An amino-acid substitution in the influenza-B NB protein affects ion-channel gating.

The effects of site-directed mutations in NB, a protein encoded by the influenza B virus that has been shown to form cation-selective ion channels at pH 6.0, were studied on ion channel characteristics in artificial lipid bilayers. It was thought that the residues in the hydrophobic region of NB we selected for mutation might be involved in the transport of cations across the channel and that changes in these residues might affect channel properties such as gating and ion-selectivity. Serine residues at positions 20 and 28, threonine at position 24 and cysteine at position 26 were replaced by alanine. We found that the mutation S20A gave channels that did not gate and that remained open most of the time. Proton permeability of NB channels, as detected by fluorescence quenching, was also altered by the mutation S20A: channels were no longer proton-permeable. The other mutations, S28A, T24A and C26A, did not have any detectable effect on the activity or proton permeability of channels formed by NB. The results indicate that serine 20 may have an important role in normal function of NB channels.

Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride.

A 63 residue peptide, p7, encoded by hepatitis C virus was synthesised and tested for ion channel activity in lipid bilayer membranes. Ion channels formed by p7 had a variable conductance: some channels had conductances as low as 14 pS. The reversal potential of currents flowing through the channels formed by p7 showed that they were permeable to potassium and sodium ions and less permeable to calcium ions. Addition of Ca(2+) to solutions made channels formed by p7 less potassium- or sodium-selective. Hexamethylene amiloride, a drug previously shown to block ion channels formed by Vpu encoded by HIV-1, blocked channels formed by p7. In view of the increasing number of peptides encoded by viruses that have been shown to form ion channels, it is suggested that ion channels may play an important role in the life cycle of many viruses and that drugs that block these channels may prove to be useful antiviral agents.

Structure of the C123S mutant of dienelactone hydrolase (DLH) bound with the PMS moiety of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF).

The structure of DLH (C123S) with PMS bound was solved to 2.5 A resolution (R factor = 15.1%). PMSF in 2-propanol was delivered directly to crystals in drops and unexpectedly caused the crystals to dissolve. New crystals displaying a different morphology emerged within 2 h in situ, a phenomenon that appears to be described for the first time. The changed crystal form reflected altered crystal-packing arrangements elicited by structural changes to the DLH (C123S) molecule on binding inhibitor. The new unit cell remained in the P2(1)2(1)2(1) space group but possessed different dimensions. The structure showed that PMS binding in DLH (C123S) caused conformational changes in the active site and in four regions of the polypeptide chain that contain reverse turns. In the active site, residues with aromatic side chains were repositioned in an edge-to-face cluster around the PMS phenyl ring. Their redistribution prevented restabilization of the triad His202 side chain, which was disordered in electron-density maps. Movements of other residues in the active site were shown to be related to the four displaced regions of the polypeptide chain. Their implied synergy suggests that DLH may be able to accommodate and catalyse a range of compounds unrelated to the natural substrate owing to an inherent coordinated flexibility in its overall structure. Implications for mechanism and further engineering studies are discussed.

Sub-cellular localisation of the white/scarlet ABC transporter to pigment granule membranes within the compound eye of Drosophila melanogaster.

The white, scarlet, and brown genes of Drosophila melanogaster encode ABC transporters involved with the uptake and storage of metabolic precursors to the red and brown eye colour pigments. It has generally been assumed that these proteins are localised in the plasma membrane and transport precursor molecules from the heamolymph into the eye pigment cells. However, the immuno-electron microscopy experiments in this study reveal that the White and Scarlet proteins are located in the membranes of pigment granules within pigment cells and retinula cells of the compound eye. No evidence of their presence in the plasma membrane was observed. This result suggests that, rather than tranporting tryptophan into the cell across the plasma membrane, the White/Scarlet complex transports a metabolic intermediate (such as 3-hydroxy kynurenine) from the cytoplasm into the pigment granules. Other functional implications of this new finding are discussed.

Mutations in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration.

