Functional characterization of a second porin isoform in Drosophila melanogaster. DmPorin2 forms voltage-independent cation-selective pores

Mitochondrial porins or voltage-dependent anion-selective channels are channel-forming proteins mainly found in the mitochondrial outer membrane. Genome sequencing of the fruit fly Drosophila melanogaster revealed the presence of three additional porin-like genes. No functional information was available for the different gene products. In this work we have studied the function of the gene product closest to the known Porin gene (CG17137 coding for DmPorin2). Its coding sequence was expressed in Escherichia coli. The recombinant DmPorin2 protein is able to form channels similar to those formed by DmPorin1 reconstituted in artificial membranes. Furthermore, DmPorin2 is clearly voltage-independent and cation-selective, whereas its counterpart isoform 1 is voltage-dependent and anion-selective. Sequence comparison of the two porin isoforms indicates the exchange of four lysines in DmPorin1 for four glutamic acids in DmPorin2. We have mutated two of them (Glu-66 and Glu-163) to lysines to investigate their role in the functional features of the pore. The mutants E163K and E66K/E163K are endowed with an almost full inversion of the ion selectivity. Both single mutations partially restore the voltage dependence of the pore. We found that an additional effect with the double mutant E66K/E163K was the restoration of voltage dependence. Protein structure predictions highlight a 16 beta-strand pattern, typical for porins. In a three-dimensional model of DmPorin2, Glu-66 and Glu-163 are close to the rim of the channel, on two opposite sides. DmPorin2 is expressed in all the fly tissues and in all the developmental stages tested. Our main conclusions are as follows. 1) The CG17137 gene may express a porin with a functional role in D. melanogaster. 2) We have identified two amino acids of major relevance for the voltage dependence of the porin pore.     

Pore forming activity of the major outer membrane protein of Rhodobacter capsulatus in lipid bilayer membranes

Porin of the outer membrane of Rhodobacter capsulatus St. Louis (ATCC 23782) was isolated and reconstituted into lipid bilayer membranes. The porin was obtained either by the sodium dodecyl sulfate treatment of cell envelopes (SDS-porin) or by saline extraction of whole cells (NaCl-porin). Nanomolar concentrations of both porin preparations resulted in a strong conductance increase of the lipid bilayer membranes by many orders of magnitude. At small protein concentrations the conductance increased in a stepwise fashion, the average single channel conductance being about 0.35 nS in 0.1 M KCl for SDS-porin and NaCl-porin as well. The single channel conductance was a linear function of the specific conductance of the aqueous phase. The results were consistent with the assumption that the porin formed large water-filled transmembrane channels in the membrane. From the average value of the single channel conductance in 0.1 M KCl an effective channel diameter of about 1.5 nm was estimated for both types of porins.Abbreviations EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate     

Identification of human porins. II. Characterization and primary structure of a 31-lDa porin from human B lymphocytes (Porin 31HL)

We characterize and describe for the first time the primary structure of a human porin with the molecular mass of 31 kDa derived from the plasmalemm of B-lymphocytes (Porin 31HL). Porin 31HL is shown to be a basic, channel forming membrane protein. The protein chain is composed of 282 amino acids with a relative molecular mass of 30641 Da without derivatisation. It is not a glycoprotein. The N-terminus is acetylated. Altogether the amino-acid sequence shows 56% hydrophilic or charged amino acids arranged in alternating regions of hydrophilic or hydrophobic character as it is typical for porins. In addition the 18 N-terminal amino acids of Porin 31HL can be arranged to an amphilic alpha-helix like in other porins. Porin 31HL shows approx. 29% or 24% identity to the primary structure of mitochondrial porins of Neurospora crassa and Saccharomyces cerevisiae. Partial data on mitochondrial porins from rat kidney and beef heart show sequence identity of about 90% to the human B cell porin elaborated here.     

Evaluation of Salmonella Porins as a Broad Spectrum Vaccine Candidate

Porins were prepared from smooth strain of Salmonella typhi 0–901 and chemotype of rough mutant of S. typhimurium Ra-30. Mice were immunized with both the porin preparations in different groups and challenged with S. typhimurium LT2–71 and S. enteritidis SH-1269. Porin immunized mice showed significant protection (P <0.01) against challenge with homologous as well as heterologous strains. Hence, the use of porins may be attempted in future to protect against salmonellosis.     

