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Absorption of dietary sphingomyelin (SM) requires its initial degradation into ceramide, a process catalyzed by the intestinal enzyme alkaline sphingomyelinase (alk-SMase, NPP7, ENPP7). alk-SMase belongs to the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, the members of which hydrolyze nucleoside phosphates, phospholipids, and other related molecules. NPP7 is the only paralog that can cleave SM, and its activity requires the presence of bile salts, a class of physiological anionic detergents. To elucidate the mechanism of substrate recognition, we determined the crystal structure of human alk-SMase in complex with phosphocholine, a reaction product. Although the overall fold and catalytic center are conserved relative to other NPPs, alk-SMase recognizes the choline moiety of its substrates via an NPP7-specific aromatic box composed of tyrosine residues. Mutational analysis and enzymatic activity assays identified features on the surface of the protein—a cationic patch and a unique hydrophobic loop—that are essential for accessing SM in bile salt micelles. These results shed new light on substrate specificity determinants within the NPP enzyme family.

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.

A family of enzymes collectively referred to as carotenoid cleavage oxygenases is responsible for oxidative conversion of carotenoids into apocarotenoids, including retinoids (vitamin A and its derivatives). A member of this family, the β-carotene 9,10-dioxygenase (BCO2), converts xanthophylls to rosafluene and ionones. Animals deficient in BCO2 highlight the critical role of the enzyme in carotenoid clearance as accumulation of these compounds occur in tissues. Inactivation of the enzyme by a four-amino acid-long insertion has recently been proposed to underlie xanthophyll concentration in the macula of the primate retina. Here, we focused on comparing the properties of primate and murine BCO2s. We demonstrate that the enzymes display a conserved structural fold and subcellular localization. Low temperature expression and detergent choice significantly affected binding and turnover rates of the recombinant enzymes with various xanthophyll substrates, including the unique macula pigment meso-zeaxanthin. Mice with genetically disrupted carotenoid cleavage oxygenases displayed adipose tissue rather than eye-specific accumulation of supplemented carotenoids. Studies in a human hepatic cell line revealed that BCO2 is expressed as an oxidative stress-induced gene. Our studies provide evidence that the enzymatic function of BCO2 is conserved in primates and link regulation of BCO2 gene expression with oxidative stress that can be caused by excessive carotenoid supplementation.

The formation of fibrillar aggregates has long been associated with neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although fibrils are still considered important to the pathology of these disorders, it is now widely understood that smaller amyloid oligomers are the toxic entities along the misfolding pathway. One characteristic shared by the majority of amyloid oligomers is the ability to disrupt membranes, a commonality proposed to be responsible for their toxicity, although the mechanisms linking this to cell death are poorly understood. Here, we describe the physical basis for the cytotoxicity of oligomers formed by the prion protein (PrP)-derived amyloid peptide PrP(106-126). We show that oligomers of this peptide kill several mammalian cells lines, as well as mouse cerebellar organotypic cultures, and we also show that they exhibit antimicrobial activity. Physical perturbation of model membranes mimicking bacterial or mammalian cells was investigated using atomic force microscopy, polarized total internal reflection fluorescence microscopy, and NMR spectroscopy. Disruption of anionic membranes proceeds through a carpet or detergent model as proposed for other antimicrobial peptides. By contrast, when added to zwitterionic membranes containing cholesterol-rich ordered domains, PrP(106-126) oligomers induce a loss of domain separation and decreased membrane disorder. Loss of raft-like domains may lead to activation of apoptotic pathways, resulting in cell death. This work sheds new light on the physical mechanisms of amyloid cytotoxicity and is the first to clearly show membrane type-specific modes of action for a cytotoxic peptide.