The white, brown and scarlet genes of Drosophila melanogaster encode proteins which transport guanine or tryptophan (precursors of the red and brown eye colour pigments) and belong to the ABC transporter superfamily. Current models envisage that the white and brown gene products interact to form a guanine specific transporter, while white and scarlet gene products interact to form a tryptophan transporter. In this study, we report the nucleotide sequence of the coding regions of five white alleles isolated from flies with partially pigmented eyes. In all cases, single amino acid changes were identified, highlighting residues with roles in structure and/or function of the transporters. Mutations in w(cf) (G589E) and w(sat) (F590G) occur at the extracellular end of predicted transmembrane helix 5 and correlate with a major decrease in red pigments in the eyes, while brown pigments are near wild-type levels. Therefore, those residues have a more significant role in the guanine transporter than the tryptophan transporter. Mutations identified in w(crr) (H298N) and w(101) (G243S) affect amino acids which are highly conserved among the ABC transporter superfamily within the nucleotide binding domain. Both cause substantial and similar decreases of red and brown pigments indicating that both tryptophan and guanine transport are impaired. The mutation identified in w(Et87) alters an amino acid within an intracellular loop between transmembrane helices 2 and 3 of the predicted structure. Red and brown pigments are reduced to very low levels by this mutation indicating this loop region is important for the function of both guanine and tryptophan transporters.

The amino-terminal region of Vpr from human immunodeficiency virus type 1 forms ion channels and kills neurons.

We have previously reported that the accessory protein Vpr from human immunodeficiency virus type 1 forms cation-selective ion channels in planar lipid bilayers and is able to depolarize intact cultured neurons by causing an inward sodium current, resulting in cell death. In this study, we used site-directed mutagenesis and synthetic peptides to identify the structural regions responsible for the above functions. Mutations in the N-terminal region of Vpr were found to affect channel activity, whereas this activity was not affected by mutations in the hydrophobic region of Vpr (amino acids 53 to 71). Analysis of mutants containing changes in the basic C terminus confirmed previous results that this region, although not necessary for ion channel function, was responsible for the observed rectification of wild-type Vpr currents. A peptide comprising the first 40 N-terminal amino acids of Vpr (N40) was found to be sufficient to form ion channels similar to those caused by wild-type Vpr in planar lipid bilayers. Furthermore, N40 was able to cause depolarization of the plasmalemma and cell death in cultured hippocampal neurons with a time course similar to that seen with wild-type Vpr, supporting the idea that this region is responsible for Vpr ion channel function and cytotoxic effects. Since Vpr is found in the serum and cerebrospinal fluids of AIDS patients, these results may have significance for AIDS pathology.

The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels.

Vpu is a small phosphorylated integral membrane protein encoded by the human immunodeficiency virus type 1 genome and found in the endoplasmic reticulum and Golgi membranes of infected cells. It has been linked to roles in virus particle budding and degradation of CD4 in the endoplasmic reticulum. However, the molecular mechanisms employed by Vpu in performance of these functions are unknown. Structural similarities between Vpu and the M2 protein of influenza A virus have raised the question of whether the two proteins are functionally analogous: M2 has been demonstrated to form cation-selective ion channels in phospholipid membranes. In this paper we provide evidence that Vpu, purified after expression in Escherichia coli, also forms ion channels in planar lipid bilayers. The channels are approximately five- to sixfold more permeable to sodium and potassium cations than to chloride or phosphate anions. A bacterial cross-feeding assay was used to demonstrate that Vpu can also form sodium-permeable channels in vivo in the E. coli plasma membrane.

Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers.

A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix. Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential. The channels were more permeable to Na+ than to Cl- ions and were inhibited when the trans potential was made positive. Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen. Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber. Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber. The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential. The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber. The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies.

Mutational analysis of the traffic ATPase (ABC) transporters involved in uptake of eye pigment precursors in Drosophila melanogaster. Implications for structure-function relationships.

The white, brown, and scarlet genes of Drosophila melanogaster encode three proteins that belong to the Traffic ATPase superfamily of transmembrane permeases and are involved in the transport of guanine and tryptophan (precursors of the red and brown eye pigments). We have determined the nucleotide sequences of two mutant white alleles (wco2 and wBwx) that cause reduced red pigmentation but have no effect on brown pigmentation. In wco2 the effect is only observed when interacting with the bw6 allele or a newly isolated allele (bwT50). These alleles of the brown gene were cloned and sequenced. In wco2 the codon for glycine 588 is changed to encode serine; in wBwx the triplet ATC encoding isoleucine 581 is deleted; asparagine 638 is changed to threonine in bw6, and glycine 578 is changed to aspartate in bwT50. No other relevant changes to the gene structures were detected. P-element-mediated germline transduction was used to construct a fly strain containing a white gene with a mutation of the nucleotide binding domain. Such flies had white eyes, indicating that the mutated white gene was unable to support either guanine or tryptophan transport. The implications of these mutations are discussed in terms of a model of the Drosophila pigment precursor transport system.

