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.     

Mitochondrial porin of Neurospora crassa: cDNA cloning, in vitro expression and import into mitochondria.

cDNA encoding porin of Neurospora crassa, the major protein component of the outer mitochondrial membrane, was isolated and the nucleotide sequence was determined. The deduced protein sequence consists of 283 amino acids (29,979 daltons) and shows sequence homology of around 43% to yeast porin; however, no significant homology to bacterial porins was apparent. According to secondary structure predictions, mitochondrial porin consists mainly of membrane-spanning sided beta-sheets. Porin was efficiently synthesized in vitro from the cDNA; this allowed us to study in detail its import into mitochondria. Thereby, three characteristics of import were defined: (i) import depended on the presence of nucleoside triphosphates; (ii) involvement of a proteinaceous receptor-like component on the surface of the mitochondria was demonstrated; (iii) insertion into the outer membrane was resolved into at least two distinct steps: specific binding to high-affinity sites and subsequent assembly to the mature form.     

Studies on human porin. VI. Production and characterization of eight monoclonal mouse antibodies against the human VDAC "Porin 31HL" and their application for histotopological studies in human skeletal muscle.

We report on the production and characterization of eight monoclonal mouse antibodies against the complete human VDAC "Porin 31HL". The antigen used was purified from a total membrane preparation of the transformed human B-lymphocyte cell line H2LCL. In Western blots all eight mAbs react with a single 31-kDa band in solubilized H2LCL membrane preparations thus demonstrating their specificity for the human VDAC "Porin 31HL". Concerning the epitope specificity we show that all eight mAbs equally react with the N-terminal part of human porin. Moreover, we demonstrate the expression of VDAC in the sarcolemma by indirect immunoenzyme labelling of cryosections of human skeletal muscle applying four of our mAbs. These data support our recent observations on the expression of porin channels in the plasmalemma of different normal and transformed human cell lines. VDAC in the plasmalemma is discussed as the molecular basis of the Blatz and Magleby channel.     

Purification and Characterization of Porin from Corn (Zea mays L.) Mitochondria

Mitochondrial porin from corn (Zea mays L. B 73) shoots was solubilized with lauryl(dimethyl)-amine oxide and purified by chromatography on a hydroxyapatite:celite column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein had an apparent molecular mass of 35 kD. When reconstituted in planar lipid bilayer membranes the porin formed ion-permeable channels with single-channel conductance of 2.0 and 4.0 nanosiemens in 1 M KCl. At low transmembrane voltages corn porin had the properties of a general diffusion pore with an estimated effective diameter of 1.6 nm and a small selectivity for anions over cations. The primary structure of corn porin seems to be quite different from that of other mitochondrial porins, because it did not cross-react with monoclonal antibodies against human porin and with polyclonal antibodies against yeast porin. Furthermore, the peptide maps of corn and bovine heart porins were very different. A sequence of 21 amino acids obtained by Edman degradation of peptides generated by porin proteolysis with Staphylococcus aureus V8 protease did not show any significant homology with known sequences of mitochondrial porins. Results of our investigation suggest that corn porin possesses functional properties similar to those of other mitochondrial porins, despite major structural differences.     

Studies on human porin. IV. The primary structures of "Porin 31HM" purified from human skeletal muscle membranes and of "Porin 31HL" derived from human B lymphocyte membranes are identical.

We report on the purification of "Porin 31HM" from the crude plasma membrane fraction of human skeletal muscle. Furthermore, all tryptic peptides of the molecule were purified and characterized by different methods. The alignment of the peptides with the complete primary structure of the human B lymphocyte plasma membrane-derived "Porin 31HL", published by us recently (Kayser, H. et al. (1989) this Journal 370, 1265-1278), proved both structures to be completely identical. Our data demonstrate that porin fractions from crude plasma membranes of different human cell types do not show any variation on the primary structure level.     

