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Query: UNIPROT:P06889 (Mol)
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We have characterized a gene encoding an Adenosine triphosphate (ATP) Binding Cassette (ABC) transmembrane protein from Trichomonas vaginalis, an early-diverging protozoan parasite. This gene, Tvpgp1, encodes a 589-amino acid protein with an amino-terminal hydrophobic region, 6 potential membrane-spanning segments and a carboxy-terminal ATP binding site. Tvpgp1 is most similar in sequence to mammalian P-glycoproteins, 170 kDa transport proteins which are frequently overexpressed in multiple drug-resistant (Mdr) tumor cell lines. However, Tvpgp1 is half the size of typical P-glycoproteins which are tandem duplications. These data suggest that the duplication/fusion events which gave rise to the bipartite structure comprised of 2 similar halves which characterize eukaryotic P-glycoproteins may have occurred after the divergence of trichomonads (Parabasalia) from the main line of eukaryotic evolutionary descent. We have examined 7 metronidazole resistant strains of T. vaginalis to determine whether the Tvpgp1 gene is overexpressed or amplified. 2 drug resistant strains show a 2-3-fold overexpression and one shows a 20-fold overexpression of Tvpgp mRNA. The gene is not amplified in any of the drug resistant strains. On the contrary, 4 of the 7 resistant strains lack one of 2 Tvpgp genes found in drug-sensitive strains.
Mol Biochem Parasitol 1994 Jul
PMID:Analysis of a single-domain P-glycoprotein-like gene in the early-diverging protist Trichomonas vaginalis. 798 75

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminal OmpA signal sequence was fused with TEM beta-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the beta-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor. In the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal beta-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the amino-terminal nuclease domain across the membrane, leaving the carboxyl terminal beta-lactamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.
Mol Microbiol 1994 Mar
PMID:Reversible topology of a bifunctional transmembrane protein depends upon the charge balance around its transmembrane domain. 802 60

The DNA sequence of the regulatory region and the structural gene, nrfA, for cytochrome c552 of Escherichia coli K-12 have been reported. We have now established that nrfA is the first gene in a seven-gene operon, designated the nrf operon, at least five of which are essential for formate-dependent nitrite reduction to ammonia. This operon terminates just upstream of the previously sequenced gltP gene encoding a sodium-independent, glutamate and aspartate transporter. Expression of lac fused to nrfA, nrfE or nrfG is regulated by oxygen repression, FNR-dependent anaerobic induction, nitrite induction and nitrate repression during anaerobic growth, exactly as previously reported for the nrfA promoter. In contrast, expression of the gltP-lac fusion was FNR-independent. The open reading frame immediately downstream of nrfA encodes NrfB, a hydrophilic, penta-haem cytochrome c with an M(r) of 20,714. The structure of the N-terminal region is typical of a signal peptide for a periplasmic protein: cleavage at the putative signal peptide cleavage site, Ala-26, would result in a periplasmic cytochrome with a molecular mass of 18 kDa. The NrfC polypeptide, M(r) 24,567, contains 16 cysteine residues arranged in four clusters typical of the CooF super-family of non-haem iron-sulphur proteins. The NrfD sequence predicts a 318-residue hydrophobic protein with a distribution of acidic and basic amino acids which suggests that NrfD is an integral transmembrane protein with loops in both the periplasm and the cytoplasm. Proteins most similar to NrfD include the PsrC subunit of polysulphide reductase from Wolinella, but, as seven of the 10 most similar proteins are NADH-ubiquinone oxidoreductases, we propose that NrfD participates in the transfer of electrons from the quinone pool into the terminal components of the Nrf pathway. NrfE, M(r) 60,851, is predicted to be another hydrophobic, integral membrane protein homologous to the CdI1 protein of Rhodobacter capsulatus, which has been implicated in the assembly of periplasmic c-type cytochromes. The sequence of the 127 residue NrfF polypeptide, M(r) 14,522, is strikingly similar to the CcI2 protein of R. capsulatus, especially in the putative haem-binding motif, RCPQCQNQN.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Microbiol 1994 Apr
PMID:A seven-gene operon essential for formate-dependent nitrite reduction to ammonia by enteric bacteria. 805 35

A novel ordered assemblage of bacteriorhodopsin, a transmembrane protein functioning as a light-driven proton pump, is found in its three-dimensional crystal. Atomic force microscope images of the crystal surface reveal that spherical protein clusters with a diameter of approximately 50 nm are hexagonally close-packed. Electron micrographs of mechanically disintegrated crystals show that the inside of the protein cluster is filled with the mother liquor. The crystal is made up of hollow protein clusters. When disintegrated crystals are illuminated in the presence of a lipophilic anion, a significant alkalization of the external medium occurs. This result indicates that the protein cluster contains native lipids and that the cytoplasmic side of the protein faces the external medium. X-ray diffraction patterns and the observed diameter of the spherical shell suggest that approximately 200 bacteriorhodopsin trimers are aligned on a polyhedral surface lattice. Another remarkable feature of the spherical assemblies of bacteriorhodopsin is that they fuse with each other at low ionic strength and occasionally form a tubular or doughnut-like structure. The concept of membrane protein polymorphism is introduced on the basis of these observations, and it is used to describe the dynamic structure of some other biological membranes.
J Mol Biol 1994 Mar 04
PMID:Polyhedral assembly of a membrane protein in its three-dimensional crystal. 812 Sep 7

