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The sterol regulatory element-binding protein (SREBP) pathway monitors the cellular cholesterol level through sterol-regulated association between the SREBP cleavage-activating protein (Scap) and the insulin-induced gene (Insig). Despite structural determination of the Scap and Insig-2 complex bound to 25-hydroxycholesterol, the luminal domains of Scap remain unresolved. In this study, combining cryogenic electron microscopy (cryo-EM) analysis and artificial intelligence-facilitated structural prediction, we report the structure of the human Scap/Insig-2 complex purified in digitonin. The luminal domain loop 1 and a co-folded segment in loop 7 of Scap resemble those of the luminal/extracellular domain in NPC1 and related proteins, providing clues to the cholesterol-regulated interaction of loop 1 and loop 7. An additional luminal interface is observed between Scap and Insig. We also show that Scap(D428A), which inhibits SREBP activation even under sterol depletion, exhibits an identical conformation with the wild-type protein when complexed with Insig-2, and its constitutive suppression of the SREBP pathway may also involve a later step in protein trafficking., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The varied applications of nanotechnology have paved way for several breakthroughs in the realm of biomedical technology. In this challenging era when illness multiplies, timely and accurate disease diagnosis is very important. Thus, well founded novel approaches matter very much in areas like disease diagnosis and monitoring. Nanomedicine has tremendous implications in the given context. An elevated cholesterol concentration in blood is risky and is associated with cardiovascular diseases (CVD). CVD remains the No. 1 global cause of death and hence there is an urge to understand cholesterol level and take preventive measures. Highly fluorescent graphene quantum dots (GQs) are well known for their biocompatibility, non toxicity and aqueous solubility. Here in we report an easy and sensitive non enzymatic based cholesterol detection using digitonin conjugated graphene quantum dots (GDG). Selectivity studies and the cholesterol detection in human blood serum suggests the probe to be reliable and selective for blood cholesterol monitoring. Digitonin conjugated fluorescent graphene quantumdots, an efficient probe for cholesterol sensing.

The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although the migration of supercomplexes has been demonstrated being real, there are still several concerns about its ability to reveal genuine interactions between respiratory complexes. Moreover, the use of different solubilization conditions generates conflicting interpretations. Here, we thoroughly compare the impact of different digitonin concentrations on the liquid dispersions' physical properties and correlate with the respiratory complexes' migration pattern and supercomplexes. Our results demonstrate that digitonin concentration generates liquid dispersions with specific size and variability critical to distinguish between a real association of complexes from being trapped in the same micelle., (Copyright © 2020. Published by Elsevier B.V.)

To conduct biochemical studies in vitro, membrane proteins (MPs) must be solubilized with detergents. While detergents are great tools, they can also inhibit the biological activity and/or perturb oligomerization of individual MPs. Nanodisc scaffold peptide (NSP r ), an amphipathic peptide analog of ApoA1, was recently shown to reconstitute detergent solubilized MPs into peptidiscs in vitro. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), also known as sterol O-acyltransferase 1 (SOAT1), plays a key role in cellular cholesterol storage in various cell types and is a drug target to treat multiple human diseases. ACAT1 contains nine transmembrane domains (TMDs) and primarily forms a homotetramer in vitro and in intact cells; deletion of the N-terminal dimerization domain produces a homodimer with full retention in catalytic activity. ACAT1 is prone to inactivation by numerous detergents. Here we pursued the use of NSP r to overcome the detergent-induced inactivation of ACAT1 by generating near detergent-free ACAT1 peptidiscs. Based on native-PAGE analysis, we showed that NSP r reconstitutes ACAT1 into soluble peptidiscs, in which ACAT1 exists predominantly in oligomeric states greater than a homotetramer. The formation of these higher-order oligomeric states was independent of the N-terminal dimerization domain, suggesting that the oligomerization is mediated through hydrophobic interactions of multiple ACAT1 subunits. ACAT1 peptidiscs were still susceptible to heat-mediated inactivation, presumably due to the residual detergent (CHAPS) bound to ACAT1. We then conditioned ACAT1 with phosphatidylcholine (PC) to replace CHAPS prior to the formation of ACAT1 peptidiscs. The results showed, when PC was included, ACAT1 was present mainly in higher-order oligomeric states with greater enzymatic activity. With PC present, the enzymatic activity of ACAT1 peptidiscs was protected from heat-mediated inactivation. These results support the use of NSP r to create a near detergent-free solution of ACAT1 in peptidiscs for various in vitro studies. Our current results also raise the possibility that, under certain conditions, ACAT1 may form higher-order oligomeric states in vivo., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Differential detergent fractionation of cells is a rapid method for extraction of cytoplasmic and nuclear proteins in preparation of an immunoprecipitation. This method can be applied for use of adherent or suspension cells and can significantly reduce nonspecific background in an immunoprecipitation by separation of cellular compartments into individual fractions. The lysis of cells by differential detergents permits the rapid extraction of proteins from the cytoplasm (digitonin), the cytoplasmic membranes, and organelles (Triton X-100), and nucleoplasm (Tween/DOC), facilitated through the use of distinct extraction buffers. Cytoplasmic and nuclear matrix proteins as well as DNA are left behind during the detergent-based extraction., (© 2020 Cold Spring Harbor Laboratory Press.)