Identification of human porins. I. Purification of a porin from human B-lymphocytes (Porin 31HL) and the topochemical proof of its expression on the plasmalemma of the progenitor cell

We describe for the first time a porin (Porin 31HL) on the plasmalemm of an eukaryontic cell line, where porins have been found only on the outer mitochondrial membranes. The expression of the porin on the plasmalemm of transformed human B-lymphocytes is demonstrated by cytotoxicity- and indirect immunofluorescence techniques with living and fixed cells. The rabbit xenoantisera used were directed against purified Porin 31HL and free or acetylated synthetic peptides of its nineteen N-terminal amino acids. The three-step purification procedure for Porin 31HL started from a total membrane fraction of the B-cell line, followed by ion-exchange chromatography on CM- and DEAE-cellulose and a final gel filtration in SDS on Sephacryl S-300.     

Molecular, antigenic, and functional analyses of Omp2b porin size variants of Brucella spp

Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various Brucella species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of Brucella melitensis reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus Brucella seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of Brucella suis biovar 3), the porin variability does not result in major antigenic variability of the Brucella surface that could help the bacteria during the reinfection of a host. Porin variation in Brucella seems to result mainly in porin conductivity modifications.     

Abnormal ovarian morphogenesis in Drosophila melanogaster after injection of embryos with conditioned media from the Daudi cell line

Summary Drosophila melanogaster embryos were injected before the blastoderm stage with conditioned media from several male Burkitt's lymphoma human cell lines and the Daudi cell line. Such injections do not have any effect on the male genital apparatus or on the female tract. The Daudi conditioned medium modifies the ovarian morphogenesis of the flies and the rudimentary ovaries obtained look like nymphal gonads. Moreover, they have a drastically reduced number of germ cells. The ovaries that looked functional contain numerous necrotic germ cells and the mean number of ovarioles per fly is significantly smaller than that of the controls. The abnormalities observed resemble the results of experimental and genetic lack of germ cells. They disappear at very high dilution (1×10^–6).     

Altered channel properties of porins from Haemophilus influenzae: isolates from cystic fibrosis patients

Changes in amino-acid sequence of the unique pore-forming protein of H. influenzae (OmpP2; porin) have been associated with increased antimicrobial resistance in H. influenzae strains isolated from cystic fibrosis patients. From patients who were subjected to long-term antimicrobial therapy, H. influenzae strains 67d and 69a (patient 27) and strains 77a and 77f (patient 30) were isolated. Strains 67d and 77a were previously shown to have elevated values for minimal inhibitory concentrations of antibiotics compared to strains 69a and 77f. Porins were extracted from all four H. influenzae strains by detergent treatment and purified to homogeneity by ion exchange chromatography. By reconstitution of the clinical Hi porins into planar lipid bilayers, single-channel conductance, ionic selectivity, and voltage-gating characteristics were assessed. Porins 77a and 77f displayed similar single-channel conductance and ionic selectivity. Current-voltage relationships were determined for the different porins: porin 77f displayed substantial voltage gating at both positive and negative polarity; porin 77a gated at negative polarity only. Porins 67d and 69a showed substantial differences in their pore-forming properties: the single-channel conductance of porin 69a was significantly increased (1.05 nS) relative to porin 67d (0.73 nS). Porin 67d was twice as permeable to cations as porin 69a, and at both positive and negative polarities the extent of voltage gating was greater for porin 67d relative to porin 69a. Expression of the porins in an isogenic, porin-deleted H. influenzae background allowed for assessment of the contribution of each porin to the minimum inhibitory concentrations of various antimicrobial compounds. Porin 67d was found to have a decreased susceptibility to the antimicrobials novobiocin and streptomycin. This decreased susceptibility of porin 67d to novobiocin and streptomycin correlates with its decrease in single-channel conductance.     