Several species of δ proteobacteria are capable of reducing insoluble metal oxides as well as other extracellular electron acceptors. These bacteria play a critical role in the cycling of minerals in subsurface environments, sediments, and groundwater. In some species of bacteria such as Geobacter sulfurreducens, the transport of electrons is proposed to be facilitated by filamentous fibers that are referred to as bacterial nanowires. These nanowires are polymeric assemblies of proteins belonging to the type IVa family of pilin proteins and are mainly comprised of one subunit protein, PilA. Here, we report the high resolution solution NMR structure of the PilA protein from G. sulfurreducens determined in detergent micelles. The protein is >85% α-helical and exhibits similar architecture to the N-terminal regions of other non-conductive type IVa pilins. The detergent micelle interacts with the first 21 amino acids of the protein, indicating that this region likely associates with the bacterial inner membrane prior to fiber formation. A model of the G. sulfurreducens pilus fiber is proposed based on docking of this structure into the fiber model of the type IVa pilin from Neisseria gonorrhoeae. This model provides insight into the organization of aromatic amino acids that are important for electrical conduction.

Background: Mitochondrial carriers were thought to be dimeric based on their migration in blue native gels. Results: The high molecular mass species observed in blue native gels are composed of protein monomers, detergent, lipid, and Coomassie stain. Conclusion: The mitochondrial carriers are monomeric not dimeric. Significance: The apparent mass of small membrane proteins in blue native gels requires significant correction., Blue native gel electrophoresis is a popular method for the determination of the oligomeric state of membrane proteins. Studies using this technique have reported that mitochondrial carriers are dimeric (composed of two ∼32-kDa monomers) and, in some cases, can form physiologically relevant associations with other proteins. Here, we have scrutinized the behavior of the yeast mitochondrial ADP/ATP carrier AAC3 in blue native gels. We find that the apparent mass of AAC3 varies in a detergent- and lipid-dependent manner (from ∼60 to ∼130 kDa) that is not related to changes in the oligomeric state of the protein, but reflects differences in the associated detergent-lipid micelle and Coomassie Blue G-250 used in this technique. Higher oligomeric state species are only observed under less favorable solubilization conditions, consistent with aggregation of the protein. Calibration with an artificial covalent AAC3 dimer indicates that the mass observed for solubilized AAC3 and other mitochondrial carriers corresponds to a monomer. Size exclusion chromatography of purified AAC3 in dodecyl maltoside under blue native gel-like conditions shows that the mass of the monomer is ∼120 kDa, but appears smaller on gels (∼60 kDa) due to the unusually high amount of bound negatively charged dye, which increases the electrophoretic mobility of the protein-detergent-dye micelle complex. Our results show that bound lipid, detergent, and Coomassie stain alter the behavior of mitochondrial carriers on gels, which is likely to be true for other small membrane proteins where the associated lipid-detergent micelle is large when compared with the mass of the protein.

Phosphatidylinositol 4-kinase IIα (PI4KIIα) is predominantly Golgi-localized, and it generates50% of the phosphatidylinositol 4-phosphate in the Golgi. The lipid kinase activity, Golgi localization, and "integral" membrane binding of PI4KIIα and its association with low buoyant density "raft" domains are critically dependent on palmitoylation of its cysteine-rich (173)CCPCC(177) motif and are also highly cholesterol-dependent. Here, we identified the palmitoyl acyltransferases (Asp-His-His-Cys (DHHC) PATs) that palmitoylate PI4KIIα and show for the first time that palmitoylation is cholesterol-dependent. DHHC3 and DHHC7 PATs, which robustly palmitoylated PI4KIIα and were colocalized with PI4KIIα in the trans-Golgi network (TGN), were characterized in detail. Overexpression of DHHC3 or DHHC7 increased PI4KIIα palmitoylation by3-fold, whereas overexpression of the dominant-negative PATs or PAT silencing by RNA interference decreased PI4KIIα palmitoylation, "integral" membrane association, and Golgi localization. Wild-type and dominant-negative DHHC3 and DHHC7 co-immunoprecipitated with PI4KIIα, whereas non-candidate DHHC18 and DHHC23 did not. The PI4KIIα (173)CCPCC(177) palmitoylation motif is required for interaction because the palmitoylation-defective SSPSS mutant did not co-immunoprecipitate with DHHC3. Cholesterol depletion and repletion with methyl-β-cyclodextrin reversibly altered PI4KIIα association with these DHHCs as well as PI4KIIα localization at the TGN and "integral" membrane association. Significantly, the Golgi phosphatidylinositol 4-phosphate level was altered in parallel with changes in PI4KIIα behavior. Our study uncovered a novel mechanism for the preferential recruitment and activation of PI4KIIα to the TGN by interaction with Golgi- and raft-localized DHHCs in a cholesterol-dependent manner.

