Mitochondrial porin regulates the sensitivity of anion carriers to inhibitors

In mitoplasts, respiratory stimulation by ADP, palmitate, DNP and CCCP and sensitivity of respiration to carboxyatractylate are considerably less pronounced than in mitochondria. Addition of porin-containing preparations (purified outer membranes or solubilized mitochondrial porin) to mitoplasts results in partial restoration of the oxygen consumption and sensitivity to carboxyatractylate (CAT). The uncoupling effect of FCCP in mitoplasts is CAT-resistant and does not depend on added porin. It is suggested that mitochondrial porin may be a natural activator of ADP/ATP antiporter and succinate carrier in mitochondria.     

The respiration of cells and mitochondria of porin deficient yeast mutants is coupled

Several mutants of yeast lacking the porin gene have been found stable and viable on glucose or glycerol media. Ethanol-supported respiration of porin-free mutant and wild cells appeared equally coupled in vivo being similarly depressed by inhibitors of ADP/ATP translocase or of ATP synthase and stimulated by the uncoupler FCCP. The absence of porin in isolated mutant mitochondria hardly impaired the electron flux but increased the requirement for Mg2+ (or Ca2+) and for ADP and carboxyatractylate concentrations necessary to drive effectively state 3 - state 4 and state 4 - state 3 transitions, respectively. The existence of another porin species, possibly controlled by bivalent cations, is postulated.     

Contributions of photosynthetic and non‐photosynthetic cell types to leaf respiration in Vicia faba L. and their responses to growth temperature

In intact leaves, mitochondrial populations are highly heterogeneous among contrasting cell types; how such contrasting populations respond to sustained changes in the environment remains, however, unclear. Here, we examined respiratory rates, mitochondrial protein composition and response to growth temperature in photosynthetic (mesophyll) and non‐photosynthetic (epidermal) cells from fully expanded leaves of warm‐developed (WD) and cold‐developed (CD) broad bean (Vicia faba L.). Rates of respiration were significantly higher in mesophyll cell protoplasts (MCPs) than epidermal cell protoplasts (ECPs), with both protoplast types exhibiting capacity for cytochrome and alternative oxidase activity. Compared with ECPs, MCPs contained greater relative quantities of porin, suggesting higher mitochondrial surface area in mesophyll cells. Nevertheless, the relative quantities of respiratory proteins (normalized to porin) were similar in MCPs and ECPs, suggesting that ECPs have lower numbers of mitochondria yet similar protein complement to MCP mitochondria (albeit with lower abundance serine hydroxymethyltransferase). Several mitochondrial proteins (both non‐photorespiratory and photorespiratory) exhibited an increased abundance in response to cold in both protoplast types. Based on estimates of individual protoplast respiration rates, combined with leaf cell abundance data, epidermal cells make a small but significant (2%) contribution to overall leaf respiration which increases twofold in the cold. Taken together, our data highlight the heterogeneous nature of mitochondrial populations in leaves, both among contrasting cell types and in how those populations respond to growth temperature.     

NADH is specifically channeled through the mitochondrial porin channel in Saccharomyces cerevisiae

In many kinds of permeabilized cells, the restriction of metabolite diffusion by a mitochondrial porin closed state has been shown to control the respiration rate. However, since in isolated mitochondria the porin appears to be always open, the physiological relevance of a putative regulation via this channel status is now a subject of discussion. In Saccharomyces cerevisiae, in which some of the NADH dehydrogenase active sites are facing the intermembrane space, this regulatory mechanism might play an important role for the regulation of the cytosolic redox status. Using permeabilized spheroplasts from wild-type and porin-deficient mutant, we show that the NADH produced in the cytosol is channeled to the mitochondrial NADH dehydrogenases through a metabolic network involving the porin channel. Thus, the control exerted by the porin (i.e., open or closed state) seems to be determined through its participation or not in organized metabolic networks.     

A water-soluble form of porin from the mitochondrial outer membrane of Neurospora crassa. Properties and relationship to the biosynthetic precursor form

Mitochondrial porin, the outer membrane pore-forming protein, was isolated in the presence of detergents and converted into a water-soluble form. This water-soluble porin existed under nondenaturing conditions as a mixture of dimers and oligomers. The proportion of dimers increased with decreasing porin concentration during conversion. Water-soluble porin inserted spontaneously into artificial bilayers as did detergent-solubilized porin. Whereas the latter form had no specific requirements for the lipid composition of the bilayer, water-soluble porin inserted only into membranes containing a sterol, and only in the presence of very low concentrations of Triton X-100 (0.001% w/v) in the solution bathing the bilayer. The channels formed by water-soluble porin were indistinguishable from those formed by detergent-purified porin with respect to specific conductance and voltage dependence of conductance. Water-soluble porin bound tightly in a saturable fashion to isolated mitochondria. The bound form was readily accessible to added protease, indicating its presence on the mitochondrial surface. The number of binding sites was in the range of 5-10 pmol/mg of mitochondrial protein. Water-soluble porin apparently binds to a site on the assembly pathway of the porin precursor, since mitochondria whose binding sites were saturated with the water-soluble form did not import porin precursor synthesized in a cell-free system.     

