytosolic tails of the proteins studied were equally important in ER export. Transport of Golgi proteins was disrupted only by mutagenesis of specific diacidic signals, suggesting that the protein environment of these signals affects their function. Our findings indicate that diacidic ER export motifs are present and functional in plant membrane proteins and that they are dominant over transmembrane domain length in determining the export of proteins from the ER in plant cells.     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.     

Membrane localization of small proteins in Escherichia coli

Escherichia coli synthesize over 60 poorly understood small proteins of less than 50 amino acids. A striking feature of these proteins is that 65% contain a predicted α-helical transmembrane (TM) domain. This prompted us to examine the localization, topology, and membrane insertion of the small proteins. Biochemical fractionation showed that, consistent with the predicted TM helix, the small proteins generally are most abundant in the inner membrane fraction. Examples of both N(in)-C(out) and N(out)-C(in) orientations were found in assays of topology-reporter fusions to representative small TM proteins. Interestingly, however, three of nine tested proteins display dual topology. Positive residues close to the transmembrane domains are conserved, and mutational analysis of one small protein, YohP, showed that the positive inside rule applies for single transmembrane domain proteins as has been observed for larger proteins. Finally, fractionation analysis of small protein localization in strains depleted of the Sec or YidC membrane insertion pathways uncovered differential requirements. Some small proteins appear to be affected by both Sec and YidC depletion, others showed more dependence on one or the other insertion pathway, whereas one protein was not affected by depletion of either Sec or YidC. Thus, despite their diminutive size, small proteins display considerable diversity in topology, biochemical features, and insertion pathways.     

Glycoproteomics and glycomics investigation of membrane N-glycosylproteins from human colon carcinoma cells

Aberrant glycosylation of proteins is known to profoundly affect cellular adhesion or motility of tumoral cells. In this study, we used HT-29 human colon epithelial cancer cells as a cellular model of cancer progression, as they can either proliferate or differentiate into enterocyte phenotype. A glycoproteomic approach based on Con A lectin-affinity chromatography, SDS-PAGE and MS analysis, allowed the identification of membrane N-glycoproteins from Triton X-100-solubilized proteins from membrane preparation. Among them, 65% were membrane proteins, and 45% were known to be N-glycosylated, such as alpha chains integrin and dipeptidyl isomerase IV. By lectin-blot analysis, significant changes of alpha-2,3- and alpha-2,6-sialylation of membrane glycoproteins were observed between proliferating and differentiated HT-29 cells. From these results, nano-LC-MS/MS analysis of the tryptic digests of the corresponding bands was performed and led to the identification of several transmembrane glycoproteins, like members of the solute carrier family and adhesion proteins. Finally, we compared N-glycans profiles and monosaccharide composition of proliferating and enterocyte-like HT-29 cells using MALDI-MS and GC-MS analyses of permethylated derivatives. This glycomic approach allowed to underscore significant changes in N-glycans structure, in particular the expression of atypical N-acetylglucosamine (GlcNAc)-ended N-glycans in enterocyte-like HT-29 cells.     

Lipopolysaccharide from Proteus mirabilis O29 induces changes in red blood cell membrane lipids and proteins

Alterations in red blood cell (RBC) plasma membranes, i.e. in lipids and proteins, and osmotic fragility of these cells after treatment with Proteus mirabilis O29 endotoxin (lipolysaccharide (LPS)) were examined using a spin labelling method. At the highest concentration of LPS, insignificantly decreased fluidity of membrane lipids was observed. Changes in conformation of membrane proteins were determined by two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (MSL) and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The analysis of spectra of MSL and ISL showed modifications in membrane proteins in red blood cells treated with the highest concentration of lipopolysaccharide. On the other hand, in the case of isolated membranes, disturbances in membrane were observed for all concentrations of LPS. The alterations in membrane lipids and proteins are paralleled in a significant rise in osmotic fragility of RBCs upon endotoxin treatment. These results provide experimental evidence that P. mirabilis O29 LPS causes deleterious changes in membranes of human red blood cells. They show that action of lipopolysaccharide mainly concerns the membrane cytoskeleton.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 degrees C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Role of sterols in modulating the human mu-opioid receptor function in Saccharomyces cerevisiae

This study provides evidence that the differences in membrane composition found from one cell type to another can represent a limiting factor to recovering the functionality of transmembrane proteins when expressed in heterologous systems. Restoring the properties of the human mu-opioid receptor in yeast (Saccharomyces cerevisiae), similar to those observed in native cells, was achieved by replacing ergosterol from yeast by cholesterol, which is normally found in mammalian plasma membranes. The results suggest that these two sterols have opposite effects with respect to the ligand binding function of the receptor. Ergosterol was found to constrain the mu-opioid receptor in an inactive state in yeast plasma membranes and cannot replace cholesterol in activating it. These data differ from previous works dealing with the function of related G-protein-coupled receptors (GPCR) in ergosterol-enriched membranes. This suggests that structural requirements of GPCR with respect to their modulation by lipid components differ from one protein to another. As a consequence, we assume that the presence of appropriate lipids around transmembrane proteins determines their function. This highlights the functional significance of lateral heterogeneities of membrane components within biological membranes.     

Claudin-2 forms homodimers and is a component of a high molecular weight protein complex

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.