Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae defective in the late steps of ergosterol biosynthesis are viable but accumulate structurally altered sterols within the plasma membrane. Despite the significance of pleiotropic abnormalities in the erg mutants, little is known about how sterol alterations mechanically affect the membrane structure and correlate with individual mutant phenotypes. Here we demonstrate that the membrane order and occurrence of voids are determinants of membrane rigidity and hypersensitivity to a drug. Among five ergΔ mutants, the erg2Δ mutant exhibited the most marked sensitivity to cycloheximide. Notably, measurement of time-resolved anisotropy indicated that the erg2Δ mutation decreased the membrane order parameter (S), and dramatically increased the rotational diffusion coefficient (D_w) of 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in the plasma membrane by 8-fold, providing evidence for the requirement of ergosterol for membrane integrity. The IC₅₀ of cycloheximide was closely correlated with S/D_w in these strains, suggesting that the membrane disorder and increasing occurrence of voids within the plasma membrane synergistically enhance passive diffusion of cycloheximide across the membrane. Exogenous ergosterol partially restored the membrane properties in the upc2-1erg2Δ strain. In this study, we describe the ability of ergosterol to adjust the dynamic properties of the plasma membrane, and consider the relevance of drug permeability.     

Localization of membrane proteins in membrane vesicles of Bacillus subtilis.

Electrons can be transferred to the respiratory chain in whole cells and in membrane vesicles of Bacillus subtilis W 23 by the membrane impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulfonate as efficiently as by the membrane permeable electron donor reduced 5-N-methyl-phenazonium methyl-sulfate, indicating that the respiratory chain is accessible from the outside of the membrane.Succinate is oxidized by whole cells and membrane vesicles at a low rate and does not energize transport of l-glutamate. In the presence of 5-N-methyl-phenazonium-3-sulfonate or 5-N-methyl-phenazonium methyl-sulfate, the oxidation rate and the rate of l-glutamate transport are increased considerably. The electrons are transferred directly from succinic dehydrogenase to these acceptors. Succinic dehydrogenase must therefore be exposed to the outside surface of the membrane in both membrane vesicles and whole cells. The exposure of succinic dehydrogenase to the outside is also indicated by the observations that only a 5% increase in the oxidation rates of succinate-5-N-methyl-phenazonium methylsulfate and succinate-5-N-methyl-phenazonium-3-sulfonate is observed upon solubilization of the membrane with the nonionic detergent Brij-58. Furthermore, treatment of membrane vesicles with trypsin decreases by more than 95% these oxidation rates.NADH is oxidized at a high rate and energizes transport of l-glutamate in whole cells and membrane vesicles effectively. The NADH-oxidation is not effected by trypsin treatment of the vesicles indicating that the oxidation occurs at the inside-surface of the membrane. Trypsin treatment of the vesicles, however, significantly decreases the rate of l-glutamate transport driven by NADH. Therefore component(s) of the transport system for l-glutamate must be effected by trypsin treatment. No apparent differences could be observed in the localization of membrane-bound functions between membrane vesicles and whole cells. This strongly supports the contention that the vesicle membrane of B. subtilis has the same orientation as the cytoplasmic membrane of whole cells.     

Indentation with atomic force microscope,Saccharomyces cerevisiae cell gains elasticity under ethanol stress

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane is the first target to be attacked by the accumulated ethanol. In such a prominent position, S. cerevisiae cell membrane could reversely provide protection through changing fluidity or elasticity secondary to remodeled membrane components or structure during the fermentation process. However, there is yet to be a direct observation of the real effect of the membrane compositional change. In this study, atomic force microscope-based strategy was performed to determine Young's modulus of S. cerevisiae to directly clarify ethanol stress-associated changes and roles of S. cerevisiae cell membrane fluidity and elasticity. Cell survival rate decreased while the cell swelling rate and membrane permeability increased as ethanol concentration increased from 0% to 20% v/v. Young's modulus decreased continuously from 3.76 MPa to 1.53 MPa while ethanol stress increased from 0% to 20% v/v, indicating that ethanol stress induced the S. cerevisiae membrane fluidity and elasticity changes. Combined with the fact that membrane composition varies under ethanol stress, to some extent, this could be considered as a forced defensive act to the ethanol stress by S. cerevisiae cells. On the other hand, the ethanol stress induced loosening of cell membrane also caused S. cerevisiae cell to proactively remodel membrane to make cell membrane more agreeable to the increase of environmental threat. Increased ethanol stress made S. cerevisiae cell membrane more fluidized and elastic, and eventually further facilitated yeast cell’s survival.     

