Isolated Membrane Fractions Research Articles

A novel potassium channel in photosynthetic cyanobacteria.

Elucidation of the structure-function relationship of a small number of prokaryotic ion channels characterized so far greatly contributed to our knowledge on basic mechanisms of ion conduction. We identified a new potassium channel (SynK) in the genome of the cyanobacterium Synechocystis sp. PCC6803, a photosynthetic model organism. SynK, when expressed in a K+-uptake-system deficient E.coli strain, was able to recover growth of these organisms. The protein functions as a potassium selective ion channel when expressed in Chinese Hamster Ovary cells. The location of SynK in cyanobacteria in both thylakoid and plasmamembranes was revealed by immunogold electron microscopy and Western blotting of isolated membrane fractions. SynK seems to be conserved during evolution, giving rise to a TPK (two-pore K+ channel) family member which is shown here to be located in the thylakoid membrane of Arabidopsis. Our work characterizes a novel cyanobacterial potassium channel and indicates the molecular nature of the first higher plant thylakoid cation channel, opening the way to functional studies.

The acrosomal membrane system of boar spermatozoa--morphological and biochemical studies.

The distribution of the cell surface saccharides with alpha-glycosidically linked mannose and terminal galactose was displayed by fluorescence microscopical examinations with FITC-conjugated concanavalin A and Ricinus communis agglutinin-120. The lectin binding sites of the inner cellular membranes--inner and outer acrosomal membrane--were ultrastructurally localized by electron microscopical studies using the lectin-peroxidase technique. It could be demonstrated that both acrosomal membranes contain covalently bound carbohydrates. In order to characterize the acrosomal membrane components which are responsible for the binding to lectins, the outer acrosomal membrane was separated after homogenization by centrifugation through modified colloidal silica. The exposed membrane proteins were labelled by galactose oxidase mediated introduction of tritium into the carbohydrate side chains of the glycoproteins. After solubilization the membrane components were analysed by affinity chromatography and electrophoresis. For a general characterization the cholesterol/phospholipid ratio of ejaculated boar spermatozoa and the isolated membrane fraction was determined and some enzymatic activities were assessed.

Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex

Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine's anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA's PPIase activity in the proper assembly and function of the HCV RC.

Regulation of callose synthase activity in situ in alamethicin-permeabilized Arabidopsis and tobacco suspension cells.

BackgroundThe cell wall component callose is mainly synthesized at certain developmental stages and after wounding or pathogen attack. Callose synthases are membrane-bound enzymes that have been relatively well characterized in vitro using isolated membrane fractions or purified enzyme. However, little is known about their functional properties in situ, under conditions when the cell wall is intact. To allow in situ investigations of the regulation of callose synthesis, cell suspensions of Arabidopsis thaliana (Col-0), and tobacco (BY-2), were permeabilized with the channel-forming peptide alamethicin.ResultsNucleic acid-binding dyes and marker enzymes demonstrated alamethicin permeabilization of plasma membrane, mitochondria and plastids, also allowing callose synthase measurements. In the presence of alamethicin, Ca2+ addition was required for callose synthase activity, and the activity was further stimulated by Mg2+ Cells pretreated with oryzalin to destabilize the microtubules prior to alamethicin permeabilization showed significantly lower callose synthase activity as compared to non-treated cells. As judged by aniline blue staining, the callose formed was deposited both at the cell walls joining adjacent cells and at discrete punctate locations earlier described as half plasmodesmata on the outer walls. This pattern was unaffected by oryzalin pretreatment, showing a quantitative rather than a qualitative effect of polymerized tubulin on callose synthase activity. No callose was deposited unless alamethicin, Ca2+ and UDP-glucose were present. Tubulin and callose synthase were furthermore part of the same plasma membrane protein complex, as judged by two-dimensional blue native SDS-PAGE.ConclusionAlamethicin permeabilization allowed determination of callose synthase regulation and tubulin interaction in the natural crowded cellular environment and under conditions where contacts between the cell wall, the plasma membrane and cytoskeletal macromolecules remained. The results also suggest that alamethicin permeabilization induces a defense response mimicking the natural physical separation of cells (for example when intercellulars are formed), during which plasmodesmata are transiently left open.