In eukaryotes, most mRNAs that encode secretory or membrane-bound proteins are translated by ribosomes associated with the surface of the endoplasmic reticulum (ER). Other such mRNAs are tethered to the ER by mRNA receptors. However, there has been much debate as to whether all mRNAs, regardless of their encoded polypeptide, are anchored to the ER at some low level. Here we describe a protocol to visualize ER-associated mRNAs in tissue culture cells by single-molecule fluorescence in situ hybridization (smFISH). Using this protocol, we have established that a subset of all mRNAs, regardless of whether they encode secretory or cytosolic proteins, are ER associated in a ribosome-dependent manner.

Zika virus (ZIKV) infection during pregnancy leads to severe congenital Zika syndrome, which includes microcephaly and other neurological malformations. No therapeutic agents have, so far, been approved for the treatment of ZIKV infection in humans; as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat ZIKV-caused diseases. After screening a natural product library, we have herein identified four natural products with anti-ZIKV activity in Vero E6 cells, including gossypol, curcumin, digitonin, and conessine. Except for curcumin, the other three natural products have not been reported before to have anti-ZIKV activity. Among them, gossypol exhibited the strongest inhibitory activity against almost all 10 ZIKV strains tested, including six recent epidemic human strains. The mechanistic study indicated that gossypol could neutralize ZIKV infection by targeting the envelope protein domain III (EDIII) of ZIKV. In contrast, the other natural products inhibited ZIKV infection by targeting the host cell or cell-associated entry and replication stages of ZIKV. A combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-ZIKV compound, exhibited significant combinatorial inhibitory effects against three ZIKV human strains tested. Importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (DENV) human strains in vitro. Taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against ZIKV and other flaviviruses, such as DENV., Competing Interests: The authors declare no competing interests.

Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the F 1 F O -ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-β-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V 2 and V 4 ) and different respiratory supercomplexes (I/IV 6 , I/III 4 , I/IV). In addition, purification/reconstitution of the supercomplexes by anion exchange chromatography was also performed. The data show that these complexes have the ability to strongly associate with each other and form DDM-stable macromolecular structures. The stable V 4 ATPase oligomer was observed by electron-microscopy and the association of the respiratory complexes in the so-called "respirasome" was able to perform in-vitro oxygen consumption., (Copyright © 2018 Elsevier B.V. All rights reserved.)

γ-secretase is an intramembrane protease complex consisting of nicastrin, presenilin-1/2, APH-1a/b, and Pen-2. Hydrolysis of the 99-residue transmembrane fragment of amyloid precursor protein (APP-C99) by γ-secretase produces β-amyloid (Aβ) peptides. Pathogenic mutations in PSEN1 and PSEN2 , which encode the catalytic subunit presenilin-1/2 of γ-secretase, lead to familial Alzheimer's disease in an autosomal dominant manner. However, the underlying mechanism of how the mutant PSEN gene may affect the function of the WT allele remains to be elucidated. Here we report that each of the loss-of-function γ-secretase variants that carries a PSEN1 mutation suppresses the protease activity of the WT γ-secretase on Aβ production. Each of these γ-secretase variants forms a stable oligomer with the WT γ-secretase in vitro in the presence of the detergent CHAPSO {3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate}, but not digitonin. Importantly, robust protease activity of γ-secretase is detectable in the presence of CHAPSO, but not digitonin. These experimental observations suggest a dominant negative effect of the γ-secretase, in which the protease activity of WT γ-secretase is suppressed by the loss-of-function γ-secretase variants through hetero-oligomerization. The relevance of this finding to the genesis of Alzheimer's disease is critically evaluated., Competing Interests: Conflict of interest statement: Y.S. and Y.-M.L. were co-authors on a 2015 paper., (Copyright © 2017 the Author(s). Published by PNAS.)

Digitonin is commonly used to permeabilize cell membranes and solubilize membrane components. It interacts specifically with cholesterol in the membrane which leads to the formation of pores. Thus far, the mechanism by which digitonin interacts with the membrane has only been described qualitatively. We investigated this interaction in model membranes that contain little or no cholesterol with a combination of isothermal titration calorimetry, dynamic light scattering, and zeta potential measurements. Digitonin partitions fully asymmetrically into large unilamellar vesicles of phosphocholine (PC) lipid at 20°C (remaining in the outer leaflet only), with a partition coefficient of 0.22±0.04mM -1 and ΔH of partitioning of 23.3±1.6kJmol -1 . Beyond a digitonin/lipid ratio of ∼0.1 in the outer leaflet, digitonin micelles coexist with vesicles without solubilizing them-even at high digitonin concentrations. This "staying out" of digitonin was also observed with phosphoserine (PS), PC/PS, and PC/PS/cholesterol vesicles. The mechanism by which digitonin perturbs and solubilizes the membrane is very different when the membrane contains little or no cholesterol as opposed to 20-30mol% cholesterol. The role of digitonin should thus be carefully considered in the design of preparative protocols and experiments in studies of cellular processes and membrane proteins., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na + ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na + /H + exchangers are required for glycosomal pH regulation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The sugar subunits of natural glycosides can be conveniently determined by acid hydrolysis and (1)H NMR spectroscopy without isolation or derivatization. The chemical shifts, coupling constants, and integral ratios of the anomeric signals allow each monosaccharide to be identified and its molar ratio to other monosaccharides to be quantified. The NMR data for the anomeric signals of 28 monosaccharides and three disaccharides are reported. Application of the method is demonstrated with the flavonoid glycoside naringin (1), the aminoglycoside antibiotics kanamycin (2) and tobramycin (3), and the saponin digitonin (4).