Background: The regulation of cell wall hydrolysis by the pneumococcal autolysin LytA is poorly understood. Results: The cell wall is susceptible to extracellular LytA only during the stationary phase or after cell wall synthesis inhibition. Conclusion: LytA is regulated on the substrate level, where peptidoglycan modifications likely prevent LytA hydrolysis. Significance: The control of amidases is essential for bacterial survival, cell-wall synthesis, and division., The pneumococcal autolysin LytA is a virulence factor involved in autolysis as well as in fratricidal- and penicillin-induced lysis. In this study, we used biochemical and molecular biological approaches to elucidate which factors control the cytoplasmic translocation and lytic activation of LytA. We show that LytA is mainly localized intracellularly, as only a small fraction was found attached to the extracellular cell wall. By manipulating the extracellular concentration of LytA, we found that the cells were protected from lysis during exponential growth, but not in the stationary phase, and that a defined threshold concentration of extracellular LytA dictates the onset of autolysis. Stalling growth through nutrient depletion, or the specific arrest of cell wall synthesis, sensitized cells for LytA-mediated lysis. Inhibition of cell wall association via the choline binding domain of an exogenously added enzymatically inactive form of LytA revealed a potential substrate for the amidase domain within the cell wall where the formation of nascent peptidoglycan occurs.

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

Integrins are bidirectional signaling molecules on the cell surface that have fundamental roles in regulating cell behavior and contribute to cell migration and adhesion. Understanding of the mechanism of integrin signaling and activation has been advanced with truncated ectodomain preparations; however, the nature of conformational change in the full-length intact integrin molecule remains an active area of research. Here we used small angle x-ray scattering and electron microscopy to study detergent-solubilized, intact platelet integrin α(IIb)β(3). In the resting state, the intact α(IIb)β(3) adopted a compact, bent conformation. Upon activation with Mn(2+), the average integrin extension increased. Further activation by addition of ligand led to stabilization of the extended state and opening of the headpiece. The observed extension and conformational rearrangement upon activation are consistent with the extension and headpiece opening model of integrin activation.

Genetic alterations of α-actinin-4 can cause podocyte injury through multiple mechanisms. Although a mechanism involving gain-of-α-actinin-4 function was well described and is responsible for a dominantly inherited form of human focal segmental glomerulosclerosis (FSGS), evidence supporting mechanisms involving loss-of-α-actinin-4 function in human glomerular diseases remains elusive. Here we show that α-actinin-4 deficiency occurs in multiple human primary glomerulopathies including sporadic FSGS, minimal change disease, and IgA nephropathy. Furthermore, we identify a close correlation between the levels of α-actinin-4 and CLP36, which form a complex in normal podocytes, in human glomerular diseases. siRNA-mediated depletion of α-actinin-4 in human podocytes resulted in a marked reduction of the CLP36 level. Additionally, two FSGS-associated α-actinin-4 mutations (R310Q and Q348R) inhibited the complex formation between α-actinin-4 and CLP36. Inhibition of the α-actinin-4-CLP36 complex, like loss of α-actinin-4, markedly reduced the level of CLP36 in podocytes. Finally, reduction of the CLP36 level or disruption of the α-actinin-4-CLP36 complex significantly inhibited RhoA activity and generation of traction force in podocytes. Our studies reveal a critical role of the α-actinin-4-CLP36 complex in podocytes and provide an explanation as to how α-actinin-4 deficiency or mutations found in human patients could contribute to podocyte defects and glomerular failure through a loss-of-function mechanism.