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering

The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni²⁺-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering.     

Cholesterol Depletion by MβCD Enhances Cell Membrane Tension and Its Variations-Reducing Integrity

Cholesterol depletion by methyl-β-cyclodextrin (MβCD) remodels the plasma membrane’s mechanics in cells and its interactions with the underlying cytoskeleton, whereas in red blood cells, it is also known to cause lysis. Currently it’s unclear if MβCD alters membrane tension or only enhances membrane-cytoskeleton interactions—and how this relates to cell lysis. We map membrane height fluctuations in single cells and observe that MβCD reduces temporal fluctuations robustly but flattens spatial membrane undulations only slightly. Utilizing models explicitly incorporating membrane confinement besides other viscoelastic factors, we estimate membrane mechanical parameters from the fluctuations’ frequency spectrum. This helps us conclude that MβCD enhances membrane tension and does so even on ATP-depleted cell membranes where this occurs despite reduction in confinement. Additionally, on cholesterol depletion, cell membranes display higher intracellular heterogeneity in the amplitude of spatial undulations and membrane tension. MβCD also has a strong impact on the cell membrane’s tenacity to mechanical stress, making cells strongly prone to rupture on hypo-osmotic shock with larger rupture diameters—an effect not hindered by actomyosin perturbations. Our study thus demonstrates that cholesterol depletion increases membrane tension and its variability, making cells prone to rupture independent of the cytoskeletal state of the cell.     

Loss of carotenoids from membranes of Pantoea sp. YR343 results in altered lipid composition and changes in membrane biophysical properties

Bacterial membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties that allows cells to respond to environmental conditions. Carotenoids are sterol analogs that are important for regulating membrane dynamics. The membrane of Pantoea sp. YR343 is characterized by the presence of the carotenoid zeaxanthin, and a carotenoid-deficient mutant, ΔcrtB, displays defects in root colonization, reduced secretion of indole-3-acetic acid, and defects in biofilm formation. Here we demonstrate that the loss of carotenoids results in changes to the membrane lipid composition in Pantoea sp. YR343, including increased amounts of unsaturated fatty acids in the ΔcrtB mutant membranes. These mutant cells displayed less fluid membranes in comparison to wild type cells as measured by fluorescence anisotropy of whole cells. Studies with artificial systems, however, have shown that carotenoids impart membrane rigidifying properties. Thus, we examined membrane fluidity using spheroplasts and vesicles composed of lipids extracted from either wild type or mutant cells. Interestingly, with the removal of the cell wall and membrane proteins, ΔcrtB vesicles were more fluid than vesicles made from lipids extracted from wild type cells. In addition, carotenoids appeared to stabilize membrane fluidity during rapidly changing temperatures. Taken together, these results suggest that Pantoea sp. YR343 compensates for the loss of carotenoids by changing lipid composition, which together with membrane proteins, results in reduced membrane fluidity. These changes may influence the abundance or function of membrane proteins that are responsible for the physiological changes observed in the ΔcrtB mutant cells.     

Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll a-depleted cytoplasmic membrane in phototrophically grown cells

The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll a-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by <9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is concluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.     

Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli

Escherichia coli is the most widely used host for producing membrane proteins. Thus far, to study the consequences of membrane protein overexpression in E. coli, we have focussed on prokaryotic membrane proteins as overexpression targets. Their overexpression results in the saturation of the Sec translocon, which is a protein-conducting channel in the cytoplasmic membrane that mediates both protein translocation and insertion. Saturation of the Sec translocon leads to (i) protein misfolding/aggregation in the cytoplasm, (ii) impaired respiration, and (iii) activation of the Arc response, which leads to inefficient ATP production and the formation of acetate. The overexpression yields of eukaryotic membrane proteins in E. coli are usually much lower than those of prokaryotic ones. This may be due to differences between the consequences of the overexpression of prokaryotic and eukaryotic membrane proteins in E. coli. Therefore, we have now also studied in detail how the overexpression of a eukaryotic membrane protein, the human KDEL receptor, affects E. coli. Surprisingly, the consequences of the overexpression of a prokaryotic and a eukaryotic membrane protein are very similar. Strain engineering and likely also protein engineering can be used to remedy the saturation of the Sec translocon upon overexpression of both prokaryotic and eukaryotic membrane proteins in E. coli.     