Structure and function of an OmpC deletion mutant porin from Escherichia coli K-12.

Escherichia coli K-12 strain RAM122 contains a mutation in the ompC gene that results in an eight amino acid deletion, delta 103-110, in the porin protein. Since this strain is capable of growing on maltodextrins in the absence of a functional lamB gene, the mutant protein is thought to have a larger channel size. The stability and structure/function properties of the mutant OmpC porin were investigated and compared to wild-type porin. Isolated unheated RAM122 porin was characterized as a trimer on sodium dodecyl sulfate-polyacrylamide gels. The RAM122 trimer was less stable to temperature when compared to the wild-type porin. In addition, the overall enthalpy for thermal denaturation was lower for the mutant than the wild-type porin as determined by using differential scanning microcalorimetry. Both the proteins' secondary structures, monitored by circular dichroism, were high in beta-sheet content, but the spectra were slightly different in their crossover points as well as their minima. When the proteins were reconstituted and channel activity was assayed by using a liposome swelling technique, the size-exclusion limit of the mutant porin was twice that of the wild-type porin. Conductance measurements across bilayer lipid membranes showed that the mutant porin was voltage gated at much lower membrane potentials, 50 and 75 mV, than the wild-type sample. The closing events of the mutant porin were predominantly of monomer size. The channels detected by using the mutant protein were larger in size than those measured for the wild-type porin monomer. These data suggest that the OmpC mutant porin has a channel size capable of allowing maltodextrins to enter and that this channel is highly voltage regulated.     

Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell

Voltage-dependent anion-selective channels (VDACs) are pore-forming proteins allowing the permeability of the mitochondrial outer membrane. The VDAC3 isoform is the least abundant and least active in a complementation assay performed in a yeast strain devoid of porin-1. We swapped the VDAC3 N-terminal 20 amino acids with homologous sequences from the other isoforms. The substitution of the VDAC3 N-terminus with the VDAC1 N-terminus caused the chimaera to become more active than VDAC1. The VDAC2 N-terminus improved VDAC3 activity, though to a lesser extent. The VDAC3 carrying the VDAC1 N-terminus was able to complement the lack of the yeast porin in mitochondrial respiration and in modulation of reactive oxygen species (ROS). This chimaera increased life span, indicating a more efficient bioenergetic metabolism and/or a better protection from ROS.     

Extramitochondrial Porin: Facts and Hypotheses

Mitochondrial porin, or VDAC, is a pore-forming protein abundant in the outer mitochondrialmembrane. Several publications have reported extramitochondrial localizations as well, butthe evidence was considered insufficient by many, and the presence of porin in nonmitochondrialcellular compartments has remained in doubt for a long time. We have now obtained newdata indicating that the plasma membrane of hematopoietic cells contains porin, probablylocated mostly in caveolae or caveolae-like domains. Porin was purified from the plasmamembrane of intact cells by a procedure utilizing the membrane-impermeable labeling reagentNH-SS-biotin and streptavidin affinity chromatography, and shown to have the same propertiesas mitochondrial porin. A channel with properties similar to that of isolated VDAC wasobserved by patch-clamping intact cells. This review discusses the evidence supportingextramitochondrial localization, the putative identification of the plasma membrane porin with themaxi chloride channel, the hypothetical mechanisms of sorting porin to various cellularmembrane structures, and its possible functions.     

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.     

Purification and properties of the voltage-dependent anion channel of the outer mitochondrial membrane

The methods for the purification of functionally active mitochondrial porin or voltage-dependent anion channel of the outer mitochondrial membrane are critically evaluated. Two rapid and efficient methods are now available. Both make use of a hydroxyapatite/celite column as a single chromatographic step. However, in one method with long polar head-group detergents, porin passes through the column, whereas in the other method, with shorter polar headgroup detergents, porin is first bound to the column and then eluted by the addition of salts. On the basis of these results, a model for the arrangement of porin in the detergent-protein micelles is proposed.     

A new mechanism of antibiotic resistance in Enterobacteriaceae induced by a structural modification of the major porin

In Enterobacter aerogenes, multidrug resistance involves a decrease in outer membrane permeability associated with changes in an as yet uncharacterized porin. We purified the major porin from the wild-type strain and a resistant strain. We characterized this porin, which was found to be an OmpC/OmpF-like protein and analysed its pore-forming properties in lipid bilayers. The porin from the resistant strain was compared with the wild-type protein and we observed (i) that its single-channel conductance was 70% lower than that of the wild type; (ii) that it was three times more selective for cations; (iii) a lack of voltage sensitivity. These results indicate that the clinical strain is able to synthesize a modified porin that decreases the permeability of the outer membrane. Mass spectrometry experiments identified a G to D mutation in the putative loop 3 of the porin. Given the known importance of this loop in determining the pore properties of porins, we suggest that this mutation is responsible for the novel resistance mechanism developed by this clinical strain, with changes in porin channel function acting as a new bacterial strategy for controlling beta-lactam diffusion via porins.     

