scholarly journals Characterization of micron-scale protein-depleted plasma membrane domains in phosphatidylserine-deficient yeast cells

2021 ◽  
Vol 135 (5) ◽  
Author(s):  
Tetsuo Mioka ◽  
Tian Guo ◽  
Shiyao Wang ◽  
Takuma Tsuji ◽  
Takuma Kishimoto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Large Scale ◽  
Living Cells ◽  
Yeast Cells ◽  
Membrane Domain ◽  
Cellular Functions ◽  
Artificial Membranes ◽  
Yeast Mutants

ABSTRACT Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the ‘void zone’. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.

scholarly journals Phosphatidylserine prevents the generation of a protein-free giant plasma membrane domain in yeast

2020 ◽  
Author(s):  
Tetsuo Mioka ◽  
Guo Tian ◽  
Wang Shiyao ◽  
Takuma Tsuji ◽  
Takuma Kishimoto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Living Cells ◽  
Yeast Cells ◽  
Membrane Domains ◽  
Membrane Domain ◽  
Artificial Membranes ◽  
Free Region ◽  
Large Phase ◽  
Plasma Membrane Domains

AbstractMembrane phase separation accompanied with micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large phase separation has not been reported in the plasma membrane of the living cells. We demonstrate here that a stable micron-scale protein-free region is generated in the plasma membrane of the yeast mutants lacking phosphatidylserine. We named this region the “void zone”. Transmembrane proteins, peripheral membrane proteins, and certain phospholipids are excluded from the void zone. The void zone is rich in ergosterol and requires ergosterol and sphingolipids for its formation. These characteristics of the void zone are similar to the properties of the cholesterol-enriched domain in phase-separated artificial membranes. We propose that phosphatidylserine prevents the formation of the void zone by preferentially interacting with ergosterol. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.Summary statementYeast cells lacking phosphatidylserine generate protein-free plasma membrane domains, and vacuoles contact with this domain. This is the first report of micron-scale plasma membrane domains in living cells.

Large scale characterization of plant plasma membrane proteins

Biochimie ◽  
1999 ◽  
Vol 81 (6) ◽  
pp. 655-661 ◽  
Author(s):  
Véronique Santoni ◽  
Patrick Doumas ◽  
David Rouquié ◽  
Monique Mansion ◽  
Thierry Rabilloud ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Large Scale ◽  
Plasma Membrane Proteins ◽  
Scale Characterization ◽  
Plant Plasma Membrane

scholarly journals Exogenous Administration of Gangliosides Displaces GPI-anchored Proteins from Lipid Microdomains in Living Cells

1999 ◽  
Vol 10 (10) ◽  
pp. 3187-3196 ◽  
Author(s):  
Mikael Simons ◽  
Tim Friedrichson ◽  
Jörg B. Schulz ◽  
Marina Pitto ◽  
Massimo Masserini ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Mdck Cells ◽  
Living Cells ◽  
Cross Linking ◽  
Cellular Functions ◽  
Exogenous Application ◽  
Triton X ◽  
Chemical Cross Linking ◽  
Darby Canine Kidney

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid–cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4°C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid–cholesterol microdomains in living cells.

scholarly journals Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles

2007 ◽  
Vol 104 (9) ◽  
pp. 3165-3170 ◽  
Author(s):  
T. Baumgart ◽  
A. T. Hammond ◽  
P. Sengupta ◽  
S. T. Hess ◽  
D. A. Holowka ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Large Scale ◽  
Membrane Vesicles ◽  
Fluid Phase ◽  
Plasma Membrane Vesicles ◽  
Separation Of Proteins

scholarly journals SugarPy facilitates the universal, discovery-driven analysis of intact glycopeptides

2020 ◽  
Author(s):  
Stefan Schulze ◽  
Anne Oltmanns ◽  
Christian Fufezan ◽  
Julia Krägenbring ◽  
Michael Mormann ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Glycosylation ◽  
Supplementary Information ◽  
Cyanidioschyzon Merolae ◽  
Human Breast Milk ◽  
The Novel ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Domains Of Life

AbstractMotivationProtein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes.ResultsTherefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii. SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae.AvailabilityThe source code is freely available on GitHub (https://github.com/SugarPy/SugarPy), and its implementation in Python ensures support for all operating [email protected] and [email protected] informationSupplementary data are available online.

scholarly journals Optogenetic inhibition and activation of Rac and Rap1 using a modified iLID system

