Cytoplasmic Leaflet Research Articles

Nickel Oxide Nanoparticles Trigger Caspase- and Mitochondria-Dependent Apoptosis in the Yeast Saccharomyces cerevisiae.

The expansion of the industrial use of nickel oxide (NiO) nanoparticles (NPs) raises concerns about their potential adverse effects. Our work aimed to investigate the mechanisms of toxicity induced by NiO NPs, using the yeast Saccharomyces cerevisiae as a cell model. Yeast cells exposed to NiO NPs exhibited typical hallmarks of regulated cell death (RCD) by apoptosis [loss of cell proliferation capacity (cell viability), exposure of phosphatidylserine at the outer cytoplasmic membrane leaflet, nuclear chromatin condensation, and DNA damage] in a process that required de novo protein synthesis. The execution of yeast cell death induced by NiO NPs is Yca1p metacaspase-dependent. NiO NPs also induced a decrease in the mitochondrial membrane potential and an increase in the frequency of respiratory-deficient mutants, which supports the involvement of mitochondria in the cell death process. Cells deficient in the apoptosis-inducing factor ( aif1Δ) displayed higher tolerance to NiO NPs, which reinforces the involvement of mitochondria in RCD by apoptosis. In summary, this study shows that NiO NPs induce caspase- and mitochondria-dependent apoptosis in yeast. Our results warn about the possible harmful effects associated with the use of NiO NPs.

Tmem30a deficiency leads to retinal rod bipolar cell degeneration.

Phospholipids are asymmetrically distributed across the mammalian plasma membrane, with phosphatidylserine (PS) and phosphatidylethanolamine concentrated in the cytoplasmic leaflet of the membrane bilayer and phosphatidylcholine in the exoplasmic leaflet. This asymmetric distribution is dependent on a group of P4 ATPases called PS flippases. The proper transport and function of PS flippases require a β-subunit transmembrane protein 30A (TMEM30A). Disruption of PS flippases leads to several human diseases. Tmem30a is essential for photoreceptor survival. However, the roles of Tmem30a in the retinal rod bipolar cells (RBC) remain elusive. To investigate the role of Tmem30a in the RBCs, we generated a RBC-specific Tmem30a knockout (cKO) mouse model using PCP2-Cre line. The Tmem30a cKO mice exhibited defect in RBC function and progressive RBC death. PKCα staining of retinal cryosections from cKO mice revealed a remarkable dendritic sprouting of rod bipolar cells during the early degenerative process. Immunostaining analysis of PSD95 and mGluT6 expression demonstrated that rod bipolar cells in Tmem30a cKO retinas exhibited aberrant dendritic sprouting as a result of impaired synaptic efficacy, which implied a crucial role for Tmem30a in synaptic transmission in the retina. In addition, loss of Tmem30a led to reactive gliosis with increased expression of glial fibrillary acidic protein and CD68. TUNEL staining suggested that apoptotic cell death occurred in the retinal inner nuclear layer (INL). Our data show that loss of Tmem30a in RBCs results in dendritic sprouting of rod bipolar cells, increased astrogliosis and RBC death. Taken together, our studies demonstrate an essential role for Tmem30a in the retinal bipolar cells. Cover Image for this issue: doi: 10.1111/jnc.14492.

Understanding phosphoinositides: rare, dynamic, and essential membrane phospholipids.

Polyphosphoinositides (PPIs) are essential phospholipids located in the cytoplasmic leaflet of eukaryotic cell membranes. Despite contributing only a small fraction to the bulk of cellular phospholipids, they make remarkable contributions to practically all aspects of a cell's life and death. They do so by recruiting cytoplasmic proteins/effectors or by interacting with cytoplasmic domains of membrane proteins at the membrane-cytoplasm interface to organize and mold organelle identity. The present study summarizes aspects of our current understanding concerning the metabolism, manipulation, measurement, and intimate roles these lipids play in regulating membrane homeostasis and vital cell signaling reactions in health and disease.

Phosphatidylinositol 4-phosphate on Rab7-positive autophagosomes revealed by the freeze-fracture replica labeling.