Demonstration and characterization of human cardiac porin: a voltage-dependent channel involved in adenine nucleotide movement across the outer mitochondrial membrane

The porins are a class of voltage-dependent, anion-selective, channel-forming proteins located in the outer mitochondrial membrane (OMM). The porins are responsible for passage of adenine nucleotides across the OMM, as well as for specific binding of hexokinase and glycerol kinase. This porin-kinase complex has direct access to ATP generated by mitochondrial oxidative phosphorylation and may be important in the regulation of glycolysis. Porin had not been described previously in humans but, due to its importance in bioenergetics, would be expected to be present, especially in organs requiring a large and constant supply of energy. We therefore postulated that porin would occur in human myocardium where it would be important in cardiac function. Polyclonal antibodies to bovine myocardial and rat liver porins were utilized in transblotting experiments after polyacrylamide gel electrophoresis of human heart preparations from atria, ventricles, papillary muscles, and interventricular septum. These immunoblots demonstrated selective staining of a 34-kDa band. This was identical to the results obtained with purified porin and the antibodies. Also notable was the finding that the vast majority of this staining was found in the homogenate pellet after high speed centrifugation (20,000g), as would be expected for a mitochondrial protein. The demonstration of human cardiac porin by immunoblotting with rat liver and bovine myocardial porin antibodies is the first demonstration of cross-species identification of the porins. The success of this approach undoubtedly occurred because of strong homology between porins from a variety of species.     

Studies on human porin. III. Does the voltage-dependent anion channel "Porin 31HL" form part of the chloride channel complex, which is observed in different cells and thought to be affected in cystic fibrosis?

"Porin 31HL", of known primary structure, is an integral protein of the plasmalemma of human B cells (Thinnes, F.P. et al. (1989) This Journal 370, 1253-1264; Kayser, H. et al. (1989) This Journal 370, 1265-1278). Purified "Porin 31HL" from human B lymphocytes was reconstituted into lipid bilayer membranes, where it formed defined voltage-dependent channels. Five minutes preincubation with 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, potent inhibitor of chloride transport, altered the channel-forming properties of the protein, so that it now showed small irregular channels instead of distinct steps. In addition, the voltage-dependence of the channel was abolished by the action of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Functional and structural similarities between "Porin 31HL" and porin preparations from other human tissues and from other species suggest that this protein may be part of the chloride channel complex, which is defective in cystic fibrosis.     

Studies on human porin. V. The expression of "porin31HL" in the plasmalemma is not by cell transformation]

In recent papers we proved "Porin 31HL" to be located on the surface of human, EBV-transformed B lymphocytes. Here we present proof of "Porin 31HL" in the plasmalemma of normal human blood lymphocytes. For this purpose B and T lymphocytes were isolated from human heparinized blood and examined by indirect immunofluorescence techniques using different monoclonal antibodies against purified "Porin 31HL" and some B and T cell markers, respectively. For comparison a number of established cell lines of different origin were employed. Hence it followed that normal B and T cells as well as transformed and leukemic cells express "Porin 31HL" in their membrane. No significant quantitative differences could be seen. Consequently, the location of "Porin 31HL" in the plasmalemma is not a product of transformation.     

Antigenic sites on porin of Haemophilus influenzae type b: mapping with synthetic peptides and evaluation of structure predictions.

The major surface-located protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin, molecular mass, 38 kDa, 341 amino acids. To define precisely the molecular reactivities of nine mouse monoclonal antibodies (MAbs) against Hib porin, overlapping hexapeptides corresponding to the entire sequence of porin were synthesized. The epitopes recognized by the MAbs were mapped by enzyme-linked immunosorbent assay to stretches of 6 to 11 amino acids. Antigenic sites between amino acids 112 and 126, 148 and 153, 162 and 172, and 318 and 325 were identified. The antigenic sites between amino acids 162 and 172 and between amino acids 318 and 325 were determined by flow cytometry to be on the bacterial cell surface. Four MAbs, POR.2, POR.3, POR.4, and POR.5, that react with amino acids 162 to 172 were able to discriminate among porins from the three major outer membrane protein subtypes of Hib, i.e., 1H, 2L, and 6U. A model for the topological organization of Hib porin was created by calculating the hydrophobicity, amphiphilicity, and turn propensity in its amino acid sequence. Determination of the molecular reactivities of the anti-Hib porin MAbs provided substantive evidence for the orientation of selected regions of porin in the outer membrane of Hib.     