Formaldehyde elimination with formaldehyde and formate oxidase in membrane of acetic acid bacteria

Formaldehyde elimination was successfully carried out with Acetobacter sp. SKU 14, having strong formaldehyde-oxidizing activity in the cytoplasmic membrane. Formaldehyde was decomposed via formate to carbon dioxide by formaldehyde- and formate-oxidizing activities. A resting-cell suspension of the organism was more convenient for practical purposes than the isolated membrane fraction. In Gluconobacter suboxydans IFO 12528, formaldehyde elimination was not so prominent when compared with that in Acetobacter sp. SKU 14.

Phosphorylation of the β2-Adrenergic Receptor in Plasma Membranes by Intrinsic GRK5

Characterization of the GRKs participating in the phosphorylation of the beta2-adrenergic receptor (beta2AR) have in part been limited by the lack of a simple cell-free assay with membrane-bound beta2AR and GRKs. We describe here a cell-free assay for GRK phosphorylation of the beta2AR in a postnuclear 600g fraction and washed membranes by intrinsic GRK activity using the GRK phosphosite-specific antibody that recognizes pS(355,356). Treatment of these cell-free preparations with 1.0 microM isoproterenol (ISO) caused a rapid maximal 10-15-fold increase in GRK site phosphorylation of the beta2AR (t1/2 = 1 min) with an EC50 for ISO stimulation of approximately 80 nM. Extensively washed plasma membrane fractions retained the 10-15-fold ISO stimulation of GRK site phosphorylation and GRK5 levels while being depleted of GRK2 and GRK6. Stimulation of GRK site phosphorylation by a range of partial agonists correlated well with their intrinsic efficacy for stimulation of adenylyl cyclase. GRK phosphorylation of the beta2AR in the washed membrane fraction caused minimal desensitization of ISO stimulation of adenylyl cyclase activity. Association of GRK5 with the beta2AR in intact cells was demonstrated by a high level of basal BRET2 using beta2AR-Rluc and GRK5-GFP2 that was not diminished by agonist stimulation. BRET2 between the beta2AR-Rluc and GFP2-betaarrestin 2 was increased by agonist, whereas BRET2 between the beta2AR and GRK2-GFP2 was not significant. On the basis of the level of GRK5-mediated phosphorylation we observe in isolated membrane fractions and co-localization of the beta2AR and GRK5, we conclude that GRK5 plays a distinctive role in the phosphorylation of the beta2AR.

DISRUPTION OF SKELETAL MYOCYTES INITIATES SUPEROXIDE RELEASE: CONTRIBUTION OF NAD(P)H OXIDASE

Generation of reactive oxygen species (ROS) as an early local reaction to muscle crush injury has frequently been predicted. However, although it is known that severe inflammatory reactions occurring after major muscle trauma originate mainly from early local incidents within the injured tissue, no detailed studies exist on the local generation of ROS in response to myocyte destruction thus far. Therefore, in this study, ROS formation after lethal mechanical damage was examined using a model of scraping injury to cultured C2C12 skeletal myocytes and superoxide detection by lucigenin chemiluminescence, nitrotetrazolium blue chloride reduction, or electron spin resonance spectroscopy. Mechanical rupture of myocytes resulted in an immediate release of superoxide from the damaged cells that could be substantially blocked by the superoxide scavengers superoxide dismutase (51%), tiron (95%), and MAMA/NO (93%) and by hypoxia (83% inhibition). Superoxide generation was primarily confined to the myocytes' membrane fraction and 7- to 8-fold enhanced by the addition of NADH or NADPH. The NADPH-enhanced superoxide generation could largely be diminished by the NAD(P)H oxidase inhibitors diphenyleneiodonium and apocynin in cell lysates (97% and 35% inhibition, respectively) and in isolated membrane fractions (61% and 63% inhibition). We thus conclude that immediately after myocyte damage, large amounts of superoxide are formed that predominantly originate from membrane-bound electron-transferring enzymes, especially NAD(P)H oxidase. This suggests a decisive role of ROS in the pathogenesis of tissue trauma, with superoxide being an initiator of the signaling mechanism from injured myocytes to the surrounding tissue and, potentially, to the whole body.