Phosphatidylinositol 4-phophate (PtdIns(4)P) is an essential signaling molecule in the Golgi body, endosomal system, and plasma membrane and functions in the regulation of membrane trafficking, cytoskeletal organization, lipid metabolism and signal transduction pathways, all mediated by direct interaction with PtdIns(4)P-binding proteins. PtdIns(4)P was recently reported to have functional roles in autophagosome biogenesis. LC3 and GABARAP subfamilies and a small GTP-binding protein, Rab7, are localized on autophagosomal membranes and participate at each stage of autophagosome formation and maturation. To better understand autophagosome biogenesis, it is essential to determine the localization of PtdIns(4)P and to examine its relationship with LC3 and GABARAP subfamilies and Rab7. To analyze PtdIns(4)P distribution, we used an electron microscopy technique that labels PtdIns(4)P on the freeze-fracture replica of intracellular biological membranes, which minimizes the possibility of artificial perturbation because molecules in the membrane are physically immobilized in situ. Using this technique, we found that PtdIns(4)P is localized on the cytoplasmic, but not the luminal (exoplasmic), leaflet of the inner and outer membranes of autophagosomes. Double labeling revealed that PtdIns(4)P mostly colocalizes with Rab7, but not with LC3B, GABARAP, GABARAPL1 and GABARAPL2. Rab7 plays essential roles in autophagosome maturation and in autophagosome-lysosome fusion events. We suggest that PtdIns(4)P is localized to the cytoplasmic leaflet of the autophagosome at later stages, which may illuminate the importance of PtdIns(4)P at the later stages of autophagosome formation.

Substrates of P4-ATPases: beyond aminophospholipids (phosphatidylserine and phosphatidylethanolamine).

P4-ATPases, a subfamily of P-type ATPases, were initially identified as aminophospholipid translocases in eukaryotic membranes. These proteins generate and maintain membrane lipid asymmetry by translocating aminophospholipids (phosphatidylserine and phosphatidylethanolamine) from the exoplasmic/lumenal leaflet to the cytoplasmic leaflet. The human genome encodes 14 P4-ATPases, and the cellular localizations, substrate specificities, and cellular roles of these proteins were recently revealed. Numerous P4-ATPases, including ATP8A1, ATP8A2, ATP11A, ATP11B, and ATP11C, transport phosphatidylserine. By contrast, ATP8B1, ATP8B2, and ATP10A transport phosphatidylcholine but not aminophospholipids, although there is a discrepancy regarding the substrate of ATP8B1 in the literature. Some yeast and plant P4-ATPases can also translocate phosphatidylcholine. At least 2 P4-ATPases (ATP8A2 and ATP8B1) are associated with severe human diseases, and other P4-ATPases are implicated in various pathophysiologic conditions in mouse models. Here, we discuss the cellular functions of phosphatidylcholine flippases and suggest a model for the phenotype of progressive familial intrahepatic cholestasis 1 caused by a defect in ATP8B1.-Shin, H.-W., Takatsu, H. Substrates of P4-ATPases: beyond aminophospholipids (phosphatidylserine and phosphatidylethanolamine).

Caveolin-1 is related to lipid droplet formation in hepatic stellate cells in human liver

Caveolins (CAVs) regulate intracellular cholesterol transport by a complex process involving caveolae, endoplasmic reticulum (ER), and the Golgi network. Hepatic stellate cells (HSCs) are the central site for retinoid storage in the liver and indeed the entire body. Herein, we attempted to elucidate the ultrastructural localization and expression of caveolin-1 (CAV-1) in human HSCs during the progression of liver cirrhosis (LC). Normal and hepatitis C-related cirrhotic liver samples were prepared using a modified perfusion-fixation method to fix organelle structures and molecules in their in vivo positions, and examined using immunoelectron microscopy. In control liver specimens, CAV-1 was minimally associated with low electron density lipid droplets (LDs) segregated around zones 1―2, and specifically associated with membranes surrounding LDs. CAV-1 was segregated in high-density LDs, consistent with the formation of membrane-enclosed lipid-rich vesicular structures, as well as caveolae on plasma membranes around zones 2―3. In cirrhotic liver specimens, CAV-1 molecules were inserted into the cytoplasmic leaflets of ER membranes for transportation to LDs. Thus, CAV-1 transport to LDs might represent an intracellular pathway from the ER in cirrhotic liver tissue.

The wheat ABC transporter Lr34 modifies the lipid environment at the plasma membrane

Phospholipids (PLs) are emerging as important factors that initiate signal transduction cascades at the plasma membrane. Their distribution within biological membranes is tightly regulated, e.g. by ATP-binding cassette (ABC) transporters, which preferably translocate PLs from the cytoplasmic to the exoplasmic membrane leaflet and are therefore called PL-floppases. Here, we demonstrate that a plant ABC transporter, Lr34 from wheat (Triticum aestivum), is involved in plasma membrane remodeling characterized by an intracellular accumulation of phosphatidic acid and enhanced outward translocation of phosphatidylserine. In addition, the content of phosphatidylinositol 4,5-bisphosphate in the cytoplasmic leaflet of the plasma membrane was reduced in the presence of the ABC transporter. When heterologously expressed in Saccharomyces cerevisiae, Lr34 promoted oil body formation in a mutant defective in PL-transfer in the secretory pathway. Our results suggest that PL redistribution by Lr34 potentially affects the membrane-bound proteome and contributes to the previously reported stimuli-independent activation of biotic and abiotic stress responses and neutral lipid accumulation in transgenic Lr34-expressing barley plants.