αB-crystallin (αB) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of αB has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that αB is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of αB in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize αB in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100–200 nm, were characterized by the presence of flotillin-1, α-enolase, and Hsp70, the same proteins that associate with detergent-resistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of αB is predominantly apical. Using OptiPrep gradients we demonstrate that αB resides in the DRM fraction. The secretion of αB is inhibited by the cholesterol-depleting drug, methyl β-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts.

BAK is a key protein mediating mitochondrial outer membrane permeabilization; however, its behavior in the membrane is poorly understood. Here, we characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment and study their interaction by in vitro pulldown experiments, size exclusion chromatography, titration calorimetry, and NMR spectroscopy. The nonionic detergent IGEPAL has little impact on the structure of MCL-1 but induces a conformational change in BAK, whereby its BH3 region is able to engage the hydrophobic groove of MCL-1. Although the zwitterionic detergent CHAPS induces only minor conformational changes in both proteins, it is still able to initiate heterodimerization. The complex of MCL-1 and BAK can be disrupted by a BID-BH3 peptide, which acts through binding to MCL-1, but a mutant peptide, BAK-BH3-L78A, with low affinity for MCL-1 failed to dissociate the complex. The mutation L78A in BAK prevented binding to MCL-1, thus demonstrating the essential role of the BH3 region of BAK in its regulation by MCL-1. Our results validate the current models for the activation of BAK and highlight the potential value of small molecule inhibitors that target MCL-1 directly.

Rare heterozygous mutations in the gene encoding surfactant protein A2 (SP-A2, SFTPA2) are associated with adult-onset pulmonary fibrosis and adenocarcinoma of the lung. We have previously shown that two recombinant SP-A2 mutant proteins (G231V and F198S) remain within the endoplasmic reticulum (ER) of A549 cells and are not secreted into the culture medium. The pathogenic mechanism of the mutant proteins is unknown. Here we analyze all common and rare variants of the surfactant protein A2, SP-A2, in both A549 cells and in primary type II alveolar epithelial cells. We show that, in contrast with all other SP-A2 variants, the mutant proteins are not secreted into the medium with wild-type SP-A isoforms, form fewer intracellular dimer and trimer oligomers, are partially insoluble in 0.5% Nonidet P-40 lysates of transfected A549 cells, and demonstrate greater protein instability in chymotrypsin proteolytic digestions. Both the G231V and F198S mutant SP-A2 proteins are destroyed via the ER-association degradation pathway. Expression of the mutant proteins increases the transcription of a BiP-reporter construct, expression of BiP protein, and production of an ER stress-induced XBP-1 spliced product. Human bronchoalveolar wash samples from individuals who are heterozygous for the G231V mutation have similar levels of total SP-A as normal family members, which suggests that the mechanism of disease does not involve an overt lack of secreted SP-A but instead involves an increase in ER stress of resident type II alveolar epithelial cells.

Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.

Pemphigus vulgaris (PV) is an autoimmune blistering disease in which antibodies against the desmosomal cadherin, DSG3 (desmoglein-3), cause acantholysis. It has become increasingly clear that loss of cell-cell adhesion in PV is a complex and active process involving multiple signaling events such as activation of p38MAPK. It has also been demonstrated that incubating keratinocytes with PV IgG causes a redistribution of DSG3 from the cell surface to endosomes, which target these proteins for degradation. This study was undertaken to determine the relationship between p38MAPK and DSG3 endocytosis in pemphigus. In this work, we confirm that PV IgG causes internalization of cell-surface DSG3 into endosomes (as early as 4 h), which are then depleted from both detergent-soluble and detergent-insoluble pools. Cell-surface DSG3 internalization and depletion from both the detergent-soluble and detergent-insoluble fractions were blocked by the p38MAPK inhibitor SB202190. These data suggest that p38MAPK is capable of regulating PV IgG-mediated DSG3 internalization and that previously isolated mechanistic observations may be linked to a common pathway by which pemphigus autoantibodies lead to acantholysis.