Early cardiac hypertrophy in mice with impaired calmodulin regulation of cardiac muscle Ca2+ release channel

Studies with isolated membrane fractions have shown that calmodulin (CaM) inhibits the activity of cardiac muscle cell Ca(2+) release channel ryanodine receptor 2 (RyR2). To determine the physiological importance of CaM regulation of RyR2, we generated a mouse with 3 amino acid substitutions (RyR2-W3587A/L3591D/F3603A) in exon 75 of the Ryr2 gene, which encodes the CaM-binding site of RyR2. Homozygous mutant mice showed an increased ratio of heart weight to body weight, greatly reduced fractional shortening of the left ventricle, and lethality at 9-16 days of age. Biochemical analysis of hearts of 7- and 10-day-old homozygous mutant mice indicated an impaired CaM inhibition of RyR2 at micromolar Ca(2+) concentrations, reduction in RyR2 protein levels and sarcoplasmic reticulum Ca(2+) sequestration, and upregulation of genes and/or proteins associated with class II histone deacetylase/myocyte enhancer factor-2 and calcineurin signaling pathways. Sustained Ca(2+) transients, often displaying repeated periods of incomplete Ca(2+) removal, were observed in homozygous cardiomyocytes. Taken together, the data indicate that impaired CaM inhibition of RyR2, associated with defective sarcoplasmic reticulum Ca(2+) release and altered gene expression, leads to cardiac hypertrophy and early death.

Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS

The core prerequisites for an efficient proteome-scale analysis of mammalian membrane proteins are effective isolation, solubilization, digestion and multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). This protocol is for analysis of the mammalian membrane proteome that relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (vol/vol) methanol. Tryptic digestion is followed by strong cation exchange (SCX) chromatography and reversed phase (RP) chromatography coupled online with MS/MS for protein identification. The use of a methanol-based buffer eliminates the need for reagents that interfere with chromatographic resolution and ionization of the peptides (e.g., detergents, chaotropes, inorganic salts). Sample losses are minimized because solubilization and digestion are carried out in a single tube avoiding any sample transfer or buffer exchange between these steps. This protocol is compatible with stable isotope labeling at the protein and peptide level, enabling identification and quantitation of integral membrane proteins. The entire procedure--beginning with isolated membrane fraction and finishing with MS data acquisition--takes 4-5 d.

Membrane Composition Changes and Physiological Adaptation byStreptococcus mutansSignal Recognition Particle Pathway Mutants

Previously, we presented evidence that the oral cariogenic species Streptococcus mutans remains viable but physiologically impaired and sensitive to environmental stress when genes encoding the minimal conserved bacterial signal recognition particle (SRP) elements are inactivated. Two-dimensional gel electrophoresis of isolated membrane fractions from strain UA159 and three mutants (Deltaffh, DeltascRNA, and DeltaftsY) grown at pH 7.0 or pH 5.0 allowed us to obtain insight into the adaptation process and the identities of potential SRP substrates. Mutant membrane preparations contained increased amounts of the chaperones DnaK and GroES and ClpP protease but decreased amounts of transcription- and translation-related proteins, the beta subunit of ATPase, HPr, and several metabolic and glycolytic enzymes. Therefore, the acid sensitivity of SRP mutants might be caused in part by diminished ATPase activity, as well as the absence of an efficient mechanism for supplying ATP quickly at the site of proton elimination. Decreased amounts of LuxS were also observed in all mutant membranes. To further define physiological changes that occur upon disruption of the SRP pathway, we studied global gene expression in S. mutans UA159 (parent strain) and AH333 (Deltaffh mutant) using microarray analysis. Transcriptome analysis revealed up-regulation of 81 genes, including genes encoding chaperones, proteases, cell envelope biosynthetic enzymes, and DNA repair and replication enzymes, and down-regulation of 35 genes, including genes concerned with competence, ribosomal proteins, and enzymes involved in amino acid and protein biosynthesis. Quantitative real-time reverse transcription-PCR analysis of eight selected genes confirmed the microarray data. Consistent with a demonstrated defect in competence and the suggested impairment of LuxS-dependent quorum sensing, biofilm formation was significantly decreased in each SRP mutant.