Disruption of Tmem30a results in cerebellar ataxia and degeneration of Purkinje cells

Phospholipids are asymmetrically distributed across mammalian plasma membrane with phosphatidylserine (PS) and phosphatidylethanolamine concentrated in the cytoplasmic leaflet of the membrane bilayer. This asymmetric distribution is dependent on a group of P4-ATPases named PS flippases. The proper transport and function of PS flippases require a β-subunit transmembrane protein 30 A (TMEM30A). Disruption of PS flippases led to several human diseases. However, the roles of TMEM30A in the central nervous system remain elusive. To investigate the role of Tmem30a in the cerebellum, we developed a Tmem30a Purkinje cell (PC)-specific knockout (KO) mouse model. The Tmem30a KO mice displayed early-onset ataxia and progressive PC death. Deficiency in Tmem30a led to an increased expression of Glial fibrillary acidic protein and astrogliosis in regions with PC loss. Elevated C/EBP homologous protein and BiP expression levels indicated the presence of endoplasmic reticulum stress in the PCs prior to visible cell loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis suggested that apoptotic cell death occurred in the cerebellum. Our data demonstrate that loss of Tmem30a in PCs results in protein folding and transport defects, a substantial decrease in dendritic spine density, increased astrogliosis and PC death. Taken together, our data demonstrate an essential role of Tmem30a in the cerebellum PCs.

Inhibiting Epidermal Growth Factor Receptor Dimerization and Signaling Through Targeted Delivery of a Juxtamembrane Domain Peptide Mimic.

Overexpression and deregulation of the epidermal growth factor receptor (EGFR) are implicated in multiple human cancers and therefore are a focus for the development of therapeutics. Current strategies aimed at inhibiting EGFR activity include monoclonal antibodies and tyrosine kinase inhibitors. However, activating mutations severely limit the efficacy of these therapeutics. There is thus a growing need for novel methods to inhibit EGFR. One promising approach involves blocking the association of the cytoplasmic juxtamembrane (JM) domain of EGFR, which has been shown to be essential for receptor dimerization and kinase function. Here, we aim to improve the selectivity and efficacy of an EGFR JM peptide mimic by utilizing the pH(low) insertion peptide (pHLIP), a unique molecule that can selectively target cancer cells solely based on their extracellular acidity. This delivery strategy potentially allows for more selective targeting to tumors than current methods and for anchoring the peptide mimic to the cytoplasmic leaflet of the plasma membrane, increasing its local concentration and thus efficacy. We show that the conjugated construct is capable of inhibiting EGFR phosphorylation and downstream signaling and of inducing concentration- and pH-dependent toxicity in cervical cancer cells. We envision that this approach could be expanded to the modulation of other single-span membrane receptors whose activity is mediated by JM domains.

Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial α-helical membrane anchors

Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayer-integrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles. Systematic cysteine-scanning mutagenesis showed the predicted TMD of one candidate MIP, DHRS3, to be a partially buried amphipathic α-helix in both lipid droplet monolayers and the cytoplasmic leaflet of endoplasmic reticulum membrane bilayers. Coarse-grained molecular dynamics simulations support these observations, suggesting that this helix is most stable at the solvent-membrane interface. The simulations also predicted similar interfacial amphipathic helices when applied to seven additional MIPs from our dataset. Our findings suggest that interfacial helices may be a common motif by which MIPs are integrated into membranes, and provide high-throughput methods to identify and study MIPs.

Transbilayer phospholipid movement facilitates the translocation of annexin across membranes.

ABSTRACTAnnexins are cytosolic phospholipid-binding proteins that can be found on the outer leaflet of the plasma membrane. The extracellular functions of annexin include modulating fibrinolysis activity and cell migration. Despite having well-described extracellular functions, the mechanism of annexin transport from the cytoplasmic inner leaflet to the extracellular outer leaflet of the plasma membrane remains unclear. Here, we show that the transbilayer movement of phospholipids facilitates the transport of annexins A2 and A5 across membranes in cells and in liposomes. We identified TMEM16F (also known as anoctamin-6, ANO6) as a lipid scramblase required for transport of these annexins to the outer leaflet of the plasma membrane. This work reveals a mechanism for annexin translocation across membranes which depends on plasma membrane phospholipid remodelling.