Dynamics of the Acinetobacter baumannii inner membrane under exogenous polyunsaturated fatty acid stress

Exogenous polyunsaturated fatty acids (PUFAs) are readily incorporated into the synthesis pathways of A. baumannii membrane phospholipids, where they contribute to reduced bacterial fitness and increased antimicrobial susceptibility. Here we examine the impact of PUFA membrane modification on membrane organisation and biophysical properties using coarse grained MARTINI simulations of chemically representative membrane models developed from mass-spectrometry datasets of an untreated, arachidonic acid (AA) treated and docosahexaenoic acid (DHA) treated A. baumannii membranes. Enzymatic integration of AA or DHA into phospholipids of the A. baumannii membrane resulted in modulation of membrane biophysical properties. Membrane thickness decreased slightly following PUFA treatment, concomitant with changes in the lateral area per lipid of each lipid headgroup class. PUFA treatment resulted in a decrease in membrane ordering and an increase in lipid lateral diffusion. Changes in lateral membrane organisation were observed in the PUFA treated membranes, with a concurrent increase in ordered cardiolipin domains and disordered PUFA-containing domains. Notably, separation between ordered and disordered domains was enhanced and was more pronounced for DHA relative to AA, providing a possible mechanism for greater antimicrobial action of DHA relative to AA observed experimentally. Furthermore, the membrane active antimicrobial, pentamidine, preferentially adsorbs to cardiolipin domains of the A. baumannii model membranes. This interaction, and membrane penetration of pentamidine, was enhanced following PUFA treatment. Cumulatively, this work explores the wide-ranging effects of PUFA incorporation on the A. baumannii membrane and provides a molecular basis for bacterial inner membrane disruption by PUFAs.     

Effects of vanadate on the plasma membrane ATPase of red beet and corn

The effect of vanadate on the plant plasma membrane ATPase were investigated in plasma membrane fractions derived from corn roots (Zea mays L.) and red beets (Beta vulgaris L.). The K_i for vanadate inhibition of the plasma membrane ATPase from corn roots and red beets was between 6 and 15 micromolar vanadate. In both membrane fractions, 80% to 90% of the total ATPase was inhibited at vanadate concentrations below 100 micromolar. Vanadate inhibition was optimal at pH 6.5, enhanced by the presence of K⁺, and was partially reversed by 1 millimolar EDTA. The Mg:ATP kinetics for the plasma membrane ATPase were hyperbolic in both the absence and presence of vanadate. Vanadate decreased both the K_m and V_max of the red beet plasma membrane ATPase, indicating that vanadate inhibits the ATPase uncompetitively. These results indicate many similarities with respect to vanadate inhibition between the plant plasma membrane ATPase and other major iontranslocating ATPases from fungal and animal cells. The high sensitivity to vanadate reported here, however, differs from other reports of vanadate inhibition of the plant plasma membrane ATPase from corn, beets, and in some instances oats.     

Cytochrome-c-assisted escape of cardiolipin from a model mitochondrial membrane

Binding of cytochrome c (Cytc) to cardiolipin (CL) in the inner mitochondrial membrane is involved with the onset of apoptosis. In this study, we used CL-containing phospholipid monolayers to mimic the inner mitochondrial membrane. Constant pressure insertion assay was employed to monitor the Cytc-induced expansion of membrane area. Simultaneous epifluorescence microscopy imaging afforded the in-situ visualization of phospholipid demixing and sorting in the membrane. The formation of a CL-rich L_d phase has been observed to prelude the insertion of Cytc. Based on the relative expansion of membrane area, a cluster of a few amino acid residues of Cytc with an area of 117 ± 7 Å² has been found to insert into the membrane. The insertion of Cytc disrupted the membrane in a way facilitating the escape of CL. When the exclusion of Cytc was induced by compression, CL molecules appeared to escape the membrane together with the protein, which resulted in a loss of more than a half of CL content from the membrane. These findings may aid in understanding the early events leading to the remodeling of inner mitochondrial membrane and loss of its function during apoptosis.     

The Pink Membrane: The Stable Photoproduct of Deionized Purple Membrane

When cations are removed from the purple membrane of Halobacterium halobium it turns blue (λ_max = 603 nm); continuous irradiation with intense red light (λ's ≥ 630 nm) converts this deionized blue membrane into a pink membrane (λ_max ≈ 491 nm). The rate and extent of the transformation from the blue to the pink membrane is facilitated by the removal of the last twenty COOH-terminal amino acids of bacteriorhodopsin. While the chromophore of the blue membrane is a 32:68 mixture of the 13-cis and all-trans isomers of retinal, the chromophore of the pink membrane is 9-cis rectinal. The quantum efficiency of the pink to blue membrane photoconversion is relatively high compared with that of the blue to pink membrane photoconversion. Proton release is observed when the pink membrane is converted to the blue form, and proton uptake occurs during the reverse transition. Unlike the blue membrane, the absorbance maximum of the pink membrane is only slightly affected by cation addition at low pH and ionic strength.     