Protective immunity induced by porin in experimental mouse salmonellosis

The induction of protective immunity to mouse salmonellosis by porin from Salmonella typhimurium LT2 was studied. The immunization with porin induced a high level of protective immunity to salmonellosis in BALB/c mice. Mice immunized with porin exhibited significant levels of delayed-type hypersensitivity response and interleukin-2 production, indicating that porin was capable of inducing cell-mediated immunity (CMI). Furthermore, we found that both T cells and sera taken from the porin-immunized mice could transfer the protection against salmonellosis into nonimmunized mice. These observations suggested that a high level of the protection to salmonellosis obtained by the porin immunization resulted from the induction of CMI in addition to humoral immunity.     

Purification procedure and monoclonal antibodies: two instruments for research on vertebrate porins.

On Western blots of skeletal muscle preparations of different vertebrate classes, four monoclonal anti-human type 1 porin antibodies recognize one single band of either 30.5 or 31 kDa, respectively. To confirm that it is eukaryotic porin which is labeled by the antibodies, we used a purification procedure developed for human type 1 porin for porins from skeletal muscle of shark, frog, and turkey. Applied to different mammalian species and tissues, this procedure exclusively provides type 1 porin. However, applied to shark skeletal muscle, it provides two porin isotypes in nearly equal amounts. In the case of frog skeletal muscle, the procedure provides mainly type 2 porin and a lower amount of type 1 porin. Applied to turkey skeletal muscle, the method provides exclusively type 2 porin. As demonstrated by two-dimensional Western blots, both shark and frog porin isotypes and the turkey type 2 porin are recognized by our antibodies. Furthermore, we elucidated the entire amino acid sequence of frog type 2 porin.     

Identification and characterization of a new porin gene of Klebsiella pneumoniae: its role in beta-lactam antibiotic resistance

Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many beta-lactam-resistant strains. PCR and Southern blot analysis revealed the existence of a third porin gene in addition to the OmpK36 and OmpK35 porin genes previously described. This new porin gene was designated ompK37 and is present in all of the clinical isolates tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed in Escherichia coli. In contrast to that of the major porins, OmpK37 porin expression was only detectable by Western blot analysis in porin-deficient beta-lactam-resistant strains, suggesting strong down regulation under standard laboratory conditions. Functional characterization suggested a narrower pore for the OmpK37 porin than for K. pneumoniae porins OmpK36 and OmpK35. This correlated with the susceptibility to certain beta-lactam antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to beta-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.     

Determinants of OmpF porin antigenicity and structure.

Sixty-six murine hybridomas raised to Escherichia coli B/r porin were used to identify and differentiate the epitopes of this outer membrane protein. Anti-porin monoclonal antibodies (mAb) were raised against outer membrane fragments, purified native trimeric porin (trimer), and purified sodium dodecyl sulfate-denatured monomeric porin (monomer). Immunochemical and flow cytometric methods identified five distinct cell surface-exposed determinants on OmpF. The peptide composition of porin epitopes was determined by analysis of mAb reactivity with cyanogen bromide-generated peptide fragments. Four of 43 anti-monomer mAb reacted with surface exposed sites on OmpF, defining epitopes that consist of residues within CNBr peptides d2, d3, and B. The anti-porin mAb panel was also used to evaluate changes in porin antigenic structure in strains with short ompF deletions. Flow cytometric experiments indicated that despite changes in porin permeability, little if any alteration of surface epitopes occurred in these strains. Western immunoblot analysis of the mutant porins showed loss of reactivity with numerous mAb, which was caused by changes in three spatially distinct epitopes at residues 108-111, 118-123, and 124-129. Our findings indicate that in these ompF mutants the residues responsible for altering porin permeability are not exposed on the cell surface, but are buried within the tertiary structure of the protein. One of these regions, which is apparently involved in the determination of channel permeability characteristics, is conserved among 15 of 16 different porin molecules which were screened with the anti-OmpF mAb panel.     

Charged Residues in Surface-located Loops Influence Voltage Gating of Porin from Haemophilus influenzae Type b

Porin of Haemophilus influenzae type b (341 amino acids; M _r 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24+/−0.41 vs. 0.85+/−0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50–55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89–91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b. Received: 11 August 2000/Revised: 8 September 2000     

Transmembrane arrangement of mitochondrial porin or voltage-dependent anion channel (VDAC)

Porin or voltage-dependent anion-selective channel (VDAC) is the main protein responsible for the high permeability of the outer mitochondrial membrane. The mitochondrial porin is mainly composed of sided -strands, in analogy with bacterial porin, whose structure has been resolved at 1.8 Å resolution. In mitochondrial porins the N-terminal region forms an amphipathic -helix, a structure conserved in organisms very distant from the evolutionary point of view. This part of the protein is exposed to the water phase, as demonstrated by ELISA assays. Various extramembranous loops have been identified by specific proteolytic cleavages. These overall, combined results were used to draw a model of the transmembrane arrangement of mammalian porin. This model is compared to other mitochondrial and bacterial porin models.