Protoporphyrin IX ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Ferrochelatase shows specificity, in vitro, for multiple metal ion substrates and exhibits substrate inhibition in the case of zinc, copper, cobalt, and nickel. Zinc is the most biologically significant of these; when iron is depleted, zinc porphyrins are formed physiologically. Examining the k(cat)/K(m)(app) ratios for zinc and iron reveals that, in vitro, zinc is the preferred substrate at all concentrations of porphyrin. This is not the observed biological specificity, where zinc porphyrins are abnormal; these data argue for the existence of a specific iron delivery mechanism in vivo. We demonstrate that zinc acts as an uncompetitive substrate inhibitor, suggesting that ferrochelatase acts via an ordered pathway. Steady-state characterization demonstrates that the apparent k(cat) depends on zinc and shows substrate inhibition. Although porphyrin substrate is not inhibitory, zinc inhibition is enhanced by increasing porphyrin concentration. This indicates that zinc inhibits by binding to an enzyme-product complex (EZnD(IX)) and is likely to be the second substrate in an ordered mechanism. Our analysis shows that substrate inhibition by zinc is not a mechanism that can promote specificity for iron over zinc, but is instead one that will reduce the production of all metalloporphyrins in the presence of high concentrations of zinc.

International audience; Secretins are an unusual and important class of bacterial outer membrane (OM) proteins. They are involved in the transport of single proteins or macromolecular structures such as pili, needle complexes, and bacteriophages across the OM. Secretins are multimeric ring-shaped structures that form large pores in the OM. The targeting of such macromolecular structures to the OM often requires special assistance, conferred by specific pilotins or pilot proteins. Here, we investigated HxcQ, the OM component of the second Pseudomonas aeruginosa type II secretion system. We found that HxcQ forms high molecular mass structures resistant to heat and SDS, revealing its secretin nature. Interestingly, we showed that HxcQ is a lipoprotein. Construction of a recombinant nonlipidated HxcQ (HxcQnl) revealed that lipidation is essential for HxcQ function. Further phenotypic analysis indicated that HxcQnl accumulates as multimers in the inner membrane of P. aeruginosa, a typical phenotype observed for secretins in the absence of their cognate pilotin. Our observations led us to the conclusion that the lipid anchor of HxcQ plays a pilotin role. The self-piloting of HxcQ to the OM was further confirmed by its correct multimeric OM localization when expressed in the heterologous host Escherichia coli. Altogether, our results reveal an original and unprecedented pathway for secretin transport to the OM.

Cellular prion proteins, PrP(C), carrying the amino acid substitutions P102L, P105L, or A117V, which confer increased susceptibility to human transmissible spongiform encephalopathies, are known to form structures that include transmembrane polypeptide segments. Herein, we investigated the interactions between dodecylphosphocholine micelles and the polypeptide fragments 90-231 of the recombinant mouse PrP variants carrying the amino acid replacements P102L, P105L, A117V, A113V/A115V/A118V, K110I/H111I, M129V, P105L/M129V, and A117V/M129V. Wild-type mPrP-(90-231) and mPrP[M129V]-(91-231) showed only weak interactions with dodecylphosphocholine micelles in aqueous solution at pH 7.0, whereas discrete interaction sites within the polypeptide segment 102-127 were identified for all other aforementioned mPrP variants by NMR chemical shift mapping. These model studies thus provide evidence that amino acid substitutions within the polypeptide segment 102-127 affect the interactions of PrP(C) with membranous structures, which might in turn modulate the physiological function of the protein in health and disease.

Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a prevalent phosphoinositide in cell membranes, with important functions in cell signaling and activation. A large fraction of PIP2 associates with the detergent-resistant membrane “raft” fraction, but the functional significance of this association remains controversial. To measure the properties of raft and nonraft PIP2 in cell signaling, we targeted the PIP2-specific phosphatase Inp54p to either the raft or nonraft membrane fraction using minimal membrane anchors. Interestingly, we observed that targeting Inp54p to the nonraft fraction resulted in an enrichment of raft-associated PIP2 and striking changes in cell morphology, including a wortmannin-sensitive increase in cell filopodia and cell spreading. In contrast, raft-targeted Inp54p depleted the raft pool of PIP2 and produced smooth T cells void of membrane ruffling and filopodia. Furthermore, raft-targeted Inp54p inhibited capping in T cells stimulated by cross-linking the T cell receptor, but without affecting the T cell receptor-dependent Ca2+ flux. Altogether, these results provide evidence of compartmentalization of PIP2-dependent signaling in cell membranes such as predicted by the membrane raft model.

Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.

The integral V0 domain of the vacuolar (H+)-ATPases (V-ATPases) provides the pathway by which protons are transported across the membrane. Subunit a is a 100-kDa integral subunit of V0 that plays an essential role in proton translocation. To better define the membrane topology of subunit a, unique cysteine residues were introduced into a Cys-less form of the yeast subunit a (Vph1p) and the accessibility of these cysteine residues to modification by the membrane permeant reagent N-ethylmaleimide (NEM) and the membrane impermeant reagent polyethyleneglycol maleimide (PEG-mal) in the presence and absence of the protein denaturant SDS was assessed. Thirty Vph1p mutants containing unique cysteine residues were constructed and analyzed. Cysteines introduced between residues 670 and 710 and between 807 and 840 were modified by PEG-mal in the absence of SDS, indicating a cytoplasmic orientation. Cysteines introduced between residues 602 and 620 and between residues 744 and 761 were modified by NEM but not PEG-mal in the absence of SDS, suggesting a lumenal orientation. Finally, cysteines introduced at residues 638, 645, 648, 723, 726, 734, and at nine positions between residue 766 and 804 were modified by NEM and PEG-mal only in the presence of SDS, consistent with their presence within the membrane or at a protein-protein interface. The results support an eight transmembrane helix (TM) model of subunit a in which the C terminus is located on the cytoplasmic side of the membrane and provide information on the location of hydrophilic loops separating TM6, 7, and 8.

alpha-Synuclein (alphaSyn) can self-associate, forming oligomers, fibrils, and Lewy bodies, the pathological hallmark of Parkinson disease. Current dogma suggests that oligomeric alphaSyn intermediates may represent the most toxic alphaSyn species. Here, we studied the effect of a potent molecular chaperone, CHIP (carboxyl terminus of Hsp70-interacting protein), on alphaSyn oligomerization using a novel bimolecular fluorescence complementation assay. CHIP is a multidomain chaperone, utilizing both a tetratricopeptide/Hsp70 binding domain and a U-box/ubiquitin ligase domain to differentially impact the fate of misfolded proteins. In the current study, we found that co-expression of CHIP selectively reduced alphaSyn oligomerization and toxicity in a tetratricopeptide domain-dependent, U-box-independent manner by specifically degrading toxic alphaSyn oligomers. We conclude that CHIP preferentially recognizes and mediates degradation of toxic, oligomeric forms of alphaSyn. Further elucidation of the mechanisms of CHIP-induced degradation of oligomeric alphaSyn may contribute to the successful development of drug therapies that target oligomeric alphaSyn by mimicking or enhancing the powerful effects of CHIP.

The lysosomal ABC transporter associated with antigen processing-like (TAPL, ABCB9) acts as an ATP-dependent polypeptide transporter with broad length selectivity. To characterize in detail its substrate specificity, a procedure for functional reconstitution of human TAPL was developed. By intensive screening of detergents, ideal solubilization conditions were evolved with respect to efficiency, long term stability, and functionality of TAPL. TAPL was isolated in a two-step procedure with high purity and, subsequently, reconstituted into proteoliposomes. The peptide transport activity of reconstituted TAPL strongly depends on the lipid composition. With the help of combinatorial peptide libraries, the key positions of the peptides were localized to the N- and C-terminal residues with respect to peptide transport. At both ends, TAPL favors positively charged, aromatic, or hydrophobic residues and disfavors negatively charged residues as well as asparagine and methionine. Besides specific interactions of both terminal residues, electrostatic interactions are important, since peptides with positive net charge are more efficiently transported than negatively charged ones.