In this article, we describe the effect of a highly hemolytic saponin, digitonin, on model lipids cholesterol and dipalmitoylphosphatidylcholine (DPPC) using a combination of tensiometric (surface pressure and dilatational surface elasticity), spectroscopic (infrared reflection absorption spectroscopy, IRRAS), microscopic (fluorescence microscopy), and scattering techniques (neutron reflectivity, NR, and grazing incidence X-ray diffraction, GIXD). The monolayers of individual lipids and their 10:9 (mol/mol) mixture were exposed to an aqueous solution of digitonin (10(-4) M) by subphase exchange using a setup developed recently in our laboratory. The results confirm that digitonin can adsorb onto both bare and lipid-covered water-air interfaces. In the case of DPPC, a relatively weak interaction can be observed, but the presence of cholesterol drastically enhances the effect of digitonin. The latter is shown to dissociate the weak cholesterol-DPPC complexes and to bind cholesterol in an additional layer attached to the original lipid monolayer.

A number of non-membranous cellular bodies have been identified in higher eukaryotes, and these bodies contain a specific set of proteins and RNAs that are used to fulfill their functions. The size of these RNA-containing cellular bodies is usually on a submicron scale, making it difficult to observe fine structures using optical microscopy due to the diffraction limitation of visible light. Recently, microscope companies have released super-resolution microscopes that were developed using different principles, enabling the observation of sub-micron structures not resolvable in conventional fluorescent microscopy. Here, we describe multi-color fluorescent in situ hybridization techniques optimized for the simultaneous detection of RNA and proteins using super-resolution microscopy, namely structured illumination microscopy (SIM)., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8-35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q10 (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q10, and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I1, III2, and IV1 using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Ess/Type VII protein secretion system, essential for virulence of pathogenic Staphylococcus aureus, is dependent upon the four core membrane proteins EssA, EssB, EssC and EsaA. Here, we use crosslinking and blue native PAGE analysis to show that the EssB, EssC and EsaA proteins individually form homomeric complexes. Surprisingly, these components appear unable to interact with each other, or with the EssA protein. We further show that two high molecular weight multimers of EssC detected in whole cells are not dependent upon the presence of EsxA, EsxB or any other Ess component for their assembly., (© 2016 Federation of European Biochemical Societies.)

Membranes prepared from rat brain were treated with increasing concentrations of cationic, neutral, anionic and zwitterionic surfactants. Potent inactivation of [(3)H]MK-801 binding to NMDA receptors (NRs) was provided by the cation cetyl pyridinium (IC50 25 μM) and the neutral digitonin (IC50 37 μM). A 2 h incubation of rat brain membranes at 24°C with 100 μM of the neutral Triton X-100 resulted in about 50% reversible inhibition (without inactivation). Reversible inhibition was also effected by the anion deoxycholate (IC50 700 μM), and by the zwitterions N-lauryl sulfobetaine (12-SB(±), 400 μM) and CHAPS (1.5 mM), with inactivation at higher concentrations. Keeping the NR cation channel in the closed state significantly protected against inactivation by cations and by 12-SB(±), but not by the other detergents. Inactivation depended differentially on the amount of the membranes, on the duration of the treatment, and on the temperature. Varying the amount of membranes by a factor 8 yielded for cetyl trimethylammonium (16-NMe3(+)) IC50s of inactivation from 10 to 80 μM, while for deoxycholate the IC50 of inactivation was 1.2 mM for all tissue quantities. Some compounds inactivated within a few min (16-NMe3(+), digitonin, CHAPS), while inactivation by others took at least half an hour (Triton X-100, deoxycholate, 12-SB(±)). These last 3 ones also exhibited the steepest temperature dependence. Knowledge about the influence of various parameters is helpful in selecting appropriate conditions allowing the treatment of brain membranes with amphiphiles without risking irreversible inactivation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In the present investigation we studied the molecular mechanisms of the monodesmosidic saponin digitonin on natural and artificial membranes. We measured the hemolytic activity of digitonin on red blood cells (RBCs). Also different lipid membrane models (large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs) in the presence and absence of cholesterol were employed. The stability and permeability of the different vesicle systems were studied by using calcein release assay, GUVs membrane permeability assay using confocal microscopy (CM) and fluorescence correlation spectroscopy (FCS) and vesicle size measurement by dynamic light scattering (DLS). The results support the essential role of cholesterol in explaining how digitonin can disintegrate biological and artificial membranes. Digitonin induces membrane permeability or causes membrane rupturing only in the presence of cholesterol in an all-or-none mechanism. This effect depends on the concentrations of both digitonin and cholesterol. At low concentrations, digitonin induces membrane permeability while keeping the membrane intact. When digitonin is combined with other drugs, a synergistic potentiation can be observed because it facilitates their uptake.

N,N'-diacetylbacillosamine is a novel sugar that plays a key role in bacterial glycosylation. Three enzymes are required for its biosynthesis in Campylobacter jejuni starting from UDP-GlcNAc. The focus of this investigation, PglE, catalyzes the second step in the pathway. It is a PLP-dependent aminotransferase that converts UDP-2-acetamido-4-keto-2,4,6-trideoxy-d-glucose to UDP-2-acetamido-4-amino-2,4,6-trideoxy-d-glucose. For this investigation, the structure of PglE in complex with an external aldimine was determined to a nominal resolution of 2.0 Å. A comparison of its structure with those of other sugar aminotransferases reveals a remarkable difference in the manner by which PglE accommodates its nucleotide-linked sugar substrate., (© 2015 The Protein Society.)

