Biomedical subjects

M Lebendiker

Publications and source records attributed to M Lebendiker.

11 recordsLinked to original sources

PubMed2001

Interactions between adaptor protein-1 of the clathrin coat and microtubules via type 1a microtubule-associated proteins.

The classical view suggests that adaptor proteins of the clathrin coat mediate the sorting of cargo protein passengers into clathrin-coated pits and the recruitment of clathrin into budding areas in the donor membrane. In the present study, we provide biochemical and morphological evidence that the adaptor protein 1 (AP-1) adaptor of the trans-Golgi network clathrin interacts with microtubules. AP-1 in cytosolic extracts interacted with in vitro assembled microtubules, and these interactions were inhibited by ATP depletion of the extracts or in the presence of 5'-adenylylimidodiphosphate. An overexpressed gamma-subunit of the AP-1 complex associated with microtubules, suggesting that this subunit may mediate the interaction of AP-1 with the cytoskeleton. Purified AP-1 did not interact with purified microtubules, but interaction occurred when an isolated microtubule-associated protein fraction was added to the reaction mix. The gamma-adaptin subunit of AP-1 specifically co-immunoprecipitated with a microtubule-associated protein of type 1a from rat brain cytosol. This suggests that type 1a microtubule-associated protein may mediate the association of AP-1 with microtubules in the cytoplasm. The microtubule binding activity of AP-1 was markedly inhibited in cytosol of mitotic cells. By means of its interaction with microtubule-associated proteins, we propose novel roles for AP-1 adaptors in modulating the dynamics of the cytoskeleton, the stability and shape of coated organelles, and the loading of nascent AP-1-coated vesicles onto appropriate microtubular tracks.

Adaptor Protein Complex gamma Subunits↗

PubMed2001

The projection structure of EmrE, a proton-linked multidrug transporter from Escherichia coli, at 7 A resolution.

EmrE belongs to a family of eubacterial multidrug transporters that confer resistance to a wide variety of toxins by coupling the influx of protons to toxin extrusion. EmrE was purified and crystallized in two dimensions by reconstitution with dimyristoylphosphatidylcholine into lipid bilayers. Images of frozen hydrated crystals were collected by cryo-electron microscopy and a projection structure of EmrE was calculated to 7 A resolution. The projection map shows an asymmetric EmrE dimer with overall dimensions approximately 31 x 40 A, comprising an arc of highly tilted helices separating two helices nearly perpendicular to the membrane from another two helices, one tilted and the other nearly perpendicular. There is no obvious 2-fold symmetry axis perpendicular to the membrane within the dimer, suggesting that the monomers may have different structures in the functional unit.

Antiporters↗

PubMed1999

Scanning cysteine accessibility of EmrE, an H+-coupled multidrug transporter from Escherichia coli, reveals a hydrophobic pathway for solutes.

EmrE is a 12-kDa Escherichia coli multidrug transporter that confers resistance to a wide variety of toxic reagents by actively removing them in exchange for hydrogen ions. The three native Cys residues in EmrE are inaccessible to N-ethylmaleimide (NEM) and a series of other sulfhydryls. In addition, each of the three residues can be replaced with Ser without significant loss of activity. A protein without all the three Cys residues (Cys-less) has been generated and shown to be functional. Using this Cys-less protein, we have now generated a series of 48 single Cys replacements throughout the protein. The majority of them (43) show transport activity as judged from the ability of the mutant proteins to confer resistance against toxic compounds and from in vitro analysis of their activity in proteoliposomes. Here we describe the use of these mutants to study the accessibility to NEM, a membrane permeant sulfhydryl reagent. The study has been done systematically so that in one transmembrane segment (TMS2) each single residue was replaced. In each of the other three transmembrane segments, at least four residues covering one turn of the helix were replaced. The results show that although the residues in putative hydrophilic loops readily react with NEM, none of the residues in putative transmembrane domains are accessible to the reagent. The results imply very tight packing of the protein without any continuous aqueous domain. Based on the findings described in this work, we conclude that in EmrE the substrates are translocated through a hydrophobic pathway.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

NMR investigation of the multidrug transporter EmrE, an integral membrane protein.