Class-3 porin protein of Neisseria meningitidis: cloning and structure of the gene.

The gene coding for the class-3 protein of Neisseria meningitidis was cloned and sequenced. The deduced amino acid (aa) sequence was highly homologous (50-78%) to those of other neisserial porin proteins. Alignment of the aa sequence of five neisserial porin proteins pinpointed several regions of identity or near identity. These are assumed to be membrane-spanning beta-strands. A comparison of the homologies between these neisserial porins showed that the class-3 protein is most closely related to the Neisseria gonorrhoeae P1A protein.     

Molecular cloning and sequencing of cDNA for yeast porin, an outer mitochondrial membrane protein: a search for targeting signal in the primary structure.

We have cloned a full-length cDNA for yeast porin, the major outer mitochondrial membrane protein from Saccharomyces cerevisiae, and determined its nucleotide sequence. The primary structure of the protein, deduced from the nucleotide sequence, consisted of 283 amino acid residues and its NH2-terminal sequence, Met-Ser-Pro-Pro-Val-Tyr-Ser, coincided with that determined by Edman degradation for yeast porin, except that the initiator methionine was missing in the mature protein. The deduced sequence had an overall polarity index of 46.3%, a value which falls in the normal range for soluble proteins. An evaluation of hydropathy of the protein indicated that the NH2-terminal one third was relatively hydrophilic and the rest of the molecule was rather hydrophobic. An interesting finding was that the NH2-terminal region of yeast porin (consisting of some 50 amino acid residues) shows structural features that resemble those of the corresponding portion of 70-kd protein, which is also a yeast outer mitochondrial membrane protein. We postulate that this NH2-terminal sequence, like that of 70-kd protein, is required for targeting the porin to the outer mitochondrial membrane.     

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.     

Cloning and Characterization of the Gene Encoding for OMP-PD Porin: The Major <Emphasis Type="Italic">Photobacterium damsela</Emphasis> Outer Membrane Protein

The outer membrane protein of Photobacterium damsela (OMP-PD) and the gene encoding for this porin protein were isolated and characterized. The deduced amino acid sequence of the OMP-PD monomer has 338 amino acids and a calculated molecular weight of 36,951 Da. This sequence includes a 22-amino acid signal peptide at the N-terminal, which is not found when the monomer is located in the outer membrane. Native OMP-PD protein forms a trimeric structure of approximately 110 kDa. It exhibits resistance to proteases, and it can be cleaved only following denaturation by SDS. The degree of identity of the OMP-PD amino acid sequence to porins from the Enterobacteriaceae was only 24%. Identity to Vibrio or Photobacterium porins was 38% and 48%, respectively. Nevertheless, the multiple alignment of this sequence with other structurally defined Enterobacteria porins demonstrated that the location of the 16 beta-strands and eight external loops, including a larger external L3 loop, are conserved in OMP-PD. These results, together with the previously known ability of OMP-PD to form an ion channel in artificial liposomes, strongly support its role as a porin in P. damsela and will help further investigations into the role of OMP-PD in P. damsela pathogenicity.     

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.     

Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum.

Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was purified by hydroxyapatite and CM-cellulose chromatography. The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis. The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component. Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials. The channel displayed a characteristic voltage dependence for potentials higher than 20 mV. It switched to substates of smaller conductance and a selectivity different to that of the open state. The closed state was stabilized at low ionic strength. The cDNA sequence of mitochondrial porin from D. discoideum was determined. It showed little sequence similarities to other known mitochondrial porins. The functional similarity, however, was striking. Localization of the porin in the mitochondrial outer membrane was confirmed by immunogold labeling of cryosections of fixed cells.     

The nuclear coded mitoribosomal proteins YmL27 and YmL31 are both essential for mitochondrial function in yeast.