Hydrophobins are small, amphiphilic proteins secreted by filamentous fungi. Their functionality arises from a patch of hydrophobic residues on the protein surface. Spontaneous self-assembly of hydrophobins leads to the formation of an amphiphilic layer that remarkably reduces the surface tension of water. We have determined by x-ray diffraction two new crystal structures of Trichoderma reesei hydrophobin HFBII in the presence of a detergent. The monoclinic crystal structure (2.2A resolution, R = 22, R(free) = 28) is composed of layers of hydrophobin molecules where the hydrophobic surface areas of the molecules are aligned within the layer. Viewed perpendicular to the aligned hydrophobic surface areas, the molecules in the layer pack together to form six-membered rings, thus leaving small pores in the layer. Similar packing has been observed in the atomic force microscopy images of the self-assembled layers of class II hydrophobin, indicating that the crystal structure resembles that of natural hydrophobin film. The orthorhombic crystal structure (1.0 A resolution, R = 13, R(free) = 15) is composed of fiber-like arrays of protein molecules. Rodlet structures have been observed on amphiphilic layers formed by class I hydrophobins; fibrils of class II hydrophobins appear by vigorous shaking. We propose that the structure of the fibrils and/or rodlets is similar to that observed in the crystal structure.

Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups ( approximately 70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 degrees C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.

The primary catabolic pathway for glucosylceramide is catalyzed by the lysosomal enzyme glucocerebrosidase that is defective in Gaucher disease patients. A distinct non-lysosomal glucosylceramidase has been described but its identity remained enigmatic for years. We here report that the non-lysosomal glucosylceramidase is identical to the earlier described bile acid beta-glucosidase, being beta-glucosidase 2 (GBA2). Expressed GBA2 is identical to the native non-lysosomal glucosylceramidase in various enzymatic features such as substrate specificity and inhibitor sensitivity. Expression of GBA2 coincides with increased non-lysosomal glucosylceramidase activity, and GBA2-targeted RNA interference reduces endogenous non-lysosomal glucosylceramidase activity in cells. GBA2 is found to be located at or close to the cell surface, and its activity is linked to sphingomyelin generation. Hydrophobic deoxynojirimycins are extremely potent inhibitors for GBA2. In mice pharmacological inhibition of GBA2 activity is associated with impaired spermatogenesis, a phenomenon also very recently reported for GBA2 knock-out mice (Yildiz, Y., Matern, H., Thompson, B., Allegood, J. C., Warren, R. L., Ramirez, D. M., Hammer, R. E., Hamra, F. K., Matern, S., and Russell, D. W. (2006) J. Clin. Invest. 116, 2985-2994). In conclusion, GBA2 plays a role in cellular glucosylceramide metabolism.

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

Tumor endothelial marker 8 (TEM8) is induced in tumor-associated vasculature and acts as a receptor for Protective Antigen (PA), the cell-binding component of the anthrax toxin determinant for toxin entrance into cells. However, the normal function for TEM8 remains unknown. We show that TEM8 functions as an adhesion molecule mediating cell spreading on immobilized PA and collagen I. The mechanism for TEM8 interaction with collagen I was cell type-specific, because binding to collagen I was abrogated by beta1 integrin function blocking antibody in HEK293 cells, but not in primary synovial rabbit fibroblasts. Binding to PA remained unaffected by the addition of beta1 integrin function blocking antibody. Whereas the extracellular and transmembrane domains of TEM8 were sufficient to provide cell attachment, the intracellular domain was critical for spreading. Fusion of the cytosolic domain of TEM8 to the IL-2 receptor, conferred cell-spreading capability on IL-2 receptor antibody substrates. The cytoplasmic domain mediated linkage with the actin cytoskeleton as it co-precipitated actin and determined partitioning of TEM8 to the actin-containing detergent insoluble cellular fraction. TEM8 anchorage to actin was relevant as spreading was inhibited by the cytoskeleton-disrupting drug cytochalasin D, but persisted in the presence of the microtubule-depolymerizing drug nocodazole, and in cells lacking intermediate filaments. Thus, our results indicate that TEM8 is a new adhesion molecule linking collagen I or PA to the actin cytoskeleton.