Induction of tyrosine phosphorylated proteins in THP-1 cells by Salmonella typhimurium, Pasteurella haemolytica and Haemophilus influenzae porins

The effect of porins purified from Salmonella typhimurium, Pasteurella haemolytica and Haemophilus influenzae on induction of tyrosine phosphorylation in THP-1 cells and C3H/HeJ macrophage was investigated. Incubation of porins at concentration of 1.0-5.0 microg ml(-1) with either THP-1 or macrophage from C3H/HeJ mice resulted in tyrosine phosphorylation of specific host cell proteins. After porin stimulation a pattern of tyrosine phosphorylated proteins appeared in the soluble cytoplasmic fraction, in the membrane fraction and in the insoluble protein fraction. The observed effects were dependent on the porin concentrations; they reached a maximal expression at 3 min and declined at 60 min. Porin and lipopolysaccharide treatments induce a similar phosphorylation pattern in all of the three cellular fractions studied. A difference can be observed in the cytoplasmic fraction bands of 50-60 kDa, which are more evident after treatment with lipopolysaccharide, and in the insoluble fraction band of 80 kDa and the cytoplasmic fraction band of 250 kDa, which are more evident after treatment with porins. The events of tyrosine protein phosphorylation were present in macrophage from lipopolysaccaride-hyporesponsive C3H/HeJ mice stimulated with porins, while they were markedly reduced when the cells were stimulated with lipopolysaccharide. Staurosporine, genistein and cytochalasin D induced in the three cellular fractions a different inhibition pattern of tyrosine protein phosphorylation in porin stimulated cells. Porins extracted from the three bacterial species investigated behave in a similar way as stimuli more or less potent; Hib porin seems to be the most powerful stimulator and, moreover, it specifically induces phosphorylation of a 55 kDa band.     

Biophysical characterization of OprB,a glucose-inducible porin ofPseudomonas aeruginosa

OprB, a glucose-inducible porin ofP. aeruginosa, was characterized by black lipid bilayer analysis and circular dichroism spectroscopy. Black lipid bilayer analysis of OprB revealed a single-channel conductance of 25 pS, the presence of a glucose binding site with aK _s for glucose of 380 ± 40 mM, and the formation of channels with a strong selection for anions. Analysis ofP. aeruginosa OprB circular dichroism spectra revealed a high sheet content (40%) which is within the range of that determined for other porins. Values obtained from black lipid bilayer analysis were compared to those previously obtained for OprB ofP. putida [Saravolacet al. (1991).J. Bacteriol. 173, 4970–4976] and indicated extensive similarities in the single-channel conductance and glucose-binding properties of these two porins. Immunological and amino terminal sequence analysis revealed a high degree of homology. Of the first 14 amino terminal residues, 12 were identical. A major difference between the two porins was found in their ion selectivity. WhereasP. aeruginosa OprB is anion selective,P. putida OprB and other carbohydrate selective porins are known to be cation selective.     

Drosophila Porin/VDAC Affects Mitochondrial Morphology

Voltage-dependent anion channel (VDAC) has been suggested to be a mediator of mitochondrial-dependent cell death induced by Ca²⁺ overload, oxidative stress and Bax-Bid activation. To confirm this hypothesis in vivo, we generated and characterized Drosophila VDAC (porin) mutants and found that Porin is not required for mitochondrial apoptosis, which is consistent with the previous mouse studies. We also reported a novel physiological role of Porin. Loss of porin resulted in locomotive defects and male sterility. Intriguingly, porin mutants exhibited elongated mitochondria in indirect flight muscle, whereas Porin overexpression produced fragmented mitochondria. Through genetic analysis with the components of mitochondrial fission and fusion, we found that the elongated mitochondria phenotype in porin mutants were suppressed by increased mitochondrial fission, but enhanced by increased mitochondrial fusion. Furthermore, increased mitochondrial fission by Drp1 expression suppressed the flight defects in the porin mutants. Collectively, our study showed that loss of Drosophila Porin results in mitochondrial morphological defects and suggested that the defective mitochondrial function by Porin deficiency affects the mitochondrial remodeling process.     