Mediation of Clathrin-Dependent Trafficking during Cytokinesis and Cell Expansion by Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE Proteins

STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) encodes a putative Rab guanine nucleotide exchange factor that functions in membrane trafficking and is required for cytokinesis and cell expansion in Arabidopsis thaliana. Here, we show that the loss of SCD2 function disrupts cytokinesis and cell expansion and impairs fertility, phenotypes similar to those observed for scd1 mutants. Genetic and biochemical analyses showed that SCD1 function is dependent upon SCD2 and that together these proteins are required for plasma membrane internalization. Further specifying the role of these proteins in membrane trafficking, SCD1 and SCD2 proteins were found to be associated with isolated clathrin-coated vesicles and to colocalize with clathrin light chain at putative sites of endocytosis at the plasma membrane. Together, these data suggest that SCD1 and SCD2 function in clathrin-mediated membrane transport, including plasma membrane endocytosis, required for cytokinesis and cell expansion.     

Orientation of the primary quinone of bacterial photosynthetic reaction centers contined in chromatophore and reconstituted membranes

The orientation of the reaction center bacteriochlorophyll dimer, (BChl)₂, and primary quinone, Q_I, has been studied by EPR in chromatophores of Rhodopseudomonas sphaeroides R26 and Chromatium vinosum and in the reconstituted membrane multilayers of the isolated Rps. sphaeroides reaction center protein. The similarity in the angular dependence of the (BChl)₂ triplet and Q_I?Fe²⁺ signals in the chromatophore and reconstituted reaction center membrane multilayers indicates that the reaction center is similarly oriented in both native and model membranes. The principle magnetic axes of the (BChl)₂ triplet are found to lie with the x direction approximately parallel to the plane of the membrane surface, and the z and y directions approx. 10–20° away from the plane of the membrane surface and membrane normal, respectively. The Q_I?Fe²⁺ signals are found to have the g 1.82 component positioned perpendicular to the plane of the membrane surface, with an orthogonal low-field transition (at g 1.68 in Rps. Sphaeroides and at g 1.62 in C. vinosum) lying parallel to the plane of the membrane surface. The orientation of Q_I was determined by the angular dependence of this signal in Fe²⁺-depleted reaction center reconstituted membrane multilayers, and it was found to be situated most likely with the plane of the quinone ring perpendicular to the plane of the membrane surface.     

NEW MEMBRANE FORMATION DURING CYTOKINESIS IN NORMAL AND CYTOCHALASIN B-TREATED EGGS OF XENOPUS LAEVIS : II. Electrophysiological Observations

The electrical membrane potential (E_m) and electrical membrane resistance (R_m) were measured continuously during the first cleavage of Xenopus eggs, using intracellular microelectrodes. A sharp hyperpolarization of E_m and decrease in R_m can be observed from 6 to 8 min after the onset of cleavage. This moment coincides with the onset of the insertion of new membrane (Bluemink and de Laat, 1973) leading to the formation of the interblastomeric membrane during normal cleavage. Removal of the vitelline membrane or exposure to cytochalasin B (CCB) leads to exposure of the entire surface area of the membrane newly formed during cleavage. These conditions allow for a direct measurement of the permeability properties of the new membrane. It was found that under these conditions E_m reaches values about 3 times more negative and R_m reaches values about 1.5–3 times smaller than during normal cleavage. The extent of reduction of R_m can be correlated with the surface area of the newly formed membrane. We conclude that the new membrane has different ionic permeability properties than the pre-existing membrane (most probably a relatively high permeability for K⁺ ions). Its mean specific resistance is 1–2 kΩ·cm², as against 74 kΩ·cm² for the pre-existing membrane. No influence of CCB on the permeability properties of the pre-existing or new membrane could be detected.     

Escherichia coli tol-pal Mutants Form Outer Membrane Vesicles

Mutations in the tol-pal genes induce pleiotropic effects such as release of periplasmic proteins into the extracellular medium and hypersensitivity to drugs and detergents. Other outer membrane defective strains such as tolC, lpp, and rfa mutations are also altered in their outer membrane permeability. In this study, electron microscopy and Western blot analyses were used to show that strains with mutations in each of the tol-pal genes formed outer membrane vesicles after growth in standard liquid or solid media. This phenotype was not observed in tolC and rfaD cells in the same conditions. A tolA deletion in three different Escherichia coli strains was shown to lead to elevated amounts of vesicles. These results, together with plasmid complementation experiments, indicated that the formation of vesicles resulted from the defect of any of the Tol-Pal proteins. The vesicles contained outer membrane trimeric porins correctly exposed at the cell surface. Pal outer membrane lipoprotein was also immunodetected in the vesicle fraction of tol strains. The results are discussed in view of the role of the Tol-Pal transenvelope proteins in maintaining outer membrane integrity by contributing to target or integrate newly synthesized components of this structure.     