Secondary bile acids have long been postulated to be tumor promoters in the colon; however, their mechanism of action remains unclear. In this study, we examined the actions of bile acids at the cell membrane and found that they can perturb membrane structure by alteration of membrane microdomains. Depletion of membrane cholesterol by treating with methyl-beta-cyclodextrin suppressed deoxycholic acid (DCA)-induced apoptosis, and staining for cholesterol with filipin showed that DCA caused a marked rearrangement of this lipid in the membrane. Likewise, DCA was found to affect membrane distribution of caveolin-1, a marker protein that is enriched in caveolae membrane microdomains. Additionally, fluorescence anisotropy revealed that DCA causes a decrease in membrane fluidity consistent with the increase in membrane cholesterol content observed after 4 h of DCA treatment of HCT116 cells. Significantly, by using radiolabeled bile acids, we found that bile acids are able to interact with and localize to microdomains differently depending on their physicochemical properties. DCA was also found to induce tyrosine phosphorylation and activate the receptor tyrosine kinase epidermal growth factor receptor in a ligand-independent manner. In contrast, ursodeoxycholic acid did not exhibit any of these effects even though it interacted significantly with the microdomains. Collectively, these data suggest that bile acid-induced signaling is initiated through alterations of the plasma membrane structure and the redistribution of cholesterol.

The amino-terminal 20.1% of apolipoprotein B (apoB20.1; residues 1-912) is sufficient to initiate and direct the formation of nascent apoB-containing lipoprotein particles. To investigate the mechanism of initial lipid acquisition by apoB, we examined the lipid binding and interfacial properties of a carboxyl-terminal His6-tagged form of apoB20.1 (apoB20.1H). ApoB20.1H was expressed in Sf9 cells and purified by nickel affinity chromatography. ApoB20.1H was produced in a folded state as characterized by formation of intramolecular disulfide bonds and resistance to chemical reduction. Dynamic light scattering in physiological buffer indicated that purified apoB20.1H formed multimers, which were readily dissociable upon the addition of nonionic detergent (0.1% Triton X-100). ApoB20.1H was incapable of binding dimyristoylphosphatidylcholine multilamellar vesicles, unless its multimeric structure was first disrupted by guanidine hydrochloride. However, apoB20.1H multimers spontaneously dissociated and bound to the interface of naked and phospholipid-coated triolein droplets. These data reveal that the initiating domain of apoB contains solvent-accessible hydrophobic sequences, which, in the absence of a hydrophobic lipid interface or detergent, engage in self-association. The high affinity of apoB20.1H for neutral lipid is consistent with the membrane binding and desorption model of apoB-containing lipoprotein assembly.

In eukaryotic cells many cell surface proteins are attached to the membrane via the glycosylphosphatidylinositol (GPI) moiety. In yeast, GPI also plays important roles in the production of mannoprotein in the cell wall. We previously isolated gwt1 mutants and found that GWT1 is required for inositol acylation in the GPI biosynthetic pathway. In this study we isolated a new gwt1 mutant allele, gwt1-10, that shows not only high temperature sensitivity but also low temperature sensitivity. The gwt1-10 cells show impaired acyltransferase activity and attachment of GPI to proteins even at the permissive temperature. We identified TAT2, which encodes a high affinity tryptophan permease, as a multicopy suppressor of cold sensitivity in gwt1-10 cells. The gwt1-10 cells were also defective in the import of tryptophan, and a lack of tryptophan caused low temperature sensitivity. Microscopic observation revealed that Tat2p is not transported to the plasma membrane but is retained in the endoplasmic reticulum in gwt1-10 cells grown under tryptophan-poor conditions. We found that Tat2p was not associated with detergent-resistant membranes (DRMs), which are required for the recruitment of Tat2p to the plasma membrane. A similar result was obtained for Fur4p, a uracil permease localized in the DRMs of the plasma membrane. These results indicate that GPI-anchored proteins are required for the recruitment of membrane proteins Tat2p and Fur4p to the plasma membrane via DRMs, suggesting that some membrane proteins are redistributed in the cell in response to environmental and nutritional conditions due to an association with DRMs that is dependent on GPI-anchored proteins.