Molecular cloning of Ian4: a BCR/ABL-induced gene that encodes an outer membrane mitochondrial protein with GTP-binding activity

Using the representation difference analysis technique, we have identified a novel gene, Ian4, which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the Bcr/Abl oncogene. Ian4 expression was undetectable in 32D cells transfected with v-src, oncogenic Ha-ras or v-Abl. Murine Ian4 maps to chromosome 6, 25 cM from the centromere. The Ian4 mRNA contains two open reading frames (ORFs) separated by 5 nt. The first ORF has the potential to encode for a polypeptide of 67 amino acids without apparent homology to known proteins. The second ORF encodes a protein of 301 amino acids with a GTP/ATP-binding site in the N-terminus and a hydrophobic domain in the extreme C-terminus. The IAN-4 protein resides in the mitochondrial outer membrane and the last 20 amino acids are necessary for this localization. The IAN-4 protein has GTP-binding activity and shares sequence homology with a novel family of putative GTP-binding proteins: the immuno-associated nucleotide (IAN) family.

Depletion of Acyl-Coenzyme A-Binding Protein Affects Sphingolipid Synthesis and Causes Vesicle Accumulation and Membrane Defects in Saccharomyces cerevisiae

Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:105. A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under control of the GAL1 promoter exhibited an altered acyl-CoA profile with a threefold increase in the relative content of C18:0-CoA, without affecting total acyl-CoA level as previously reported for an adapted acb1Δ strain. Depletion of Acb1p did not affect the general phospholipid pattern, the rate of phospholipid synthesis, or the turnover of individual phospholipid classes, indicating that Acb1p is not required for general glycerolipid synthesis. In contrast, cells depleted for Acb1p showed a dramatically reduced content of C26:0 in total fatty acids and the sphingolipid synthesis was reduced by 50–70%. The reduced incorporation of [3H]myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport. The plasma membrane of the Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids. Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures. The present results strongly suggest that Acb1p plays an important role in fatty acid elongation and membrane assembly and organization.

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BCθ) of perfringolysin O (θ-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BCθ on a sucrose gradient. BCθ was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCθ binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BCθ binding to FLDF. The staining patterns of BCθ and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCθ binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCθ binding does not cause any damage to cell membranes, indicating that BCθ is a useful probe for the detection of membrane rafts in living cells.

Characterization of Spermidine Binding to Solubilized Plasma Membrane Proteins from Zucchini Hypocotyls1

In this work [14C]spermidine binding to total proteins solubilized from plasma membrane purified from zucchini (Cucurbita pepo L.) hypocotyls was investigated. Proteins were solubilized using octyl glucoside as a detergent. Specific polyamine binding was thermolabile, reversible, pH dependent with an optimum at pH 8.0, and had a Kd value of 5 μm, as determined by glass-fiber-filter assays. Sephadex G-25 M gel-filtration assays confirmed the presence of a spermidine-protein(s) complex with a specific binding activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native polyacrylamide gel electrophoresis of collected fractions having the highest specific spermidine-binding activity, several protein bands (113, 75, 66, and 44 kD) were identified. The specificity of spermidine binding was examined by gel-filtration competition experiments performed using other polyamines and compounds structurally related to spermidine. Partial purification on Sephadex G-200 led to the identification of 66- and 44-kD protein bands, which may represent the putative spermidine-binding protein(s) on the plasmalemma.

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase1 : IV. Fusicoccin Induces the Association between the Plasma Membrane H+-ATPase and the Fusicoccin Receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H+-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H+-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H+-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H+-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H+-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H+-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H+-ATPase. Analysis of the activation state of PM H+-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.

Specific transcellular binding between membrane proteins crucial to Alzheimer disease.

