A fundamental feature of cellular plasma membranes (PMs) is an asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM ...leaflets nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being approximately twofold more unsaturated than the exoplasmic leaflet. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in the asymmetric structures of protein transmembrane domains. These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.
A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) ...and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast ...mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.
Many cellular processes rely on precise and timely deformation of the cell membrane. While many proteins participate in membrane reshaping and scission, usually in highly specialized ways, Bin ...amphiphysin Rvs (BAR) domain proteins play a pervasive role, as they not only participate in many aspects of cell trafficking but also are highly versatile membrane remodelers. Subtle changes in the shape and size of the BAR domain can greatly impact the way in which BAR domain proteins interact with the membrane. Furthermore, the activity of BAR domain proteins can be tuned by external physical parameters, and so they behave differently depending on protein surface density, membrane tension, or membrane shape. These proteins can form 3D structures that mold the membrane and alter its liquid properties, even promoting scission under various circumstances.As such, BAR domain proteins have numerous roles within the cell. Endocytosis is among the most highly studied processes in which BAR domain proteins take on important roles. Over the years, a more complete picture has emerged in which BAR domain proteins are tied to almost all intracellular compartments; examples include endosomal sorting and tubular networks in the endoplasmic reticulum and T-tubules. These proteins also have a role in autophagy, and their activity has been linked with cancer. Here, we briefly review the history of BAR domain protein discovery, discuss the mechanisms by which BAR domain proteins induce curvature, and attempt to settle important controversies in the field. Finally, we review BAR domain proteins in the context of a cell, highlighting their emerging roles in cell signaling and organelle shaping.
Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively ...native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.
A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in ...animal cells. This anomalous diffusion results from a combination of structuring factors including protein–protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.
β-Barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and ...morphology, nutrient acquisition, colonization and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs must fold and insert into a crowded and asymmetric OM that lacks much freely accessible lipid. This feat is accomplished in the absence of an external energy source and is thought to be driven by the high thermodynamic stability of folded OMPs in the OM. With such a stable fold, the challenge that bacteria face in assembling OMPs into the OM is how to overcome the initial energy barrier of membrane insertion. In this review, we highlight the roles of the lipid environment and the OM in modulating the OMP-folding landscape and discuss the factors that guide folding in vitro and in vivo. We particularly focus on the composition, architecture, and physical properties of the OM and how an understanding of the folding properties of OMPs in vitro can help explain the challenges they encounter during folding in vivo. Current models of OMP biogenesis in the cellular environment are still in flux, but the stakes for improving the accuracy of these models are high. OMP folding is an essential process in all Gram-negative bacteria, and considering the looming crisis of widespread microbial drug resistance it is an attractive target. To bring down this vital OMP-supported barrier to antibiotics, we must first understand how bacterial cells build it.
Damage to mitochondria can lead to the depolarization of the inner mitochondrial membrane, thereby sensitizing impaired mitochondria for selective elimination by autophagy. However, fusion of ...uncoupled mitochondria with polarized mitochondria can compensate for damage, reverse membrane depolarization, and obviate mitophagy. Parkin, an E3 ubiquitin ligase that is mutated in monogenic forms of Parkinson's disease, was recently found to induce selective autophagy of damaged mitochondria. Here we show that ubiquitination of mitofusins Mfn1 and Mfn2, large GTPases that mediate mitochondrial fusion, is induced by Parkin upon membrane depolarization and leads to their degradation in a proteasome- and p97-dependent manner. p97, a AAA+ ATPase, accumulates on mitochondria upon uncoupling of Parkin-expressing cells, and both p97 and proteasome activity are required for Parkin-mediated mitophagy. After mitochondrial fission upon depolarization, Parkin prevents or delays refusion of mitochondria, likely by the elimination of mitofusins. Inhibition of Drp1-mediated mitochondrial fission, the proteasome, or p97 prevents Parkin-induced mitophagy.
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Introduction
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Membrane lipid composition
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Membrane lipid structure
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Membrane lipid organization
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Why so many different lipids?
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Lipid mixing and demixing
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Lateral pressure
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Surface ...electrostatics
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Role of lipids in cell functions
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Lipid influence in transmembrane protein function
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Prokaryotic potassium channel (KcsA)
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Mechanosensitive channels
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Voltage‐gated potassium channel (KvAP)
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Nicotinic acetylcholine receptor (nAcChR)
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G protein‐coupled receptors
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Other examples
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Non‐permanent proteins in membranes
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Proteins that interact reversibly with the bilayers
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Proteins that interact irreversibly with the bilayers
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Proteins that interact weakly with the membrane
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Proteins that interact strongly with the membrane
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G proteins and their interactions with membranes
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Small monomeric G proteins: the Ras and Ras‐like family
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Protein kinase C
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Membrane microdomains and lipid mediators in the control of heat‐shock protein response
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Stress sensing and signalling: the membrane sensor theory
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Hsp signalling in cancer and diabetes
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The role of membrane microdomains
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Lipid mediators of the stress response
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A subpopulation of Hsps can interact with and translocate through membranes
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Hsp90 in eukaryotic membranes
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Hsp70 in cell membranes
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Hsp27‐membrane interactions
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Secreted Hsps
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Representative cases where Hsps interact with membranes or release from the cells
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Concluding remarks
Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio‐temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co‐/post‐translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane‐lipid therapy.
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