Saponins, naturally occurring plant compounds are known for their biological and pharmacological activity. This activity is strongly related to the amphiphilic character of saponins that allows them to aggregate in aqueous solution and interact with membrane components. In this work, Langmuir monolayer techniques combined with polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and Brewster angle microscopy were used to study the interaction of selected saponins with lipid model membranes. Two structurally different saponins were used: digitonin and a commercial Merck Saponin. Membranes of different composition, namely, cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) were formed at the air/water and air/saponin solution interfaces. The saponin-lipid interaction was characterized by changes in surface pressure, surface potential, surface morphology and PM-IRRAS signal. Both saponins interact with model membranes and change the physical state of membranes by perturbing the lipid acyl chain orientation. The changes in membrane fluidity were more significant upon the interaction with Merck Saponin. A higher affinity of saponins for cholesterol than phosphatidylglycerols was observed. Moreover, our results indicate that digitonin interacts strongly with cholesterol and solubilize the cholesterol monolayer at higher surface pressures. It was shown, that digitonin easily penetrate to the cholesterol monolayer and forms a hydrogen bond with the hydroxyl groups. These findings might be useful in further understanding of the saponin action at the membrane interface and of the mechanism of membrane lysis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer's disease. Despite recent structural advances, the transmembrane segments (TMs) of γ-secretase remain to be specifically assigned. Here we report a 3D structure of human γ-secretase at 4.32-Å resolution, determined by single-particle, electron cryomicroscopy in the presence of digitonin and with a T4 lysozyme fused to the amino terminus of presenilin 1 (PS1). The overall structure of this human γ-secretase is very similar to that of wild-type γ-secretase determined in the presence of amphipols. The 20 TMs are unambiguously assigned to the four components, revealing principles of subunit assembly. Within the transmembrane region, PS1 is centrally located, with its amino-terminal fragment (NTF) packing against Pen-2 and its carboxyl-terminal fragment (CTF) interacting with Aph-1. The only TM of nicastrin associates with Aph-1 at the thick end of the TM horseshoe, and the extracellular domain of nicastrin directly binds Pen-2 at the thin end. TM6 and TM7 in PS1, which harbor the catalytic aspartate residues, are located on the convex side of the TM horseshoe. This structure serves as an important framework for understanding the function and mechanism of γ-secretase.

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Platelet secretion not only drives thrombosis and hemostasis, but also mediates a variety of other physiological and pathological processes. The ubiquitous SNARE machinery and a number of accessory proteins have been implicated in regulating secretion in platelet. Although several platelet SNAREs have been identified, further members of the SNARE family may be needed to fine-tune platelet secretion. In this study we identified expression of the t-SNARE syntaxin 8 (STX8) (Qc SNARE) in mouse and human platelets. In mouse studies, whereas STX8 was not essential for α-granule or lysosome secretion, Stx8(-/-) platelets showed a significant defect in dense granule secretion in response to thrombin and CRP. This was most pronounced at intermediate concentrations of agonists. They also showed an aggregation defect that could be rescued with exogenous ADP and increased embolization in Stx8(-/-) mice in vivo consistent with an important autocrine and paracrine role for ADP in aggregation and thrombus stabilization. STX8 therefore specifically contributes to dense granule secretion and represents another member of a growing family of genes that play distinct roles in regulating granule release from platelets and thus platelet function in thrombosis and hemostasis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Digitonin is an amphiphilic steroidal saponin, a class of natural products that can bind to cholesterol and lyse cells. Despite the known cell membrane lysis activity, it remains unclear how it interacts with cell membranes. In the present work, the interaction mechanism between digitonin and cell membrane models has quantitatively been investigated using a combination of physical techniques. It has been demonstrated that digitonin molecules bind specifically to cholesterol in the membrane, resulting in the formation of cholesterol-digitonin complexes on the membrane surface by removing cholesterol from the membrane core. Changes in the mass density and the film mechanics caused by the digitonin were determined by using quartz crystal microbalance with dissipation (QCM-D), and the combination of X-ray reflectivity (XRR) and dual polarization interferometry (DPI) yielded the hydration level of the cholesterol-digitonin complexes. From differential scanning calorimetry (DSC) analysis, supporting evidence was obtained that cholesterol was removed from the membrane core.

Optically active deuterated primary amines have been obtained with 78-98% ee's from chiral N-tert-butanesulfinyl aldimines via reduction with N-Selectride and subsequent alcoholysis.

Real-time imaging coupled with a permeabilized cell system presents a very versatile platform to visualize the dynamic and intricate nature of nuclear envelope breakdown, one of the major morphological changes of mitosis. Here, we describe such a strategy in which the plasma membrane of cells expressing fluorescently tagged nucleoporin POM121 and Histone H2B is permeabilized with digitonin. These cells are then incubated with mitotic Xenopus egg extract to create conditions that recapitulate the major events of mitotic nuclear remodeling seen in live-cell imaging, providing the opportunity to probe mechanisms and pathways that coordinate nuclear disassembly.