Diffusion of β-Lactam Antibiotics Through Oligomeric or Monomeric Porin Channels of Some Gram-Negative Bacteria

The penetration of anionic β-lactam antibiotics through porins was evaluated as a mechanism of drug resistance. The major proteins with porin activity were purified from the outer membranes of six bacteria. Three of the six porins were oligomeric porins. The molecular weights of their monomers were 37 kDa from Photobacterium damsela, 42 kDa from Serratia liquefaciens, and 36 kDa from E. coli B. The other three porins were heat-modifiable monomeric porins with molecular weights of 43 kDa from Porphyromonas asaccharolytica and Acinetobacter baumannii, and 37 kDa from Escherichia coli K12. Comparison of the six porin proteins revealed that, independent of their aggregation state, their amino acid content is similar but not identical. All have double the amount of negatively charged amino acids compared with positively charged amino acids. They have a similar polarity and polarity index. Two of the six tested bacteria do not produce β-lactamase. These two bacteria were sensitive to the different β-lactams tested. The other four bacteria were resistant to all or to several β-lactams. A modified liposome swelling method was used for determining the rate of penetration of charged β-lactam antibiotics. Zwitterionic β-lactams were found to penetrate into liposomes at a rate that more or less fits their molecular weight, whether the porins are monomeric or oligomeric. The penetration rates of negatively charged β-lactams are different for oligomeric and monomeric porins. Negatively charged β-lactams penetrate through oligomeric porins better than estimated by their molecular weight, whereas monomeric porins are less penetrable to negatively charged β-lactams than estimated by their molecular weight. The contribution of all types of porins to the susceptibility of bacteria to β-lactam antibiotics (zwitterionic or negatively charged) is apparently doubtful. The porins may decrease or increase bacterial penetration rates to β-lactams, and only the existence of a potential β-lactamase that can destroy the penetrating drug will cause resistance. Received: 28 January 2002 / Accepted: 4 May 2002     

Identification and expression analysis of rainbow trout <Emphasis Type="Italic">pumilio</Emphasis>-1 and <Emphasis Type="Italic">pumilio</Emphasis>-2

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.This work was in part supported by a grant from the Akiyama Foundation to E.I. Nucleotide sequence data for rainbow trout pumilio-1 and pumilio-2 have been deposited in the DDBJ/EMBL/GenBank databases.     

Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells

dead end (dnd) was identified in zebrafish as a gene encoding an RNA-binding protein essential for primordial germ cell (PGC) development and gametogenesis in vertebrates. The adult dnd RNA expression has been restricted to the ovary in Xenopus or to the testis in mouse. Its protein product is nuclear in chicken germ cells but both cytosolic and nuclear in mouse cell cultures. Here we report the cloning and expression pattern of Odnd, the medakafish (Oryzias latipes) dnd gene. Sequence comparison, gene structure, linkage analysis and expression demonstrate that Odnd encodes the medaka Dnd orthologue. A systematic comparison of Dnd proteins from five fishes and tetrapod representatives led to the identification of five previously unidentified conserved regions besides the RNA recognition motif. The Odnd RNA is maternally supplied and preferentially segregated with PGCs. Its adult expression occurs in both sexes and is restricted to germ cells. In the testis, Odnd is abundant in spermatogonia and meiotic cells but absent in sperm. In the ovary, Odnd RNA persists throughout oogenesis. Furthermore, we developed a dual color fluorescent in situ hybridization procedure allowing for precise comparisons of expression and distribution patterns between two genes in medaka embryos and adult tissues. Importantly, this procedure co-localized Odnd and Ovasa in testicular germ cells and PGCs. Surprisingly, by cell transfection and embryo RNA injection we show that ODnd is cytoplasmic in cell cultures, cleavage embryos and PGCs. Therefore, medaka dnd encodes a cytoplasmic protein and identifies embryonic and adult germ cells of both sexes.     