C24 Sphingolipids Govern the Transbilayer Asymmetry of Cholesterol and Lateral Organization of Model and Live-Cell Plasma Membranes

Mammalian sphingolipids, primarily with C24 or C16 acyl chains, reside in the outer leaflet of the plasma membrane. Curiously, little is known how C24 sphingolipids impact cholesterol and membrane microdomains. Here, we present evidence that C24 sphingomyelin, when placed in the outer leaflet, suppresses microdomains in giant unilamellar vesicles and also suppresses submicron domains in the plasma membrane of HeLa cells. Free energy calculations suggested that cholesterol has a preference for the inner leaflet if C24 sphingomyelin is in the outer leaflet. We indeed observe that cholesterol enriches in the inner leaflet (80%) if C24 sphingomyelin is in the outer leaflet. Similarly, cholesterol primarily resides in the cytoplasmic leaflet (80%) in the plasma membrane of human erythrocytes where C24 sphingolipids are naturally abundant in the outer leaflet. We conclude that C24 sphingomyelin uniquely interacts with cholesterol and regulates the lateral organization in asymmetric membranes, potentially by generating cholesterol asymmetry.

Interaction of Myelin Basic Protein with Myelin-like Lipid Monolayers at Air-Water Interface.

Interaction of myelin basic protein (MBP) and the cytoplasmic leaflets of the oligodendrocyte membrane is essential for the formation and compaction of the myelin sheath of the central nervous system and is altered aberrantly and implicated in the pathogenesis of neurodegenerative diseases like multiple sclerosis. To gain more detailed insights into this interaction, the adsorption of MBP to model lipid monolayers of similar composition to the myelin of the central nervous system was studied at the air-water interface with monolayer adsorption experiments. Measuring the surface pressure and the related maximum insertion pressure of MBP for different myelin-like lipid monolayers provided information about the specific role of each of the single lipids in the myelin. Depending on the ratio of negatively charged lipids to uncharged lipids and the distance between charges, the adsorption process was found to be determined by two counteracting effects: (i) protein incorporation, resulting in an increasing surface pressure and (ii) lipid condensation due to electrostatic interaction between the positively charged protein and negatively charged lipids, resulting in a decreasing surface pressure. Although electrostatic interactions led to high insertion pressures, the associated lipid condensation lowered the fluidity of the myelin-like monolayer.

Phospholipid-flipping activity of P4-ATPase drives membrane curvature.

P4-ATPases are phospholipid flippases that translocate phospholipids from the exoplasmic/luminal to the cytoplasmic leaflet of biological membranes. All P4-ATPases in yeast and some in other organisms are required for membrane trafficking; therefore, changes in the transbilayer lipid composition induced by flippases are thought to be crucial for membrane deformation. However, it is poorly understood whether the phospholipid-flipping activity of P4-ATPases can promote membrane deformation. In this study, we assessed membrane deformation induced by flippase activity via monitoring the extent of membrane tubulation using a system that allows inducible recruitment of Bin/amphiphysin/Rvs (BAR) domains to the plasma membrane (PM). Enhanced phosphatidylcholine-flippase activity at the PM due to expression of ATP10A, a member of the P4-ATPase family, promoted membrane tubulation upon recruitment of BAR domains to the PM This is the important evidence that changes in the transbilayer lipid composition induced by P4-ATPases can deform biological membranes.

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane.

Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane.

Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE-containing cells were exposed to membrane-impermeant collisional quenchers (spin-labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass-bead lysis or repeated freeze-thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.

Scramblase Activities of Transmembrane Peptides Depend on Relative Position of Hydrophilic Amino Acid Residues and their Depth in the Membrane