The two novel cyanobacterial cyclic lipopeptides, anabaenolysin (Abl) A and B permeabilised mammalian cells, leading to necrotic death. Abl A was a more potent haemolysin than other known biodetergents, including digitonin, and induced discocyte-echinocyte transformation in erythrocytes. The mitochondria of the dead cells appeared intact with regard to both ultrastructure and membrane potential. Also isolated rat liver mitochondria were resistant to Abl, judged by their ultrastructure and lack of cytochrome c release. The sparing of the mitochondria could be related to the low cholesterol content of their outer membrane. In fact, a supplement of cholesterol in liposomes sensitised them to Abl. In contrast, the prokaryote-directed cyclic lipopeptide surfactin lysed preferentially non-cholesterol-containing membranes. In silico comparison of the positions of relevant functional chemical structures revealed that Abl A matched poorly with surfactin in spite of the common cyclic lipopeptide structure. Abl A and the plant-derived glycolipid digitonin had, however, predicted overlaps of functional groups, particularly in the cholesterol-binding tail of digitonin. This may suggest independent evolution of Abl and digitonin to target eukaryotic cholesterol-containing membranes. Sub-lytic concentrations of Abl A or B allowed influx of propidium iodide into cells without interfering with their long-term cell viability. The transient permeability increase allowed the influx of enough of the cyanobacterial cyclic peptide toxin nodularin to induce apoptosis. The anabaenolysins might therefore not only act solely as lysins, but also as cofactors for the internalisation of other toxins. They represent a potent alternative to digitonin to selectively disrupt cholesterol-containing biological membranes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Muramyl peptides derived from bacterial peptidoglycan have long been known for their ability to trigger host innate immune responses, including inflammation and antimicrobial defense. Muramyl peptides have also been widely studied for their role as immune adjuvants. In mammals, the nucleotide-binding oligomerization domain (Nod) proteins Nod1 and Nod2 detect distinct muramyl peptide structures and mediate their biological activity. Because of the poor immunogenicity of these small peptidoglycan derivatives, research in this field is currently limited by the lack of reagents to track or immobilize specific muramyl peptides. We present here the generation and initial biological characterization of synthetic muramyl peptides covalently coupled to dansyl or biotinyl derivatives and demonstrate that biotinyl coupling on the muramyl moiety results in derivatives that can be tracked by immunofluorescence and maintain full biological activity, as observed by their capacity to trigger Nod signaling. Moreover, using digitonin-mediated permeabilization techniques on live cells, we also demonstrate that biotinylated muramyl peptides efficiently reach the host cytosol, where they activate Nod signaling. Therefore, these derivatives represent useful probes to study the cell biology and the biochemistry of host responses to muramyl peptides., (© 2011 John Wiley & Sons A/S.)

The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit.

Functionalized colloidal gold is widely used for qualitative and quantitative detection of specific analytes. We report here a novel modification of gold nanoparticles by digitonin, a glycoside used for precipitating membrane cholesterol. The specific molecular recognition of cholesterol by digitonin gold nanoparticles (DGNP), make it an attractive alternative to the existing enzymatic methods for cholesterol sensing. To enable cholesterol binding, we modified mercapto modified GNPs with digitonin, by a simple esterification reaction. The blue shift in the plasmon absorption spectra of DGNP with different cholesterol concentrations accompanied by a decrease in the absorbance is the principle applied here for the estimation. The observed size reduction followed by cholesterol binding is reasoned due to the enhanced hydophobicity of the surface which in turn expels the water layers associated with the particles prior to cholesterol binding. The method exhibited linearity between concentration of cholesterol and the corresponding absorbance of the plasmon peak, in the range of 160-600 ng/mL with a detection limit of 100±9 ng/mL. Other steroids did not show any binding affinity towards DGNP. The method depicted here has potential for development as an enzyme free sensor for cholesterol although many factors need to be addressed to transform it for assaying samples like blood., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Permeabilization was evaluated as a rapid method to prepare mammalian cells for subcellular enzyme activity measurement. It was observed that enzymes can be measured directly in cell suspensions permeabilized by Triton X-100 and digitonin with various concentrations. Total enzyme activities measured in permeabilized cells were identical to those measured in sonicated cells showing that permeabilization can replace the more complicated sonication method. Tuning of digitonin concentration allowed selective permeabilization of plasma and mitochondrial membranes. This was studied by analyzing the release of extramitochondrial and mitochondrial marker enzymes on treatment with different concentrations of the agent. Solely the plasma membrane was permeabilized by using 0.01-0.02% (w/v) digitonin. Access to all cellular enzymes was achieved by using 0.05% (v/v) Triton X-100. This selective permeabilization was further evaluated in a 96-well plate format by testing additional marker enzymes and additional cell lines, Hep G2 and CHO-K1, applying the developed protocol. The presented method is well suited for the high-throughput analysis of subcellular localization and activity of enzymes. The method is simple and enables one to distinguish between mitochondrial and extramitochondrial activities, which is usually achieved only by much more complicated and time-consuming cell preparation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: The estrogen receptor alpha (ERα) is found predominately in the nucleus, both in hormone stimulated and untreated cells. Intracellular distribution of the ERα changes in the presence of agonists but the impact of different antiestrogens on the fate of ERα is a matter of debate., Results: A MCF-7 cell line stably expressing GFP-tagged human ERα (SK19 cell line) was created to examine the localization of ligand-bound GFP-ERα. We combined digitonin-based cell fractionation analyses with fluorescence and immuno-electron microscopy to determine the intracellular distribution of ligand-bound ERα and/or GFP-ERα.Using fluorescence- and electron microscopy we demonstrate that both endogenous ERα and GFP-ERα form numerous nuclear focal accumulations upon addition of agonist, 17β-estradiol (E2), and pure antagonists (selective estrogen regulator disruptor; SERD), ICI 182,780 or RU58,668, while in the presence of partial antagonists (selective estrogen regulator modulator; SERM), 4-hydroxytamoxifen (OHT) or RU39,411, diffuse nuclear staining persisted.Digitonin based cell fractionation analyses confirmed that endogenous ERα and GFP-ERα predominantly reside in the nuclear fraction. Overall ERα protein levels were reduced after estradiol treatment. In the presence of SERMs ERα was stabilized in the nuclear soluble fraction, while in the presence of SERDs protein levels decreased drastically and the remaining ERα was largely found in a nuclear insoluble fraction. mRNA levels of ESR1 were reduced compared to untreated cells in the presence of all ligands tested, including E2. E2 and SERDs induced ERα degradation occurred in distinct nuclear foci composed of ERα and the proteasome providing a simple explanation for ERα sequestration in the nucleus., Conclusions: Our results indicate that chemical structure of ligands directly affect the nuclear fate and protein turnover of the estrogen receptor alpha independently of their impact on transcription. These findings provide a molecular basis for the selection of antiestrogen compounds issue from pharmacological studies aimed at improving treatment of breast cancer.

Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

Import of exogenous plasmid DNA (pDNA) into mammalian cell nuclei represents a key intracellular obstacle to efficient non-viral gene delivery. This includes access of the pDNA to the nuclei of non-dividing cells where the presence of an intact nuclear membrane is limiting for gene transfer. Here we identify, isolate, and characterize, cytoplasmic determinants of pDNA nuclear import into digitonin-permeabilized HeLa cells. Depletion of putative DNA-binding proteins, on the basis of their ability to bind immobilized pDNA, abolished pDNA nuclear import supporting the critical role of cytoplasmic factors in this process. Elution of pDNA-bound proteins, followed by two-dimensional sodium dodecyl polyacrylamide gel electrophoresis identified several candidate DNA shuttle proteins. We show that two of these, NM23-H2, a ubiquitous c-Myc transcription-activating nucleoside diphosphate kinase, and the core histone H2B can both reconstitute pDNA nuclear import. Further, we demonstrate a significant increase in gene transfer in non-dividing HeLa cells transiently transfected with pDNA containing binding sequences from two of the DNA shuttle proteins, NM23-H2 and the homeobox transcription factor Chx10. These data support the hypothesis that exogenous pDNA binds to cytoplasmic shuttle proteins and is then translocated to the nucleus using the minimal import machinery. Importantly, increasing the binding of pDNA to shuttle proteins by re-engineering reporter plasmids with shuttle binding sequences enhances gene transfer. Increasing the potential for exogenously added pDNA to bind intracellular transport cofactors may enhance the potency of non-viral gene transfer.

Phosphoarginine is a cell energy buffer molecule synthesized by the enzyme arginine kinase. In Trypanosoma cruzi, the aetiological agent of Chagas' disease, 2 different isoforms were identified by data mining, but only 1 was expressed during the parasite life cycle. The digitonin extraction pattern of arginine kinase differed from those obtained for reservosomes, glycosomes and mitochondrial markers, and similar to the cytosolic marker. Immunofluorescence analysis revealed that although arginine kinase is localized mainly in unknown punctuated structures and also in the cytosol, it did not co-localize with any of the subcelular markers. This punctuated pattern has previously been observed in many cytosolic proteins of trypanosomatids. The knowledge of the subcellular localization of phosphagen kinases is a crucial issue to understand their physiological role in protozoan parasites.

The effect of Thapsigargicin (TGC), a non-phorbol ester type tumor promoter, on Ca2+ movements has been investigated using L1210 mouse lymphoma cells. Ca2+ release from intact and digitonin permeabilized cells was evaluated using Fura-2 and Fura-3. TGC like Thapsigargin (TG) has the ability to discharge the intracellular Ca2+ stores and to increase intracellular free Ca2+ concentrations. TGC in a concentration dependent manner (0.16-16 nM) also inhibited cell growth and this effect was at least partially reversed by arachidonate.

Abnormal forms of prion protein (PrP(Sc)) accumulate via structural conversion of normal PrP (PrP(C)) in the progression of transmissible spongiform encephalopathy. Under cell-free conditions, the process can be efficiently replicated using in vitro PrP(Sc) amplification methods, including protein misfolding cyclic amplification. These methods enable ultrasensitive detection of PrP(Sc); however, there remain difficulties in utilizing them in practice. For example, to date, several rounds of protein misfolding cyclic amplification have been necessary to reach maximal sensitivity, which not only take several weeks, but also result in an increased risk of contamination. In this study, we sought to further promote the rate of PrP(Sc) amplification in the protein misfolding cyclic amplification technique using mouse transmissible spongiform encephalopathy models infected with either mouse-adapted bovine spongiform encephalopathy or mouse-adapted scrapie, Chandler strain. Here, we demonstrate that appropriate regulation of sonication dramatically accelerates PrP(Sc) amplification in both strains. In fact, we reached maximum sensitivity, allowing the ultrasensitive detection of < 1 LD(50) of PrP(Sc) in the diluted brain homogenates, after only one or two reaction rounds, and in addition, we detected PrP(Sc) in the plasma of mouse-adapted bovine spongiform encephalopathy-infected mice. We believe that these results will advance the establishment of a fast, ultrasensitive diagnostic test for transmissible spongiform encephalopathies.