Using synthetic oligonucleotides deduced from the N-terminal amino acid sequence of purified mitoribosomal protein (mt r-protein) YmL27, the corresponding nuclear gene MRP-L27 of the yeast Saccharomyces cerevisiae has been cloned and sequenced. The MRP-L27 gene codes for 146 amino acids and is located on chromosome X. The mature YmL27 protein consists of 130 amino acids - after cleaving the putative mitochondrial signal peptide - with a net charge of +17 and a calculated relative molecular mass of 14,798 Da. The YmL27 protein as well as the yeast mitoribosomal protein YmL31, which had been characterized and its gene (MRP-L31) cloned previously, is essential for mitochondrial function as shown by the inability of gene disrupted mutants for the MRP-L27 or MRP-L31 genes to grow on non-fermentable carbon sources.     

Purification, amino acid composition and N-terminal sequence of the major protein (protein H) of the outer membrane of Pasteurella multocida]

The major protein (protein H) of the outer membrane of Pasteurella multocida was purified by size-exclusion chromatography after selective extraction with detergents. The protein forms homotrimers which are stable in the presence of SDS at room temperature. Upon treatment at 100 degrees C, the protein is fully dissociated by the detergent into monomers exhibiting an apparent molecular mass of 37 kDa as estimated by electrophoresis. The amino acid composition of protein H is characterized by a low hydropathy index (HI = -0.40) and is strongly related to the compositions of bacterial porins, notably porins P2 (Haemophilus influenzae), PIA (Neisseria gonorrhoeae) and Cl.2 ("class 2 porin" of N. meningitidis). The N-terminal amino acid sequence of protein H shares a strong homology with those of porins OmpC (Escherichia coli) and P2. These data indicate that protein H of P. multocida is a porin belonging to the superfamily of the non-specific porins of Gram-negative eubacteria outer membrane.     

Homology models of the Yersinia pseudotuberculosis and Yersinia pestis general porins and comparative analysis of their functional and antigenic regions

The amino acid sequences of the Yersinia pseudotuberculosis porin (YPS) and Y. pestis porin (YPT) have recently deduced but their three-dimensional structures were not known. These sequences were analyzed using the servers 3D-PSSM and PredPort. The YPS and YPT porins were shown to have a high degree of identity (above 50%) in primary and secondary structures. The three-dimensional models of the Yersinia pseudotuberculosis porin (YPS) and Y. pestis porin (YPT) were obtained using the homology modeling approach, SWISS-MODEL Protein Modeling Server and 3-D structure of PhoE porin from E. coli as template. The superposition of the Calpha-atoms of the monomers of the Yersinia porins and PhoE porin gave a root mean square deviations of 0.47 A and 0.43 A for YPS and YPT respectively. Yersinia porins were found to be very similar in their three-dimensional structure to other non-specific enterobacterial porins, having the same features of overall fold and disposition of loop L3. The intrinsic structures of the monomer pores of YPS and YPT were investigated and their conductances were predicted with the program HOLE. The good correspondence between the theoretical and experimental magnitudes of YPS conductance was found. The Yersinia porins were determined to be unusual in containing the substitution, Glu replaced by Val, in a highly conserved pentapeptide (Pro-Glu-Phe-Gly-Gly-Asp), located in the loop L3 tip that disturbs the functionally important cluster of the acidic amino acids in the constriction site. Comparative analysis of structural organization of YPS and E. coli OmpF porin in the regions involved in subunit association and pore lumen was performed. The YPS porin functional properties were predicted. The differences between these porins in polar interactions playing a significant role in stabilization of the porin trimers were found and discussed in term of the variations in trimer stability. The Yersinia porins were shown to have the highest degree of the structural similarity. The differences between the porins were observed in their external loops. Their loops L6 and loops L8 showed 71.4 and 52.9% of sequence identity, respectively. The arrangement of charged residues clustered in the channel external vestibule of these porins was found to be also different suggesting the possible differences in their functional properties. The surface exposed regions of Yersinia porins involved in their potential sequential antigenic determinants were compared. The structural basis of their cross reactivity and antigenic differences is discussed.