A putative alpha-helical porin from Corynebacterium glutamicum

The cell wall of Corynebacterium glutamicum contains a mycolic acid layer, which is a protective nonpolar barrier similar to the outer membrane of Gram-negative bacteria. The exchange of material across this barrier requires porins. Porin B (PorB) is one of them. Recombinant PorB has been produced in Escherichia coli, purified, crystallized and analyzed by X-ray diffraction, yielding 16 independent molecular structures in four different crystal forms at resolutions up to 1.8 Å. All 16 molecules have the same globular core, which consists of 70 residues forming four α-helices tied together by a disulfide bridge. The 16 structures vary greatly with respect to the 29 residues in the N- and C-terminal extensions. Since corynebacteria belong to the group of mycolata that includes some prominent human pathogens, the observed structure may be of medical relevance. Due to the clearly established solid structure of the core, the native porin has to be oligomeric, and the reported structure is one of the subunits. An α-helical porin in a bacterial outer envelope is surprising because all presently known structures of such porins consist of β-barrels. Since none of the four crystal packing arrangements was compatible with an oligomeric membrane channel, we constructed a model of such an oligomer that was consistent with all available data of native PorB. The proposed model is based on the required polar interior and nonpolar exterior of the porin, on a recurring crystal packing contact around a 2-fold axis, on the assumption of a simple C_n symmetry (a symmetric arrangement around an n-fold axis), on the experimentally established electric conductivity and anion selectivity and on the generally observed shape of porin channels.     

Binding of Rat Brain Hexokinase to Recombinant Yeast Mitochondria: Identification of Necessary Molecular Determinants

The association in vitro of rat brain hexokinase to mitochondria from rat liver or yeast (wildtype, porinless, or expressing recombinant human porin) was studied in an effort to identifyminimal requirements for each component. A short hydrophobic N-terminal peptide ofhexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria.Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loopsaround residues 110 and 200, as determined by proteolytic-fragment identification usingantibodies. Recombinant human porin in yeast mitochondria is more sensitive to proteolysisthan wild-type porin in rat liver mitochondria. Recombinant yeast mitochondria, harboringseveral natural or engineered porins from various sources, bind hexokinase to variable extentwith marked preference for the mammalian porin1 isoform. Genetic alteration of this isoformat the C-, but not the N-terminal, results in a significant reduction of hexokinase bindingability. Macromolecular crowding (dextran) promotes a stronger association of the enzyme toall recombinant mitochondria, as well as to proteolytically digested organelles. Consequently,brain hexokinase association with heterologous mitochondria (yeast) in these conditions occursto an extent comparable to that with homologous (rat) mitochondria. The study, also pertinentto the topology and organization of porin in the membrane, represents a necessary first stepin the functional investigation of the physiological role of mammalian hexokinase binding tomitochondria in reconstituted heterologous recombinant systems, as models to cellularmetabolism.     

Distinct expression patterns for two Xenopus Bar homeobox genes

The BarH1 and BarH2 homeobox genes are coexpressed in cells of the fly retina and in the central and peripheral nervous systems. The fly Bar genes are required for normal development of the eye and external sensory organs. In Xenopus we have identified two distinct vertebrate Bar-related homeobox genes, XBH1 and XBH2. XBH1 is highly related in sequence and expression pattern to a mammalian gene, MBH1, suggesting that they are orthologues. XBH2 has not previously been identified but is clearly related to the Drosophila Bar genes. During early Xenopus embryogenesis XBH1 and XBH2 are expressed in overlapping regions of the central nervous system. XBH1, but not XBH2, is expressed in the developing retina. By comparing the expression of XBH1 with that of hermes, a marker of differentiated retinal ganglion cells, we show that XBH1 is expressed in retinal ganglion cells during the differentiation process, but is down-regulated as cells become terminally differentiated. Received: 12 August 1999 / Accepted: 5 October 1999     

Primordial follicle assembly was regulated by notch signaling pathway in the mice

Notch signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms. The objective of this study was to examine Notch signaling pathway in germ cell cyst breakdown and primordial follicle formation. The receptor and ligand genes of Notch pathway (Notch1, Notch2, Jagged1, Jagged2 and Hes1) were extremely down-regulated after newborn mouse ovaries were cultured then exposed to DAPT or L-685,458 in vitro (P < 0.01). Since DAPT or L-685,548 inhibits Notch signaling pathway, the expression of protein LHX8 and NOBOX was significantly reduced during the formation of the primordial follicles. Down-regulated mRNA expression of specific genes including Lhx8, Figla, Sohlh2 and Nobox, were also observed. The percentages of female germ cells in germ cell cysts and primordial follicles were counted after culture of newborn ovaries for 3 days in vitro. The result showed female germ cells in cysts was remarkably up-regulated while as the oocytes in primordial follicles was significantly down-regulated (P < 0.05). In conclusion, Notch signaling pathway may regulate the formation of primordial follicle in mice.