The isoprenoid biosynthetic pathway is a very complex route that entails multiple steps and generates a high number of end-products that are essential for cell viability such as sterols, dolichols, coenzyme Q, heme and prenylated proteins. In parasites from the Trypanosomatidae family this pathway provides new potential drug targets for exploitation in the search for improved therapies, and indeed compounds such as ketoconazole, aminobisphosphonates or terbinafine have been shown to have antiprotozoal activity both in vitro and in vivo. However, despite the high therapeutic importance of the pathway, the subcellular compartmentalization of the different steps of isoprenoid biosynthesis is not known in detail. Here we have analysed the intracellular location of the enzymes 3-hydroxy-3-methyl-glutaryl Coenzyme A (HMG-CoA) synthase (HMGS) and mevalonate kinase (MVAK) in Leishmania major promastigotes as well as in Trypanosoma brucei procyclic and bloodstream forms. For this purpose we generated specific polyclonal antibodies against both highly purified recombinant proteins and used those in indirect immunofluorescence and digitonin titration experiments. Results show that sterol biosynthesis is distributed in multiple intracellular compartments and provide evidence indicating that in trypanosomatids the production of HMG-CoA from acetyl Coenzyme A and generation of mevalonate occur mainly in the mitochondrion while further mevalonate phosphorylation is almost exclusively located in glycosomes. Furthermore, we have determined that peroxin 2 (PEX2) is involved in efficient targeting of MVAK and that the enzyme is relocated to the cytosol upon depletion of this peroxin involved in glycosomal matrix protein import.

The enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) are coassembled into higher ordered supercomplexes within the mitochondrial inner membrane. The cytochrome bc(1)-cytochrome c oxidase (COX) supercomplex is formed by the coassociation of the two electron transport chain complexes, the cytochrome bc(1) (cytochrome c reductase) and the COX complex. Recent evidence indicates that a diversity in the populations of the cytochrome bc(1)-COX supercomplexes exists within the mitochondria, because different subpopulations of this supercomplex have been shown to further interact with distinct partner complexes (e.g., the TIM23 machinery and also the Shy1/Cox14 proteins). By use of native gel electrophoresis and affinity purification approaches, the abundant ADP/ATP carrier protein (AAC) isoform in the yeast Saccharomyces cerevisiae, the Aac2 isoform, has recently been found to also exist in physical association with the cytochrome bc(1)-COX supercomplex and its associated TIM23 machinery. The AAC proteins play a central role in cellular metabolism, because they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. The method used to analyze the cytochrome bc(1)-COX-AAC supercomplex and to affinity purify the Aac2 isoform and its associating proteins from S. cerevisiae mitochondria will be outlined in this chapter.

The present chapter describes methods for the separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts by using different detergents, high-performance liquid chromatography and mass spectrometry. Thylakoid membranes represent a good model for setting up analytical methods suitable for membrane protein characterization.The first step in the procedure is the preparation of purified membrane fractions from plant tissues, followed by the fractionation of membrane proteins by differential solubilization using different detergents. Thus, several protein complexes can be isolated, collected, separated by ion-pair reversed-phase chromatography and detected online by UV-absorption and/or mass spectrometry. Finally, identification of the eluting proteins is accomplished by comparing the molecular mass determined in silico with the molecular mass measured by mass spectrometry.

A hybrid quadrupole orthogonal time-of-flight mass spectrometer optimized for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization has been equipped with a C 60 cluster ion source. This configuration is shown to exhibit a number of characteristics that improve the performance of traditional time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments for the analysis of complex organic materials and, potentially, for chemical imaging. Specifically, the primary ion beam is operated as a continuous rather than a pulsed beam, resulting in up to 4 orders of magnitude greater ion fluence on the target. The secondary ions are extracted at very low voltage into 8 mTorr of N 2 gas introduced for collisional focusing and cooling purposes. This extraction configuration is shown to yield secondary ions that rapidly lose memory of the mechanism of their birth, yielding tandem mass spectra that are identical for SIMS and MALDI. With implementation of ion trapping, the extraction efficiency is shown to be equivalent to that found in traditional TOF-SIMS machines. Examples are given, for a variety of substrates that illustrate mass resolution of 12,000-15,600 with a mass range for inorganic compounds to m/ z 40,000. Preliminary chemical mapping experiments show that with added sensitivity, imaging in the MS/MS mode of operation is straightforward. In general, the combination of MALDI and SIMS is shown to add capabilities to each technique, providing a robust platform for TOF-SIMS experiments that already exists in a large number of laboratories.

The fluorescent carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) has been widely used for tracing of neuronal pathways. To examine identities of the DiI-labeled neuronal pathways, it is desirable to combine DiI labeling with immunofluorescent staining. However, DiI labeling and immunofluorescent staining are not well compatible, mainly because treatment of DiI-labeled neurons with detergents, which are commonly used for immunohistochemistry, results in high levels of diffusion of the DiI label. In this study, we searched for detergents that are compatible with DiI labeling, and found that a cholesterol-specific detergent digitonin is useful for fluorescent double-labeling with DiI tracing and immunohistochemistry. We show that digitonin treatment, in contrast to Triton X-100, methanol and Nonidet P-40 treatment, preserves DiI labeling, even at higher concentrations. We also show that digitonin also preserves the signal of a DiI derivative CM-DiI. Moreover, we demonstrate that digitonin efficiently increases antibody penetration into brain sections. As a result, immunohistochemical images obtained with digitonin treatment are as good as those obtained with Triton X-100 treatment. In addition, we also try another cholesterol-specific detergent quillaja saponin, but find that it degrades the DiI label. Our simple double-labeling protocol using digitonin should prove useful in enabling detailed examination of the neuronal circuitry of the nervous system.