Transmembrane localization of cis-isomers of zeaxanthin in the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the 9-cis and 13-cis isomers of zeaxanthin on the molecular organization and dynamics of dimyristoylphosphatidylcholine (DMPC) membranes were investigated using conventional and saturation recovery EPR observations of the 1-palmitoyl-2-(14-doxylstearoyl)phosphatidylcholine (14-PC) spin label. The results were compared with the effects caused by the all-trans isomer of zeaxanthin. Effects on membrane fluidity, order, hydrophobicity, and the oxygen transport parameter were monitored at the center of the fluid phase DMPC membrane. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane center, studied by saturation-recovery EPR, decreased by 47% and 27% by including 10 mol% 13-cis and 9-cis zeaxanthin, respectively; whereas, incorporation of all-trans zeaxanthin decreased this parameter by only 11%. At a zeaxanthin-to-DMPC mole ratio of 1:9, all investigated isomers decreased the membrane fluidity and increased the alkyl chain order in the membrane center. They also increased the hydrophobicity of the membrane interior. The effects of these isomers of zeaxanthin on the membrane properties mentioned above increase as: all-trans < 9-cis ≤ 13-cis. Obtained results suggest that the investigated cis-isomers of zeaxanthin, similar to the all-trans isomer, are located in the membrane interior, adopting transmembrane orientation with the polar terminal hydroxyl groups located in the opposite leaflets of the bilayer. However, the existence of the second pool of cis-zeaxanthin molecules located in the one leaflet and anchored by the terminal hydroxyl groups in the same polar headgroup region cannot be completely ruled out.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. II

Frog (Rana catesbeiana) rod outer segment membrane contains cyclic GMP phosphodiesterase (EC 3.1.4.1). Irradiation of dark-adapted rod outer segment membrane increased the enzyme activity by 5–20-fold in the presence of GTP. The phosphodiesterase in rod outer segment membrane is also activated by mixing a photo-product of 11-cis (regenerated), 9-cis or 7-cis rhodopsin which is stable at 0°C. However, neither opsin in the membrane nor all-trans retinal activates the enzyme. The phosphodiesterase in rod outer segment membrane is also activated by irradiation at ?4°C. Thus, we conclude that the phosphodiesterase is activated by a common photolysis intermediate of these rhodopsin isomers, perhaps before metarhodopsin II decays.     

Bacterial-based membrane protein production

Escherichia coli is by far the most widely used bacterial host for the production of membrane proteins. Usually, different strains, culture conditions and production regimes are screened for to design the optimal production process. However, these E. coli-based screening approaches often do not result in satisfactory membrane protein production yields. Recently, it has been shown that (i) E. coli strains with strongly improved membrane protein production characteristics can be engineered or selected for, (ii) many membrane proteins can be efficiently produced in E. coli-based cell-free systems, (iii) bacteria other than E. coli can be used for the efficient production of membrane proteins, and, (iv) membrane protein variants that retain functionality but are produced at higher yields than the wild-type protein can be engineered or selected for. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Crystal Structures of CusC Review Conformational Changes Accompanying Folding and Transmembrane Channel Formation

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the RND (resistance–nodulation–cell division) family to expel diverse toxic compounds from the cell. These complexes span both the inner and outer membranes of the bacterium via an α-helical, inner membrane transporter; a periplasmic membrane fusion protein; and a β-barrel, outer membrane channel. One such efflux system, CusCBA, is responsible for extruding biocidal Cu(I) and Ag(I) ions. To remove these toxic ions, the CusC outer membrane channel must form a β-barrel structural domain, which creates a pore and spans the entire outer membrane. We here report the crystal structures of wild-type CusC, as well as two CusC mutants, suggesting that the first N-terminal cysteine residue plays an important role in protein–membrane interactions and is critical for the insertion of this channel protein into the outer membrane. These structures provide insight into the mechanisms on CusC folding and transmembrane channel formation. It is found that the interactions between CusC and membrane may be crucial for controlling the opening and closing of this β-barrel, outer membrane channel.