Phospholipid transbilayer movement (flip-flop) is very rapid in the ER of eucaryotes, where phospholipids in the cell are mainly synthesized only at the cytoplasmic leaflet. However, the mechanism to promote the phospholipid flip-flop in the ER remains unknown. We have previously revealed that a peptide with a membrane-spanning sequence of a human ER protein, EDEM1, promotes the flip-flop, and that both Arg and His residues in the sequence are necessary for the peptide's activity. Interestingly, these hydrophilic residues are located at the same side of an α-helix structure of EDEM1 peptide. Therefore, we hypothesized that relative position of two hydrophilic residues in the helix may be important for the flip-promotion ability of peptides. In this study, we synthesized model transmembrane peptides containing two hydrophilic residues at various positions in the sequences and evaluated their activity. We demonstrated that the activities of the peptides depended on the turn angle between Arg and His in the helices, and significantly increased when two hydrophilic residues are located at the same side of the helix. The peptides containing other high hydrophilic residues at the same side of the helices also had flip-promotion abilities, whereas the substitution of either Arg or His with a lower hydrophilic or hydrophobic residue inhibited the activities. Therefore, the scramblase activities of the peptides were attributed to the hydrophilicity of the residues at the same side. We also found that the peptides with hydrophilic residues at the deeper position in the membrane showed higher flip-promotion ability. These results provided the possibility that the proteins whose transmembrane regions have similar physicochemical properties as those of the peptides used in this study might be involved in the flip-flop promotion in the ER.

Is ABCA1 a lipid transfer protein?

ABCA1 functions as a lipid transporter because it mediates the transfer of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apoA-I and related proteins present in the extracellular medium. ABCA1 is a membrane PL translocase and its enzymatic activity leads to transfer of PL molecules from the cytoplasmic leaflet to the exofacial leaflet of a cell plasma membrane (PM). The presence of active ABCA1 in the PM promotes binding of apoA-I to the cell surface. About 10% of this bound apoA-I interacts directly with ABCA1 and stabilizes the transporter. Most of the pool of cell surface-associated apoA-I is bound to lipid domains in the PM that are created by the activity of ABCA1. The amphipathic α-helices in apoA-I confer detergent-like properties on the protein enabling it to solubilize PL and FC in these membrane domains to create a heterogeneous population of discoidal nascent HDL particles. This review focuses on current understanding of the structure-function relationships of human ABCA1 and the molecular mechanisms underlying HDL particle production.

Phospholipid flippase ATP11C is endocytosed and downregulated following Ca2+-mediated protein kinase C activation

We and others showed that ATP11A and ATP11C, members of the P4-ATPase family, translocate phosphatidylserine (PS) and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflets at the plasma membrane. PS exposure on the outer leaflet of the plasma membrane in activated platelets, erythrocytes, and apoptotic cells was proposed to require the inhibition of PS-flippases, as well as activation of scramblases. Although ATP11A and ATP11C are cleaved by caspases in apoptotic cells, it remains unclear how PS-flippase activity is regulated in non-apoptotic cells. Here we report that the PS-flippase ATP11C, but not ATP11A, is sequestered from the plasma membrane via clathrin-mediated endocytosis upon Ca2+-mediated PKC activation. Importantly, we show that a characteristic di-leucine motif (SVRPLL) in the C-terminal cytoplasmic region of ATP11C becomes functional upon PKC activation. Moreover endocytosis of ATP11C is induced by Ca2+-signaling via Gq-coupled receptors. Our data provide the first evidence for signal-dependent regulation of mammalian P4-ATPase.

Evolution and adaptation of single-pass transmembrane proteins

A comparative analysis of 6039 single-pass (bitopic) membrane proteins from six evolutionarily distant organisms was performed based on data from the Membranome database. The observed repertoire of bitopic proteins is significantly enlarged in eukaryotic cells and especially in multicellular organisms due to the diversification of enzymes, emergence of proteins involved in vesicular trafficking, and expansion of receptors, structural, and adhesion proteins. The majority of bitopic proteins in multicellular organisms are located in the plasma membrane (PM) and involved in cell communication. Bitopic proteins from different membranes significantly diverge in terms of their biological functions, size, topology, domain architecture, physical properties of transmembrane (TM) helices and propensity to form homodimers. Most proteins from eukaryotic PM and endoplasmic reticulum (ER) have the N-out topology. The predicted lengths of TM helices and hydrophobic thicknesses, stabilities and hydrophobicities of TM α-helices are the highest for proteins from eukaryotic PM, intermediate for proteins from prokaryotic cells, ER and Golgi apparatus, and lowest for proteins from mitochondria, chloroplasts, and peroxisomes. Tyr and Phe residues accumulate at the cytoplasmic leaflet of PM and at the outer leaflet of membranes of bacteria, Golgi apparatus, and nucleus. The propensity for dimerization increases from unicellular to multicellular eukaryotes, from enzymes to receptors, and from intracellular membrane proteins to PM proteins. More than half of PM proteins form homodimers with a 2:1 ratio of right-handed to left-handed helix packing arrangements. The inverse ratio (1:2) was observed for dimers from the ER, Golgi and vesicles.