Molecular cloning of the prolactin (PRL) receptor cDNA has revealed different forms of the receptor: among them, the longest form encodes a transmembrane protein of 592-598 amino acids and was originally found in rabbit mammary gland as well as in human and rat tissues. It contains a cytoplasmic domain of 358 amino acids. In CHO cells transfected with the PRL receptor cDNA, PRL is able to induce the specific expression of a reporter gene provided with the promoter of the milk protein gene beta-lactoglobulin. The cDNA encoding this long receptor form has been expressed permanently after stable transfection of Chinese hamster ovary (CHO) cells. In these cells, we have determined the fate of the bound hormone and of the receptor. At 37 degrees C, transfected cells were able to endocytose 125I-labeled human growth hormone (hGH) or ovine prolactin (oPRL) at an initial rate of about 1 fmol/h at 100 pM labeled hormone and 10(6) cells/well. Lowering the temperature to 15 degrees C slowed the endocytosis of [125I]hGH by a factor of 5. These results were confirmed by electron microscopy with oPRL labeled with colloidal gold. At 37 degrees C, the receptor underwent rapid insertion to the cell surface and constitutive endocytosis (half-life 80 min). This rate of endocytosis was enhanced in the presence of 10 nM oPRL (half-life 8 min), leading to down-regulation of the receptor by exhaustion of the intracellular receptor pool. After down-regulation, the cell surface was replenished with newly synthesized PRL receptor with a half-time of 8-10 min. If cycloheximide was added, almost no receptors could be found on the cell surface. These results indicate that in transfected cells the PRL receptor behaved largely as in classical target cells. A "conveyor belt" endocytosis behavior was found, with degradation of the endocytosed receptors, and occupation by the hormone enhancing this process. Moreover, since the PRL receptor belongs to a family of receptors in which companion protein(s) seem to play important roles, transfected CHO cells appear to provide the expressed receptors with the necessary element(s) to function as in normal PRL target cells.
Mol Cell Endocrinol 1994 Mar
PMID:Endocytosis and degradation of prolactin and its receptor in Chinese hamster ovary cells stably transfected with prolactin receptor cDNA. 820 30

The chromosomal ampC beta-lactamase in Citrobacter freundii and Enterobacter cloacae is inducible by beta-lactam antibiotics. When an inducible ampC gene is introduced on a plasmid into Escherichia coli together with its transcriptional regulator ampR, the plasmid-borne beta-lactamase is still inducible. We have isolated mutants, containing alterations in a novel E. coli gene, ampG, in which a cloned C. freundii ampC gene is unable to respond to beta-lactam inducers. The ampG gene was cloned, sequenced and mapped to minute 9.6 on the E. coli chromosome. The deduced amino acid sequence predicted AmpG to be a 53 kDa, transmembrane protein, which we propose acts as a signal transducer or permease in the beta-lactamase induction system. Immediately upstream of ampG there is another 579-base-pair-long open reading frame (ORF) encoding a putative lipoprotein shown to be non-essential for beta-lactamase induction. We have found that ampG and this ORF form an operon, whose promoter is located in front of the ORF. Located closely upstream of the putative promoter is the morphogene bolA, which is transcribed in the opposite orientation. However, using transcription fusions, we have found that the ampG transcription is not regulated by bolA. In addition, we show that transcription is probably not regulated by either the starvation specific sigma factor RpoS, which controls bolA, or by AmpD the negative regulator for ampC transcription.
Mol Microbiol 1993 Aug
PMID:AmpG, a signal transducer in chromosomal beta-lactamase induction. 823 4

A transmembrane protein serine/threonine kinase, Atr-I, that is structurally related to receptors for members of the transforming growth factor-beta (TGF-beta) family has been cloned from Drosophila melanogaster. The spacing of extracellular cysteines and the cytoplasmic domain of Atr-I resemble most closely those of the recently described mammalian type I receptors for TGF-beta and activin. When expressed alone in test cells, Atr-I is unable to bind TGF-beta, activin, or bone morphogenetic protein 2. However, Atr-I binds activin efficiently when coexpressed with the distantly related Drosophila activin receptor Atr-II, with which it forms a heteromeric complex. Atr-I can also bind activin in concert with mammalian activin type II receptors. Two alternative forms of Atr-I have been identified that differ in an ectodomain region encompassing the cysteine box motif characteristic of receptors in this family. Comparison of Atr-I with other type I receptors reveals the presence of a characteristic 30-amino-acid domain immediately upstream of the kinase region in all these receptors. This domain, of unknown function, contains a repeated Gly-Ser sequence and is therefore referred to as the GS domain. Maternal Atr-I transcripts are abundant in the oocyte and widespread during embryo development and in the imaginal discs of the larva. The structural properties, binding specificity, and dependence on type II receptors define Atr-I as an activin type I receptor from D. melanogaster. These results indicate that the heteromeric kinase structure is a general feature of this receptor family.
Mol Cell Biol 1994 Feb
PMID:Two distinct transmembrane serine/threonine kinases from Drosophila melanogaster form an activin receptor complex. 828 34