2020 ◽  
Author(s):  
Nick R. Elston ◽  
Michael Pablo ◽  
Fred Pimenta ◽  
Klaus M. Hahn ◽  
Takashi Watanabe
Keyword(s):  
Plasma Membrane ◽  
Living Cells ◽  
Small Gtpases ◽  
Spatiotemporal Dynamics ◽  
Membrane Localization ◽  
Cellular Functions ◽  
Activation Kinetics ◽  
System 1 ◽  
Control Plasma

The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatiotemporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light induced dimerization (iLID) system [1] was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1 [2, 3, 4, 5]). Irradiation yielded a 50% to 230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.

scholarly journals ProXs drive the formation of membraneless organelles in plants

2021 ◽  
Author(s):  
Honghong Zhang ◽  
Fangyu Peng ◽  
Yan Liu ◽  
Haiteng Deng ◽  
Xiaofeng Fang
Keyword(s):  
Phase Separation ◽  
Higher Plants ◽  
Research Community ◽  
Yeast Cells ◽  
Cellular Functions ◽  
Crop Species ◽  
Cellular Space ◽  
High Degree ◽  
Heterologous Expression In Yeast ◽  
Liquid Liquid Phase Separation

Membraneless organelles (MLOs) are non-membranous structures inside cells that organize cellular space and processes. The recent discovery that MLOs can be assembled via liquid-liquid phase separation (LLPS) advanced our understanding of these structures. However, the proteins that are capable of forming MLOs are largely unknown, especially in plants. In this study, we developed a method to identify proteins that we referred as ProXs (Proteins enriched by b-isoX) in Arabidopsis. Heterologous expression in yeast cells showed that most ProXs were capable of forming MLOs autonomously. We applied this method to several model and crop species including early and higher plants. This allowed us to generate an atlas of ProXs for studying plant MLOs. Analysis of ProXs from different species revealed high degree of conservation, supporting that they play important roles in cellular functions and are positively selected during evolution. Our method will be a valuable tool to characterize novel MLOs from desired cells and the data generated in present study will be instrumental for the plant research community to investigate MLO biology.

scholarly journals Cation/Ca2+ Exchanger 1 (MdCCX1), a Plasma Membrane-Localized Na+ Transporter, Enhances Plant Salt Tolerance by Inhibiting Excessive Accumulation of Na+ and Reactive Oxygen Species

2021 ◽  
Vol 12 ◽  
Author(s):  
Jie Yang ◽  
Weihan Li ◽  
Xin Guo ◽  
Peihong Chen ◽  
Yunpeng Cheng ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Salt Tolerance ◽  
Crop Yields ◽  
Yeast Cells ◽  
Ros Scavenging ◽  
Severe Damage ◽  
Family Gene ◽  
Plant Salt Tolerance ◽  
Yeast Mutants ◽  
Excessive Accumulation

High salinity causes severe damage to plant growth and significantly reduces crop yields. The CCX family proteins can facilitate the transport of multiple ions to prevent toxicity. CCX proteins play an important role in regulating plant salt tolerance, but no detailed studies on CCX proteins in apples have been reported. Here, the CCX family gene MdCCX1 was cloned from apple (Malus domestica). It is constitutively expressed in various apple tissues and is significantly induced by salt stress. As a plasma membrane-localized protein, MdCCX1-overexpression could complement the Na+-sensitive phenotype of yeast mutants and reduce the Na+ content in yeast cells under NaCl treatment, suggesting that MdCCX1 could be a plasma membrane-localized Na+ transporter. To identify the function of MdCCX1 in salt response, we transformed this gene into Arabidopsis, apple calli, and apple plants. Overexpression of MdCCX1 significantly improved the salt tolerance of these transgenic materials. The significantly reduced Na+ content under NaCl treatment indicated that MdCCX1 overexpression could enhance plant salt tolerance by inhibiting the excessive accumulation of Na+. Besides, MdCCX1 overexpression could also enhance plant salt tolerance by promoting ROS scavenging. These findings provide new insight and rich resources for future studies of CCX proteins in plant species.

scholarly journals Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids

2020 ◽  
Vol 61 (5) ◽  
pp. 758-766 ◽  
Author(s):  
Guangtao Li ◽  
Qing Wang ◽  
Shinako Kakuda ◽  
Erwin London
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Light Microscopy ◽  
Large Scale ◽  
Membrane Vesicles ◽  
Lipid Domains ◽  
Domain Formation ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Wide Range

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.

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