Proteomic analysis of isolated membrane fractions from superinvasive cancer cells

The superinvasive phenotype exhibited by paclitaxel-selected variants of an in vitro invasive clonal population of the human cancer cell line, MDA-MB-435S were examined using DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry. Isolation of membrane proteins from the MDA-MB-435S-F/Taxol-10p4p and parental populations was performed by temperature-dependent phase partitioning using the detergent Triton X-114. Subsequent DIGE-generated data analysed using Decyder software showed many differentially-expressed proteins in the membrane fraction. 16 proteins showing statistically significant upregulation in the superinvasive cells were identified by MALDI-ToF. Proteins upregulated in the superinvasive population include Galectin-3, Cofilin, ATP synthase beta subunit, voltage-dependent anion channel 1, voltage dependent anion channel 2, ER-60 protein, MHC class II antigen DR52, Beta actin, TOMM40 protein, Enolase 1, Prohibitin, Guanine nucleotide-binding protein, Annexin II, Heat shock 70 kDa protein, Stomatin-like protein 2 and Chaperonin. Many of these proteins are associated with inhibition of apoptosis, the progression of cancer, tumourigenicity, metastasis, actin remodelling at the leading edge of cells, polarized cell growth, endocytosis, phagocytosis, cellular activation, cytokinesis, and pathogen intracellular motility. These results suggest a correlation between the increased abundance of these proteins with the superinvasive phenotype of the paclitaxel-selected MDA-MB-435S-F/Taxol-10p4p population.

In vitro assays differentially recapitulate protein export from the trans-Golgi network

In vitro assays have been broadly applied to reconstitute traYcking events along the biosynthetic and endocytic pathways of eukaryotic cells [1]. Cell-based assays employ mild detergents or mechanical means to perforate the plasma membrane and release cytosolic components while maintaining the integrity of intracellular membrane compartments (Supplementary Fig. 1). In contrast, cell-free assays are performed using isolated membrane fractions enriched in a particular organelle. Cell-based assays can allow analysis of multiple steps along a traYcking pathway, while cell-free assays are generally designed to reconstitute an individual membrane Wssion or fusion step. Both cellbased and cell-free assays have proven useful for dissecting the roles of various components in intracellular traYcking events. In many instances, these assays have been utilized to dissect the requirements for polarized biosynthetic sorting of cargo molecules from the trans-Golgi network (TGN)1[2–5]. Interestingly, segregation of apical and basolateral marker proteins into distinct transport carriers that bud from the TGN appears to occur even in nonpolarized cells, enabling the use of nondiVerentiated cell types for such assays [6–8]. However, the extent to which these assays recapitulate in vivo sorting and regulation of membrane traYc has not been systematically examined. Here we have compared cell-based and a cell-free in vitro assays for their ability to reconstitute polarized protein sorting from the TGN. We previously found that eYcient apical delivery of the marker protein inXuenza hemagglutinin (HA) in polarized Madin–Darby canine kidney (MDCK) cells requires TGN acidiWcation [9]. In particular,

Detection of superoxide anion generation by equine spermatozoa

To identify the generation of the superoxide anion by equine spermatozoa. Multiple ejaculates collected from 3 Thoroughbred stallions. Induced superoxide production by reduced nicotinamide adenine dinucleotides (NAD[P]H; ie, reduced nicotinamide adenine dinucleotide [NADH] and reduced nicotinamide adenine dinucleotide phosphate [NADPH]) was measured by use of a nitroblue tetrazolium (NBT) reduction assay on whole spermatozoa and a cytochrome c reduction assay on isolated membrane fractions of spermatozoa. Localization of superoxide generation was determined by use of NBT cytochemistry. A dose-dependent increase in NBT reduction was found in the presence of NADPH, which was inhibited by superoxide dismutase (SOD). The flavoprotein inhibitor, diphenyleneiodonium (DPI; 5 or 15 microM), significantly decreased NBT reduction. Cytochrome c reduction by plasma membranes of spermatozoa was significantly higher in the presence of NADPH than in its absence. Cytochemical staining of equine spermatozoa in the presence of NADPH and NADH revealed diaphorase labeling in the spermatozoon midpiece and head. This staining was inhibited by DPI and SOD. Results of our study indicate that superoxide generation is associated with a membrane-associated NAD(P)H oxidase present in equine spermatozoa, although mitochondrial generation of superoxide is also detected. This oxidase may play a role in cell signaling or may also contribute to cytopathic effects associated with oxidative stress in equine spermatozoa.