Molecular genetic studies of families suffering from genetic forms of early onset Alzheimer disease (AD) have identified three genes and their protein products as being crucially involved in the etiology of AD. The three proteins are all integral membrane proteins. One of them is beta-APP, the precursor of the beta-amyloid found in the characteristic neuritic plaques present in the brains of AD patients. The other two, S182 and STM2, are homologous in amino acid sequence to one another but are unrelated to beta-APP. It is shown here, using cultured cells transfected for each of these proteins, that beta-APP binds specifically and transcellularly to either S182 or STM2. We propose that this transcellular binding may not only be important in normal neuronal physiology and development but may be directly involved in the process of formation of beta-amyloid from beta-APP.

3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake.

Alpha 1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex.

Calcium-dependent homotypic cell-cell adhesion, mediated by molecules such as E-cadherin, guides the establishment of classical epithelial cell polarity and contributes to the control of migration, growth, and differentiation. These actions involve additional proteins, including alpha- and beta-catenin (or plakoglobin) and p120, as well as linkage to the cortical actin cytoskeleton. The molecular basis for these interactions and their hierarchy of interaction remain controversial. We demonstrate a direct interaction between F-actin and alpha (E)-catenin, an activity not shared by either the cytoplasmic domain of E-cadherin or beta-catenin. Sedimentation assays and direct visualization by transmission electron microscopy reveal that alpha 1(E)-catenin binds and bundles F-actin in vitro with micromolar affinity at a catenin/G-actin monomer ratio of approximately 1:7 (mol/mol). Recombinant human beta-catenin can simultaneously bind to the alpha-catenin/actin complex but does not bind actin directly. Recombinant fragments encompassing the amino-terminal 228 residues of alpha 1(E)-catenin or the carboxyl-terminal 447 residues individually bind actin in cosedimentation assays with reduced affinity compared with the full-length protein, and neither fragment bundles actin. Except for similarities to vinculin, neither region contains sequences homologous to established actin-binding proteins. Collectively these data indicate that alpha 1 (E)-catenin is a novel actin-binding and -bundling protein and support a model in which alpha 1(E)-catenin is responsible for organizing and tethering actin filaments at the zones of E-cadherin-mediated cell-cell contact.

AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA ( ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multi-vesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.

Dapsone hydroxylamine induces premature removal of human erythrocytes by membrane reorganization and antibody binding

N-hydroxylation of dapsone leads to the formation of the toxic hydroxylamines responsible for the clinical methaemoglobinaemia associated with dapsone therapy. Dapsone has been associated with decreased lifespan of erythrocytes, with consequences such as anaemia and morbidity in patients treated with dapsone for malaria. Here, we investigated how dapsone and/or its hydroxylamine derivative (DDS-NHOH) induced erythrocyte membrane alterations that could lead to premature cell removal.

Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.

The Cratylia Mollis Seed Lectin Induces Membrane Permeability Transition In Isolated Rat Liver Mitochondria And A Cyclosporine A-insensitive Permeability Transition In Trypanosoma Cruzi Mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca(2+) was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca(2+) this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.

Galectin-1 Induces Reversible Phosphatidylserine Exposure at the Plasma Membrane

Cells normally undergo physiological turnover through the induction of apoptosis and phagocytic removal, partly through exposure of cell surface phosphatidylserine (PS). In contrast, neutrophils appear to possess apoptosis-independent mechanisms of removal. Here we show that Galectin-1 (Gal-1) induces PS exposure independent of alterations in mitochondrial potential, caspase activation, or cell death. Furthermore, Gal-1-induced PS exposure reverts after Gal-1 removal without altering cell viability. Gal-1-induced PS exposure is uniquely microdomain restricted, yet cells exposing PS do not display evident alterations in membrane morphology nor do they exhibit bleb formation, typically seen in apoptotic cells. Long-term exposure to Gal-1 prolongs PS exposure with no alteration in cell cycle progression or cell growth. These results demonstrate that Gal-1-induced PS exposure and subsequent phagocytic removal of living cells represents a new paradigm in cellular turnover.