Occurrence,localization, and possible significance of an ornithine-containing lipid in Paracoccus denitrificans

A ninhydrin-positive, phosphorus-negative lipid from Paracoccus denitrificans ATCC 13543 has been isolated and purified by mild alkaline methanolysis followed by silicic acid column chromatography and preparative thin-layer chromatography. The lipid was identified as an ornithine-containing lipid. The major ester-linked fatty acid was cis vaccenic acid. Major amide-linked fatty acids were 3-OH-20:1 and 3-OH-18:0. Ornithine-containing lipid was a major lipid component of P. denitrificans. Phospholipids made up about 57% and ornithine-containing lipid about 14% of the weight of the total lipid of the organism. The ratios of lipid ornithine: lipid phosphorus were 0.23, 0.65 and 0.58 in cytoplasmic membrane, outer membrane, and an NaCl extract, which is thought to represent chiefly outer membrane, respectively. Thus ornithine-containing lipid appears to be present in larger amounts in outer membrane than cytoplasmic membrane. No substantial variations in lipid ornithine levels were noted in stationary phase versus exposnential phase organisms, organisms grown in complex medium versus organisms grown in minimal medium with and without amino acid supplements, or in organisms grown in low phosphate-containing medium.Non standard abbreviations TLC thin-layer chromatography - Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride - TMS trimethylsilyl - TFA triluoroacetyl - NPPN ninhydrin-positive, phosphorus-negative - ECL equivalent chain length     

Effect of interferon on phospholipid methylation by peripheral blood mononuclear cells

The effect of human interferon (IFN) preparations on the metabolic pathway leading to the synthesis of phosphatidylcholine (PC) by a stepwise addition of methyl groups to phosphatidylethanolamine (PE) was investigated in human peripheral blood mononuclear (PBMN) cells. An inhibition of the synthesis of PC via this pathway was regularly observed with both alpha- (recombinant or natural) and beta-IFN. This inhibition was apparent within the first 5 min of treatment, reached its maximum between 15 min and 1 hr, and persisted at the same level until 6 hr, the last time point examined. Each of the transmethylated products of PE underwent a similar inhibition, as measured by the turnover rate of individual products. The intracellular pool of the methyl donors, methionine and S-adenosyl-methionine (SAM), was shown to be unaffected. The methyltransferase activity of IFN-pretreated cell extracts was unchanged. These findings support the hypothesis that IFN induces a functional change in phospholipid methylation at the level of organized membrane-bound phospholipid methyltransferase enzymes in intact cells.     

Identification of surface autoantigens which appear during spermatogenesis

Autoantigens that appear during spermatogenesis in the rabbit were identified using immunoadsorbent chromatography and SDS-PAGE. To identify cell-surface proteins, samples of freshly isolated, staged cells were labeled by the lactoperoxidase or Iodo-Gen iodination procedure and run on SDS-PAGE. Autoradiograms of the stained, dried gels were prepared. By correlating the band patterns in the SDS gels of immunocolumn and surface-labeled samples with the band patterns in the autoradiograms, it was possible to show when the autoantigenic proteins appeared on the cell surface. To further support the identification of membrane autoantigens, surface-labeled, staged cell samples were lysed in Triton X-100 and immunoprecipitated with antitestis cell autoantisera. Three types of autoantigens have been identified: (1) late class antigens that are present only on late spermatids and epididymal spermatozoa, but are intracellular in early stages, (2) early class antigens which occur on the surface of pachytene spermatocytes and are present throughout subsequent stages of development, and (3) early class, transient antigens, which appear on spermatogenic cells but are not present on epididymal spermatozoa.     

Simulation of grana stacking in a model membrane system. Mediation by a purified light-harvesting pigment-protein complex from chloroplasts

An isolated light-harvesting pigment-protein complex contains polypeptides which bind chlorophyll a and b. The individual complexes can be purified from detergent-solubilized membranes. The isolated light-harvesting complex, when dialyzed to remove detergents, was examined by freeze-fracture electron microscopy. The material consisted of planar sheets of 80-Å subunits which interacted via an edge-to-edge contact. Addition of cations caused the planar light-harvesting complex sheets to become tightly appressed in multilamellar stacks, with distinct subunits still visible within each lamellar sheet. A transition of particle organization from random to crystalline occurred in parallel with the cation-induced lamellar association. Treatment of the dialyzed light-harvesting complex subunits with low levels of the proteolytic enzyme trypsin removed a 2000 molecular weight segment of the major polypeptide of the light-harvesting complex and blocked all subsequent cation-induced changes in structural organization of the isolated light-harvesting complex lamellar sheets.To gain further evidence for mechanisms of cation effects upon the organization of the light-harvesting complex in native membranes, the light-harvesting complex was incorporated into uncharged (phosphatidylcholine) lipid vesicles. The protein complexes spanned the lipid bilayer and were arranged in either a random pattern or in hexagonal crystalline lattices. Addition of either monovalent or divalent cations to ‘low-salt’ (20 mM monovalent cation) vesicles containing light-harvesting complex caused extensive regions of membrane appression to appear. It is concluded that this cation-induced membrane appression is mediated by surface-exposed segments of the light-harvesting complex since (a) phosphatidylcholine vesicles themselves did not undergo cation-induced aggregation, and (b) mild trypsin digestion of the surface-exposed regions of the light-harvesting complex blocked cation-induced lamellar appression. The particles in the appressed vesicle membranes tended to form long, linear arrays of particles, with occasional mixed quasi-crystalline arrays with an angular displacement near 72°. Surface-mediated interactions among light-harvesting complex subunits of different membranes are, therefore, related to changes in structural organization and interaction of the particles within the lipid phase of the membrane.Numerous previous studies have implicated the involvement of the light-harvesting complex in mediating grana stocking in intact chloroplast membranes. The data presented herein provide a simulation of the membrane appression phenomena using a single class of chloroplast-derived membrane subunits. The data demonstrate that specific surface-localized regions of the light-harvesting complex are involved in membrane-membrane interactions.     