PIN2 Polarity Requires Vesicular Cycling and Proteasomal Degradation

PIN2 is part of the auxin efflux carrier that controls the asymmetric distribution of auxin, which is a key regulator of root gravitropism. Abas et al. show that root meristems reorient to point their tips down in response to being positioned horizontally in the gravitational field (gravistimulation) by generating an asymmetric distribution of PIN2 such that PIN2 was more abundant on the lower side of the root tip. Before positioning the root horizontally, PIN2 abundance was equal in the lateral sides of the root. Gravistimulation initially caused PIN2 to be removed from the upper root cells' plasma membrane and appear in intracellular vesicles. The decrease in PIN2 abundance (measured in isolated membrane fractions or from the upper cells by microscopic techniques) and gravitropic root bending was blocked if the root meristems were treated with an inhibitor of the proteasome (MG132) or brefeldin A (BFA), which inhibits vesicular trafficking. Plants expressing a mutant PIN2 (wav6-52) do not respond to gravity, and Abas et al. showed that wav6-52 root meristems had increased abundance of PIN2, which accumulated in intracellular vesicles and the plasma membrane in unstimulated roots and did not redistribute in response to gravistimulation. Furthermore, wav6-52 roots exhibited aberrant auxin activity in the lateral root cap cells and elongation zone, which is not seen in wild-type roots. Auxin also exhibited feedback onto PIN2, with application of an auxin decreasing PIN2 abundance. Uniform pretreatment of the roots with an auxin analog followed by gravistimulation caused the internalization of PIN2 from both the upper and lower root cells. Auxin would only accumulate in the lower cells and thus may have served as a signal to reset the system once the root bent and was oriented vertically again. The constitutive cycling of PIN2 provides an opportunity for the cells of the upper root to selectively trigger degradation of PIN2, thereby decreasing its abundance and decreasing auxin flow to the upper side of the root to mediate bending in response to gravity. L. Abas, R. Benjamins, N. Malenica, T. Paciorek, J. Wiřniewska, J. C. Moulinier-Anzola, T. Sieberer, J. Friml, C. Luschnig, Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat. Cell Biol. 8 , 249-256 (2006). [PubMed]

Wnt Signaling Inhibits Adipogenesis through β-Catenin-dependent and -independent Mechanisms

Wnt signaling has been reported to block apoptosis and regulate differentiation of mesenchymal progenitors through inhibition of glycogen synthase kinase 3 and stabilization of beta-catenin. The effects of Wnt in preadipocytes may be mediated through Frizzled (Fz) 1 and/or Fz2 as these Wnt receptors are expressed in preadipocytes and their expression declines upon induction of differentiation. We ectopically expressed constitutively active chimeras between Wnt8 and Fz1 or Fz2 in preadipocytes and mesenchymal precursor cells. Our results indicated that activated Fz1 increases stability of beta-catenin, inhibits apoptosis, induces osteoblastogenesis, and inhibits adipogenesis. Although activated Fz2 does not influence apoptosis or osteoblastogenesis, it inhibits adipogenesis through a mechanism independent of beta-catenin. An important mediator of the beta-catenin-independent pathway appears to be calcineurin because inhibitors of this serine/threonine phosphatase partially rescue the block to adipogenesis caused by Wnt3a or activated Fz2. These data supported a model in which Wnt signaling inhibits adipogenesis through both beta-catenin-dependent and beta-catenin-independent mechanisms.

Natriuretic peptide interaction with [ 3H]isatin binding sites in rat brain

Isatin is an endogenous indole, which has a distinct and discontinuous distribution in the brain and exhibits a wide range of physiological and pharmacological effects. In the present study, we have demonstrated that atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) inhibited [ 3H]isatin binding to rat brain sections and isolated membrane fractions. Isatin itself antagonised not only natriuretic peptide receptor type A (NPR-A) (ANP-stimulation of guanylyl cyclase) but also NPR-C (ANP and CNP mediated inhibition of adenylyl cyclase) signalling. These results suggest that some [ 3H]isatin binding in the brain may be to NPR-A and NPR-C. Competitive interactions between isatin and natriuretic peptides and their receptors give a possible explanation of the known anxiogenic effect of low doses of isatin, interacting at NPR-A, and the sedative effects of higher doses, antagonising respectively the anxiolytic effect of ANP and the anxiogenic effect of CNP.