Obtaining sufficient amount of purified G-protein coupled receptors (GPCRs) is almost always one of the major challenges for their structural studies. CB2(271-326), a human cannabinoid receptor 2 (CB2) fragment comprising part of the third extracellular loop (EL3), the seventh transmembrane domain (TM7) and C-terminal juxtamembrane region of the receptor, was over-expressed as a fusion protein into inclusion body (IB) of Escherichia coli. The fusion protein was purified by histidine-selected nickel affinity chromatography under denaturing conditions. Then, the fusion protein IBs were solubilized in detergent (Brij58) and the expression fusion leader sequence (TrpLE) was specifically cleaved with tobacco etch virus (TEV) protease. The target fragment, CB2(271-326), was subsequently purified by reverse-phase HPLC and confirmed by SDS-PAGE and mass spectrometry. This hydrophobic fragment can refold in mild detergents digitonin and Brij58. Circular dichroism (CD) spectroscopy of CB2(271-326) in digitonin and Brij58 micelles showed that the fragment adopts a more than 75% alpha-helical structure, with the remainder having beta-strand structure. Fluorescence spectroscopy and quenching studies suggested that the C-terminal region lies near the surface of the digitonin micelles and the TM7 region is folded relatively close to the center of the micelles. This study may provide an alternative strategy for the production and structure/functional studies of GPCRs such as CB2 receptor protein produced in the form of IBs.

We introduce high resolution clear native electrophoresis (CNE) as a powerful technique to resolve enzymatically active mitochondrial complexes from cultured human cell lines and skeletal muscle biopsy samples. Quantitative enzymatic assays can be performed using small amounts of cultured cells with low mitochondria content, for example, around 10 mg of sedimented osteosarcoma cells (wet weight) which is equivalent to around 10 million cells. High resolution CNE offers general advantages for in-gel catalytic activity assays compared to blue native electrophoresis. It seems especially suited for assaying mitochondrial ATP synthase and respiratory chain complexes I and II in cell models of human mitochondrial disorders and for detailed analyses of patient cells and tissues with defects in oxidative phosphorylation.

Heavy fragments were isolated from pea chloroplasts using digitonin treatment and differential centrifugation. The particles were characterized by a significantly lowered chlorophyll a/b ratio, contents of photosystem I (PS I) proteins and ATPase, as well as of amount of P700. The content of photosystem II (PS II) proteins decreased insignificantly, whereas that of proteins of the light-harvesting complex II did not change. The absorption and low-temperature fluorescence spectra were indicative of a decreased content of PS I. Electron microscopy of ultrathin sections of heavy fragment preparations identified them as grana with reduced content of thylakoids. The diameter of these particles was practically the same as within chloroplasts. Comparison of various characteristics of the fragments and chloroplasts from which the fragments were isolated allowed us to define a high degree of preservation of marginal regions in thylakoids present in the heavy fragment particles. Analysis of the results shows that the procedure of fragmentation produces grana with high extent of thylakoid integrity. The phenomenon of reduction of the thylakoid content in grana, occurring as our heavy fragments, is considered in the frame of our previous hypothesis concerning the peculiarities of grana organization in the transversal direction.

Within recent years, the advances in proteomics techniques have resulted in considerable novel insights into the protein expression patterns of specific tissues, cells, and organelles. The information acquired from large-scale proteomics approaches indicated, however, that the proteomic analysis of whole cells or tissues is often not suited to fully unravel the proteomes of individual organellar constituents or to identify proteins that are present at low copy numbers. In addition, the identification of hydrophobic proteins is still a challenge. Therefore, the development of techniques applicable for the enrichment of low-abundance membrane proteins is essential for a comprehensive proteomic analysis. In addition to the enrichment of particular subcellular structures by subcellular fractionation, the spectrum of techniques applicable for proteomics research can be extended toward the separation of integral and peripheral membrane proteins using organic solvents, detergents, and detergent-based aqueous two-phase systems with water-soluble polymers. Here, we discuss the efficacy of a number of experimental protocols. We demonstrate that the appropriate selection of physicochemical conditions results in the isolation of synaptic vesicles of high purity whose proteome can be subfractionated into integral membrane proteins and soluble proteins by several phase separation techniques.

Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.

Nitroxide sensors were placed in rhodopsin at sites 140, 227, 250, and 316 to monitor the dynamics and conformation of the receptor at the cytoplasmic surface in solutions of dodecyl maltoside (DM), digitonin, and phospholipid bilayers of two compositions. The EPR spectra reveal a remarkable similarity of rhodopsin structure and the activating conformational change in DM and bilayers, the hallmark of which is an outward tilt of transmembrane helix VI. This conformational change is blocked in solutions of digitonin, although changes in optical absorbance accompany activation, showing that absorbance and structural changes are not necessarily coupled. In DM and bilayers, the receptor is apparently in equilibrium between conformational substates whose populations are modulated by activation. Despite the general similarity in the two environments, the receptor conformations have increased flexibility in DM relative to bilayers. For the activated receptor in DM and bilayers, a pH-dependent conformational equilibrium is identified that may correspond to the optically characterized MII(a)()-MII(b)() equilibrium. No specific effects of headgroup composition on receptor conformation in lipid bilayers were found.