Membrane surface properties of sheep erythrocytes,an immunological reagent,after different treatments as reflected by partition in two-polymer aqueous phases

Sheep erythrocytes (E) which, with or without certain treatments, are currently used as “immunological reagents” to detect cells with specific receptors (by rosette-formation) have been partitioned in two-polymer aqueousphase systems selected so as to reflect charge-associatedor lipid-related membrane surface properties. We have found that the partitioning behavior of E is not affected in these phases by reacting the cells with anti-E antibody (either IgG or IgM), forming EA. The additional binding of complement to the cell-antibody complex, forming EAC, results, however, in a marked decrease in the partition coefficient,K. Apparently both the charge-associated and hydrophobic properties reflected by partitioning remain accessible to the phase polymers when the cells are coated with antibody, but are not with the addition of complement. It is interesting that EA can still rosette with T-lymphocytes (14), a property of E, while the additional coating with complement results in EAC which does not appreciably do so (26). Neuraminidase or trypsin treatments of E, which yield Es having quite different rosetting properties with T-lymphocytes (14), cause increasedKs and unchangedKs, respectively, in phases reflecting lipid-related surface properties. Either treatment causes reducedKs of E in charged-phase systems. Neuraminidase treatment also results in a reduced electrophoretic mobility of E, while trypsin treatment is not detectable by cell electrophoresis (25). We are currently studying the possible usefulness of employing cell electrophoresis and cell partitioning in charged-phase systems jointly to obtain information on events occurring at the shear plane versus those occurring deeper in the membrane.     

Perturbation of the chemotactic tumbling of bacteria

The bacterial sensing system has been studied on three levels. First, a quantitative method has been devised for measuring the “action spectrum” of the bacterium in response to a sudden addition of attractant. Second, a technique has been developed for the rapid isolation of mutants defective in the transmission part of the sensing system. Third, a study of the effects of light on the transmission system reveals two components, one which generates tumbling and another which inhibits it.     

Human C1orf27 protein interacts with α2A-adrenergic receptor and regulates its anterograde transport

The molecular mechanisms underlying the anterograde surface transport of G protein–coupled receptors (GPCRs) after their synthesis in the endoplasmic reticulum (ER) are not well defined. In C. elegans, odorant response abnormal 4 has been implicated in the delivery of olfactory GPCRs to the cilia of chemosensory neurons. However, the function and regulation of its human homolog, C1orf27, in GPCR transport or in general membrane trafficking remain unknown. Here, we demonstrate that siRNA-mediated knockdown of C1orf27 markedly impedes the ER-to-Golgi export kinetics of newly synthesized α_2A-adrenergic receptor (α_2A-AR), a prototypic GPCR, with the half-time being prolonged by more than 65%, in mammalian cells in retention using the selective hooks assays. Using modified bioluminescence resonance energy transfer assays and ELISAs, we also show that C1orf27 knockdown significantly inhibits the surface transport of α_2A-AR. Similarly, C1orf27 knockout by CRISPR-Cas9 markedly suppresses the ER–Golgi-surface transport of α_2A-AR. In addition, we demonstrate that C1orf27 depletion attenuates the export of β₂-AR and dopamine D2 receptor but not of epidermal growth factor receptor. We further show that C1orf27 physically associates with α_2A-AR, specifically via its third intracellular loop and C terminus. Taken together, these data demonstrate an important role of C1orf27 in the trafficking of nascent GPCRs from the ER to the cell surface through the Golgi and provide novel insights into the regulation of the biosynthesis and anterograde transport of the GPCR family members.     

Cell specific expression of three genes involved in plasma membrane sucrose transport in developingVicia faba seed

Summary In developing seeds ofVicia faba, transfer cells line the inner surface of the seed coat and the juxtaposed epidermal surface of the cotyledons. Circumstantial evidence, derived from anatomical and physiological studies, indicates that these cells are the likely sites of sucrose efflux to, and influx from, the seed apoplasm, respectively. In this study, expression of an H⁺/sucrose symporter-gene was found to be localised to the epidermal-transfer cell complexes of the cotyledons. The sucrose binding protein (SBP) gene was expressed in these cells as well as in the thin-walled parenchyma transfer cells of the seed coat. SBP was immunolocalised exclusively to the plasma membranes located in the wall ingrowth regions of the transfer cells. In addition, a plasma membrane H⁺-ATPase was most abundant in the wall ingrowth regions with decreasing levels of expression at increasing distance from the transfer cell layers. The observed co-localisation of high densities of a plasma membrane H⁺-ATPase and sucrose transport proteins to the wall ingrowths of the seed coat and cotyledon transfer cells provides strong evidence that these regions are the principal sites of facilitated membrane transport of sucrose to and from the seed apoplasm.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - DIG digoxigenin - H⁺-ATPase plasma membrane H⁺-translocating adenosine triphosphatase - Ig immunoglobulin - LeSUT1 tomato H⁺/sucrose symporter - SBP sucrose binding protein