EmrE is an Escherichia coli multidrug transport protein that confers resistance to a wide range of toxicants by active transport across the bacterial cell membrane. The highly hydrophobic polytopic integral membrane protein has been purified and studied in its full-length form by high-resolution NMR spectroscopy in a mixture of chloroform/methanol/water (6:6:1, by vol.). Full activity is maintained after reconstitution of the protein into proteoliposomes from this solvent mixture. A series of heteronuclear (1H-15N) two- and three-dimensional experiments, as well as triple resonance experiments, were applied to the 110-residue protein and led to the assignment of the 1H, 15N and a large part of the 13C backbone resonances as well as many of the sidechain resonances. A preliminary analysis of the secondary structure, based on sequential NOE connectivities, deviation of chemical shifts from random coil values and 3J(NH-H alpha) coupling constants supports a model where the protein forms four alpha-helices between residues 4-26 (TM1), 32-53 (TM2), 58-76 (TM3) and 85-106 (TM4). For the residues of helices TM2 and TM3 a significant line broadening occurs due to slow conformational processes.

Amino Acid Sequence↗

PubMed1997

EmrE, the smallest ion-coupled transporter, provides a unique paradigm for structure-function studies.

EmrE is an Escherichia coli multidrug transporter which confers resistance to a wide variety of toxicants by actively removing them in exchange for hydrogen ions. EmrE is a highly hydrophobic 12 kDa protein which has been purified by taking advantage of its unique solubility in organic solvents. After solubilization and purification, the protein retains its ability to transport as judged from the fact that it can be reconstituted in a functional form. Hydrophobicity analysis of the sequence yielded four putative transmembrane domains of similar sizes. Results from transmission Fourier transform infrared measurements agree remarkably well with this hypothesis and yielded alpha-helical estimates of 78% and 80% for EmrE in CHCl3:MeOH and 1,2-dimyristoyl phosphocholine, respectively. Furthermore, the fact that most of the amide groups in the protein do not undergo amide-proton H/D exchange implies that most (approximately 80%) of the residues are embedded in the bilayer. These observations are only consistent with four transmembrane helices. A domain lined by Cys41 and Cys95 accessible only to substrates such as the organic mercurial 4-(chloromercuri)benzoic acid has been identified. Both residues are asymmetric in their location with respect to the plane of the membrane, Cys95 being closer than Cys41 to the outside face of the membrane. In co-reconstitution experiments of wild-type protein with three different inactive mutants, negative dominance has been observed. This phenomenon suggests that EmrE is functional as a homo-oligomer.

Amino Acid Sequence↗

PubMed1996

Negative dominance studies demonstrate the oligomeric structure of EmrE, a multidrug antiporter from Escherichia coli.

EmrE, the smallest known ion-coupled transporter, is an Escherichia coli 12-kDa protein 80% helical and soluble in organic solvents. EmrE is a polyspecific antiporter that exchanges hydrogen ions with aromatic toxic cations such as methyl viologen. Since it is many times smaller than the classical consensus 12 transmembrane segments transporters, it was particularly interesting to determine its oligomeric state. For this purpose, a series of nonfunctional mutants has been generated and characterized to test their effect on the activity of the wild-type protein upon mixing. As opposed to the wild type, these mutants do not confer resistance to methyl viologen, ethidium bromide, or a series of other toxicants. Co-expression of each of the nonfunctional mutants with the wild-type protein results in a reduction in the ability of the functional transporter to confer resistance to several toxicants. To perform mixing experiments in vitro, all the mutants have been purified by extraction with organic solvents, reconstituted in proteoliposomes, and found to be inactive. When co-reconstituted with wild-type protein, they inhibit the activity of the latter in a dose-dependent form up to full inhibition. We assume that this inhibition is due to the formation of mixed oligomers in which the presence of one nonfunctional subunit causes full inactivation. A binomial analysis of the results based on the latter assumptions do not provide statistically significant answers but suggests that the oligomer is composed of three subunits. The results described provide the first in vitro demonstration of the functional oligomeric structure of an ion-coupled transporter.