Overexpression of calsenilin enhances γ-secretase activity

Presenilin/γ-secretase is a membrane-associated protease that cleaves within the transmembrane region of the amyloid precursor protein (APP) to generate amyloid-β peptide (Aβ) whose deposition in the brain is a characteristic of Alzheimer's disease (AD). Calsenilin, a calcium binding protein that has been shown to interact with the C-termini of both presenilin 1 (PS1) and presenilin 2 (PS2), appears to play a role in transcriptional regulation and apoptosis and to bind to A-type voltage-gated potassium channels. Here, we report that overexpression of calsenilin enhanced γ-secretase activity in cells. The effect of calsenilin on the γ-cleavage of substrates was blocked by the selective γ-secretase inhibitor L-685,458. We also employed a cellular γ-cleavage GFP-reporter assay to demonstrate the effect of calsenilin on γ-secretase activity. To establish a direct role for calsenilin in regulating γ-secretase activity, we incubated purified calsenilin with isolated membrane fractions and found increased Aβ production in a cell free system. These data suggest that calsenilin may be one of the regulatory factors for γ-secretase.

Structural organization of the twin-arginine translocation system in Streptomyces lividans

The twin-arginine translocation (Tat) system exports folded proteins across bacterial cytoplasmic membranes. Recently, genes encoding TatA, TatB and TatC homologues were identified in Streptomyces lividans and the functionality of the Tat pathway was demonstrated. Here, we have examined the localization and structural organization of the Tat components in S. lividans. Interestingly, besides being membrane-associated proteins, S. lividans TatA and TatB were also detected in the cytoplasm. TatC could only be detected in isolated membrane fractions. Whereas all TatC was found to be stably inserted in the membrane, part of membrane-associated TatA and TatB could be extracted following high salt, sodium carbonate or urea treatment suggesting a more loose association with the membrane. Finally, we have analyzed Tat complexes that could be purified from an S. lividans TatABC overproducing strain. From the cytoplasmic membrane, two types of high molecular mass Tat complexes could be isolated having a similar composition as those isolated from Escherichia coli. In the cytoplasm, TatA and TatB were detected as monomer or as homo-oligomeric complexes.

Triacylglycerol Is Synthesized in a Specific Subclass of Caveolae in Primary Adipocytes

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.

Mechanisms of Innate and Adaptive Immune Responsiveness to Bacterial Infection in Cornea

Purpose. The dysfunctional tear syndrome of Sjogren’s syndrome (SjS) is associated with focal CD4+ cell infiltration of the lacrimal glands and serum autoantibodies to intracellular proteins. To learn how lacrimal acinar cells (AC) expose or present autoantigen epitopes, we wish to identify autoantigens in amenable experimental models. Methods. Subcellular fractions from rat lacrimal glands were analyzed by SDS-PAGE. Western blots were probed with sera from NOD mice with diagnosed SjS-like disease, and IgG1 heavy chain was detected with isotype specific secondary antibody. Labeled bands were analyzed using matrix-associated laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry. Tryptic peptides were identified by Mascot Matrix Science search software. Compartmental distributions of candidate B cell autoantigens were determined by Western blotting of isolated membrane fractions from rabbit AC cultured with and without chronic carbachol (CCh) stimulation. Results. Subcellular fractionation analysis indicated that the candidate autoantigens were primarily associated with the membrane phase; most were primarily in compartments of the biosynthetic and lysosomal pathways. Some also were in secretory vesicle-related compartments of control cells but re-distributed to the endosomes after chronic CCh stimulation. The proteomic analysis identified Grp 78, rab3D, and ribosomal protein L7 as candidate autoantigens. Western blotting with monoclonal antibodies confirmed the presence and compartmental distributions of Grp 78 and rab3D. Protein sequence analysis with TEPITOPE confirmed that an identified B cell epitope at the N-terminal end of L7 also is a likely T cell epitope. Conclusion Grp78 and L7 are known to be antigens in other autoimmune diseases but have not previously been known to be associated with SjS. Rab3D has not previously been recognized as a disease-associated autoantigen. These and other AC autoantigens are present in intracellular compartments where they may be subject to aberrant proteolytic processing that we theorize generates otherwise cryptic epitopes. Commercial Relationship(s): None; Support: EY13720, EY05801, DK48522