Switching of bovine cytochrome c oxidase subunit VIa isoforms in skeletal muscle during development.

A cDNA encoding the liver isoform of bovine cytochrome c oxidase subunit VIa (VIaL) was cloned from bovine liver RNA by reverse transcription and the polymerase chain reaction. The nucleotide and deduced amino acid sequences show high conservation with the corresponding rat and human liver subunits. The sequence similarity between beef heart and beef liver VIa is 60%. Northern analyses of the steady-state levels of the VIa-heart (VIaH) and VIa-liver (VIaL) transcripts showed that adult liver and brain contained only VIaL transcripts, the VIaH transcript predominated in heart with a small amount of VIaL also present, while in adult skeletal muscle VIaH was present exclusively. The VIaL transcript was found in heart with a small amount of VIaL also present, while in adult skeletal muscle VIaH was present exclusively. The VIaL transcript was found in fetal heart and skeletal muscle from 104-215-day-old fetuses, in as much as 25% of the amount of VIaH transcript. The down-regulation of VIaL transcript in skeletal muscle at or close to birth may be correlated with a change in amount of cytochrome c oxidase relative to the bc1 complex (complex III) observed spectrally when fetal and adult muscle samples were compared.

Soluble hydrogenase of Anabaena cylindrica. Cloning and sequencing of a potential gene encoding the tritium exchange subunit.

A gene potentially encoding a subunit of the soluble hydrogenase of Anabaena cylindrica was isolated from a genomic library by screening with a set of redundant oligonucleotides, the sequence of which was deduced from the amino acid sequence of the purified hydrogenase subunit that catalyses tritium exchange. The nucleotide sequence of the potential gene was determined from two overlapping DNA fragments spanning 7237 bp of the A. cylindrica genome. The region sequenced contained an open reading frame encoding a protein of 383 amino acids with a predicted molecular mass of 41,108 Da. The NH2-terminal amino acid sequence of the purified enzyme, determined by Edman degradation, corresponds exactly with that deduced from the nucleic acid sequence. No significant amino acid or nucleotide similarity is evident between this gene and the periplasmic hydrogenases from three species of Desulfovibrio (D. vulgaris, D. baculatus and D. gigas), or with the membrane-bound 'uptake' hydrogenases of Bradyrhizobium japonicum and Rhodobacter capsulatus. This suggests that the soluble enzyme from cyanobacteria represents a discrete class of hydrogenase. The gene encoding the second subunit (m = 50 kDa) of the soluble hydrogenase, which is required for the catalysis of hydrogen production from dithionite-reduced methyl viologen [Ewart, G. D. & Smith, G. D. (1989) Arch. Biochem. Biophys. 268, 327-337], apparently comprises a separate transcription unit since it appears not to be located adjacent to that for the 42-kDa subunit.

Purification and properties of soluble hydrogenase from the cyanobacterium Anabaena cylindrica.

Two soluble hydrogenase activities were separable from cell extracts of the cyanobacterium Anabaena cylindrica, one detectable by the tritium exchange assay, the other having a relatively low tritium exchange activity but catalyzing methyl viologen-dependent hydrogen formation. Their molecular weights, by gel filtration chromatography, were 42,000 and 100,000, respectively. The two hydrogenase activities were differentially inhibited. The methyl viologen-dependent activity has been purified to homogeneity from cells in which the enzyme was induced by gassing the growing cells with N2/H2/CO2 (95.7%/4%/0.3%, v/v/v). The procedure involved French pressure cell disruption of the cells, differential precipitation with ZnCl2, heat treatment (50 degrees C), and lyophilization of the heat-step supernatant. It was then subjected to DEAE-Sephacel chromatography, dye-ligand chromatography on Procion Red, and HPLC anion exchange on QMA-Accel. Polyacrylamide gel electrophoresis on both native and denaturing gels revealed two peptides with Mr's 42,000 and 50,000. The 42,000 protein alone catalyzed tritium exchange activity; both proteins appeared to be necessary for the methyl viologen activity. The native enzyme appears to be a readily dissociable dimer of two nonidentical subunits, one of which contains the hydrogen binding site and the other providing the ability to utilize electrons from a reductant for hydrogen formation.