Antiporters↗

PubMed1996

Identification of residues in the translocation pathway of EmrE, a multidrug antiporter from Escherichia coli.

EmrE is a small, 12-kDa, highly polyspecific antiporter, which exchanges hydrogen ions with aromatic cations such as methyl viologen. EmrE-mediated transport is inhibited by the sulfhydryl-reactive reagent 4-(chloromercuri)benzoic acid (PCMB) but not by a variety of other sulfhydryl reagents. This differential effect is due to the fact that the organic mercurial is a substrate of the transporter and can reach domains otherwise inaccessible to the different reagents. To find out which of the three cysteine residues in EmrE is reacting with PCMB, each was replaced with serine and it was shown that none of them is essential for transport activity. A protein completely devoid of Cys residues (CL) is also capable of substrate accumulation albeit at a slower rate. Mutated proteins in which only one of the native cysteines was left whereas the other changed to serine were also constructed. The use of these proteins demonstrated that two of the three Cys in EmrE, Cys-41 and Cys-95, but not Cys-39, react with PCMB. A related mercurial, 4-(chloromercuri)benzenesulfonic acid (PCMBS), is only a very poor inhibitor, probably because of the negative charge it bears. PCMBS reacts with EmrE in an asymmetric and unique way. It reacts with the mutant bearing a single Cys residue in position 95 (CL-C95) only when the reagent is present in the outside face of the membrane and with the mutant CL-C41 only when allowed to permeate to the cell interior; as expected, it does not react with the mutant protein bearing a single Cys at position 39 (CL-C39). It is concluded that PCMB permeates through the substrate pathway of EmrE and covalently reacts with the two exposed residues, Cys-95 and Cys-41, but not with Cys-39, located on the opposite face of the helix relative to residue 41. In addition, because of the asymmetric reactivity to PCMBS, an inhibitor that does not permeate through the protein, it is concluded that Cys-41 is closer to the cytoplasmic face than Cys-95. The results demonstrate the existence of a domain accessible only to substrates and provide a unique tool for studying the substrate permeation pathway of an ion-coupled transporter.

4-Chloromercuribenzenesulfonate↗

PubMed1996

Determining the secondary structure and orientation of EmrE, a multi-drug transporter, indicates a transmembrane four-helix bundle.

EmrE is a member of a newly emerging family of MiniTEXANS, a family of multi-drug antiporters from bacteria characterized by their small size of roughly 100 amino acids. In this report we have obtained transmission FTIR spectra of EmrE in CHCl3:MeOH, DMPC vesicles, and Escherichia coli lipid vesicles. Secondary structure analysis has shown that both in DMPC vesicles and in CHCl3: MeOH the protein adopts a highly helical secondary structure that correlates remarkably well with that predicted by hydropathy analysis. The protein was shown to be resistant to amide proton H/D exchange, providing evidence that most of the protein is embedded in the lipid bilayer. Polarized ATR-FTIR spectra of the protein in DMPC vesicles have shown that the helices are oriented with an average tilt angle of 27 degrees from the bilayer normal. The protein was found to be less oriented in E. coli lipid vesicles, most likely as a result of the poor orientation of the bilayer lipids themselves. Thus, the protein is identified as a transmembrane four-helix bundle providing valuable structural data for this family of multi-drug transporters. The results set the stage for further studies aimed at deriving a detailed model for this protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

EmrE, an Escherichia coli 12-kDa multidrug transporter, exchanges toxic cations and H+ and is soluble in organic solvents.