Wheat germ agglutinin (WGA) elicits a number of biological effects in erythrocytes as a result of specific binding to the transmembrane protein glycophorin A. The structure of co-crystals of WGA (isolectin 1: WGA1) with a bivalent sialoglycopeptide fragment of glycophorin A (T5), determined at 2.0 A resolution, has been further refined and analyzed with respect to ligand-induced changes in the tertiary structure, mobility, solvation, saccharide conformation and protein/saccharide interactions at three independent N-acetyl-D-neuraminic (NeuNAc) binding sites. The final model, which includes the two independent WGA1 monomers (composed of domains A, B, C and D), two positions for bound T5 sialo-tetrasaccharide (NeuNAc-alpha 2,3-Gal-beta 1,3-(alpha 2,6-NeuNAc)GalNAc) and 386 water molecules, refined to a crystallographic R-factor of 17.1% (Fo > 1.0 sigma) and an average temperature factor of 31.99 A2. Comparisons between the tertiary structures of the liganded and unliganded WGA1 dimers indicate that the largest deviations from 2-fold symmetry are localized in domains engaged in sugar binding (B1 and C2) and at the C-terminal domain of monomer II (D2), forming a strong lattice contact. Interactions of the tetrasaccharide with amino acid ligands in the three binding sites and with water were carefully analyzed and compared. Bound conformations of terminal NeuNAc match to within a root-mean-square delta r of 0.3 A. The specificity-determining N-acetyl group superimposes best in comparison with other substituents of the sugar ring. Of the five domain binding sites that are not occupied in this dimeric crosslinked complex, only one is accessible to the NeuNAc monosaccharide as determined from a difference Fourier map at 3.0 A resolution.
J Mol Biol 1993 Jul 20
PMID:Crystallographic refinement and structure analysis of the complex of wheat germ agglutinin with a bivalent sialoglycopeptide from glycophorin A. 834 26

A defect in the map3 gene of the fission yeast Schizosaccharomyces pombe causes h+ mating-type-specific sterility. This gene was cloned by complementation. Nucleotide sequence analysis showed that it has a coding capacity of 365 amino acids. The deduced map3 gene product is a putative seven-transmembrane protein and has 20.0% amino acid identity with the a-factor receptor of Saccharomyces cerevisiae, encoded by STE3. It is also homologous with the Ustilago maydis mating pheromone receptors. The map3 gene is expressed in h+ cells but not in h- cells, and the transcripts are induced in response to nitrogen starvation. h+ cells defective in map3 do not respond to purified M-factor. When map3 is expressed ectopically in h- cells, they apparently acquire the ability to respond to the M-factor produced by themselves. The gpa1 gene, which encodes the alpha-subunit of a G-protein presumed to couple with the mating pheromone receptors, is essential for this function of map3. These observations strongly suggest that map3 encodes the M-factor receptor. Furthermore, this study provides strong support for the notion that pheromone signaling is essential for initiation of meiosis in S. pombe and that either M-factor signaling or P-factor signaling alone is sufficient.
Mol Cell Biol 1993 Jan
PMID:Schizosaccharomyces pombe map3+ encodes the putative M-factor receptor. 838 Feb 33

The retinal protein halorhodopsin (HR), a light-driven chloride pump from Halobacterium halobium, was homologously overexpressed in this archaebacterium. Two DNA expression systems differing in their promoter region were investigated. The halopsin, hop, promoter coupled to the hop gene gave an increased level of HR synthesis. However, the extent of expression was driven by the copy number of the shuttle vector and did not reach the magnitude of the bacterio-opsin, bop, promoter system. Employing a gene fusion approach, the promoter for the bop gene was used to drive expression of the hop gene. A shuttle vector containing a bop-hop-cartridge was transformed into a HR-deficient strain and blueish-coloured transformants were obtained. The bop promoter expressed HR to an extent where a specific membrane fraction resembled the crystalline purple membrane of BR in terms of the lipid to protein ratio. HR could, therefore, be easily isolated in a natural membrane-bound state. This allows for direct use in biophysical studies without the application of detergents. This was the first successful overexpression of a 7-helical transmembrane protein and may be extended to other proteins of this family.
Mol Microbiol 1993 Feb
PMID:Homologous overexpression of a light-driven anion pump in an archaebacterium. 838 88


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