The smallest membrane protein shown to catalyze ion-coupled transport is documented in this report. A gene coding for a small 110-amino acid membrane protein (emrE or mvrC) has been previously identified and cloned and shown to render Escherichia coli cells resistant to methyl viologen and to ethidium. In this report, it is shown that the resistance is due to extrusion of the toxic compounds in a process that requires a proton electrochemical gradient rather than ATP. For this purpose, cells in which the unc gene was inactivated were used so that the interconversion between the proton gradient and ATP is not possible, and the effect of agents, which specifically affect either of them, was tested on transport of ethidium in the intact cell. In addition, EmrE has been overexpressed and metabolically labeled with [35S]methionine. Strikingly, the protein can be quantitatively extracted with a mixture of organic solvents such as chloroform:methanol and is practically pure after this extraction. Moreover, after addition of E. coli lipids to the chloroform:methanol extract, EmrE has been reconstituted in proteoliposomes loaded with ammonium chloride. Upon dilution of the proteoliposomes in ammonium-free medium, a pH gradient was formed that drove transport of ethidium and methyl viologen into the proteoliposome. Both substrates compete with each other and exchange with previously transported solute. EmrE is a multidrug transporter of a novel type, and, because of its size and its solubility properties, it provides a unique model to study structure-function aspects of transport reactions in ion-coupled processes.

Bacterial Proteins↗

PubMed1984

The nature of the interaction of eukaryotic initiation factor 2 with double-stranded RNA.

In addition to binding messenger RNA molecules at specific sequences, eukaryotic initiation factor 2 (eIF-2) also binds to double-stranded RNA (dsRNA). The dsRNA is a powerful inhibitor of initiation of eukaryotic translation, causing the inactivation of eIF-2, but in the presence of certain mRNA templates, dsRNA fails to establish inhibition. Such mRNA templates bind to eIF-2 with higher affinity than does dsRNA, while globin mRNA, a template sensitive to inhibition, binds with lower affinity. Here, the nature of the interaction between dsRNA and eIF-2 was studied by examining both the binding of eIF-2 to Penicillium chrysogenum dsRNA molecules carrying 32P label at their 5' ends, and the ability of eIF-2 to protect such label against pancreatic ribonuclease digestion. The results reveal binding sites for eIF-2 at the 5' ends, as well as throughout internal regions of the dsRNA molecule. At least 15 molecules of eIF-2 can be accommodated on a 3000-base molecule of P. chrysogenum dsRNA. eIF-2 protects a 105-base-pair 5'-terminal fragment in dsRNA against digestion, but exhibits no noticeable preference for the 5' ends. By contrast, eIF-2 fails to protect label at the 5' ends of denatured dsRNA molecules, even though it binds to them at internal sites more avidly than to native dsRNA. Binding of eIF-2 to dsRNA is not restricted to specific sequences: eIF-2 binds with equal affinity to the synthetic dsRNA sequence, poly(rI . rC). The data support the interpretation that eIF-2 recognizes the A conformation in dsRNA rather than sequence. Apparently binding of eIF-2 at sites spaced 200 base pairs apart prevents relaxation of the intervening length of the double helix, thereby stabilizing the dsRNA molecule against ribonuclease attack. These results show that, even though dsRNA and mRNA compete in their binding to eIF-2, the structural features recognized by eIF-2 in these RNA species are distinct.

Animals↗

PubMed1982

Application of RNase T1 one- and two-dimensional analyses to the rapid identification of foot-and-mouth disease viruses.

The analysis of several isolates of foot-and-mouth disease virus by RNase T1 fingerprinting of the 32P-labeled RNA is described. It has been shown that use of the 35S induced RNA instead of the virus particle RNA has two advantages. (i) About 40 times more radioactivity is incorporated into the induced RNA. (ii) The RNA can be prepared much more rapidly, thus increasing the value of the technique in rapid diagnosis. One-dimensional maps, in which the RNase T1 oligonucleotides are separated according to size, have been shown to provide a valuable screening method for distinguishing between viruses. Those viruses giving similar one-dimensional maps also gave similar two-dimensional maps. The value of using the length of the polycytidylic acid tract of foot-and-mouth disease virus as a diagnostic tool is also discussed.

Aphthovirus↗