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M Klingenberg

Publications and source records attributed to M Klingenberg.

At least 55 records · Page 3Linked to original sources
PubMed1994

The reconstituted ADP/ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine mutants.

Although the site-directed C73S mutation in the ADP/ATP carrier (AAC) AAC2 gene from Saccharomyces cerevisiae produced a glycerol-positive strain, indicating that the mutant AAC is active, on isolation and reconstitution in egg yolk phosphatidylcholine, the C73S AAC had no transport activity, whereas the wild-type AAC was fully active. Only on addition of cardiolipin was an exchange activity with the C73S AAC obtained. The AACs isolated from the other cysteine mutants did not (C244S) or only marginally (C271S) require cardiolipin for transport on reconstitution. [3H]Carboxyatractylate binding as a measure of incorporated AAC molecules was unchanged on addition of cardiolipin in all mutants, indicating that cardiolipin does not increase the incorporation of the AAC. It also shows that cardiolipin is required only for translocation and not for binding. The activity of the C73S mutant AAC shows half-saturation with cardiolipin at 2% by weight or at 1.15 mol % in the phosphatidylcholine vesicles. Other acidic phospholipids tested such as phosphatidylserine and phosphatidic acid did not activate. Among various cardiolipin derivatives, the selectivity for cardiolipin is high. Only monolysocardiolipin still retains 12% activity. After removal of the bulk of phospholipid, the content of bound phospholipids was assayed by 31P NMR. By unmasking with SDS, in the wild-type AAC and in the C73S AAC, 6.4 mol and only 1.3 and 2.9 mol of bound cardiolipin/mol of AAC dimer are found, respectively. Presumably, on isolation, cardiolipin is lost from the more labile C73S mutant AAC. Although the absolute requirement for cardiolipin is unique for the C73S AAC, it is concluded that in this mutant, the unmasking of the cardiolipin requirement demonstrates a general cardiolipin requirement of the wild-type AAC and of AACs from other sources.

Adenosine Diphosphate↗
PubMed1993

Site-directed mutagenesis of the yeast mitochondrial ADP/ATP translocator. Six arginines and one lysine are essential.

The ADP/ATP translocator mediates adenine nucleotide exchange across the inner mitochondrial membrane. ADP/ATP exchange is essential when yeast are grown on a non-fermentable carbon source such as glycerol, but it is not required for growth on glucose. Failure to grow on glycerol is therefore a phenotypic indicator of protein function, and it has been used here to screen site-directed mutants to identify functionally important amino acids in the yeast adenine nucleotide translocator (AAC2). Single mutations of all four charged amino acids in the transmembrane segments of AAC2 (K38A, R96D, R96H, R96L, R96P, R204L, R294A) resulted in loss of function, as did mutations in the matrix arginine cluster (R252I, R253I, R254I). Seven other residues were mutated without affecting growth on glycerol (C73S, C244S, C271S, K179M, K182I, P247G, W235F). The non-functional mutants have been used to select intragenic suppressors to gain further insight into the structure of this membrane transport protein.

Amino Acid Sequence↗
PubMed1993

Dialectics in carrier research: the ADP/ATP carrier and the uncoupling protein.

A concise review is given of the research in our laboratory on the ADP/ATP carrier (AAC) and the uncoupling protein (UCP). Although homologous proteins, their widely different functions and contrasts are stressed. The pioneer role of research on the AAC, not only for the mitochondrial but also for other carriers, and the present state of their structure-function relationship is reviewed. The function of UCP as a highly regulated H+ carrier is described in contrast to the largely unregulated ADP/ATP exchange in AAC. General principles of carrier catalysis as derived from studies on the AAC and UCP are elucidated.

Adenosine Diphosphate↗
PubMed1992

Labeling of two different regions of the nucleotide binding site of the uncoupling protein from brown adipose tissue mitochondria with two ATP analogs.

The nucleotide binding site of the uncoupling protein (UCP) from brown adipose tissue was mapped by photoaffinity labeling with 2-azidoadenosine 5'-triphosphate (2-azido-ATP) and by affinity labeling with 3'-O-(5-fluoro-2,4-dinitrophenyl)adenosine 5'-triphosphate (FDNP-ATP). Both analogs bind with high affinity and specificity to the UCP in intact mitochondria, as well as to the isolated solubilized protein. Reversible binding at 4 degrees C in the dark is competitively blocked by GTP. Like the natural ligands ATP and GTP, both analogs are capable of inhibiting the H+/OH- conductance of the UCP as measured in proteoliposomes with reconstituted UCP. 2-azido-ATP was incorporated into UCP in mitochondria in the presence of carboxyatractylate, while FDNP-ATP was inserted into isolated UCP by prolonged incubation at room temperature under pH variation. Both reactions can be blocked by GTP. The incorporation of 2-azido-ATP could be localized between residues 258 and 283 by cleavage with CNBr. Solid-phase sequencing of the homoserine-linked radioactive peptide indicated that the 2-azido-ATP was linked to threonine-263. The incorporation of FDNP-ATP could be assigned by cleavage with CNBr and alternatively with trypsin at a locus of covalent attachment between residues 238 and 255. On the basis of published data that no tyrosine participates in nucleotide binding of the UCP, the probable residue reacting with FDNP-ATP is cysteine-253.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

An improved procedure for reconstitution of the uncoupling protein and in-depth analysis of H+/OH- transport.

An improved procedure for reincorporation of isolated uncoupling protein (UCP) from brown adipose tissue into phospholipid vesicles is reported and H+ uptake in K(+)-driven exchange diffusion quantitatively analyzed. UCP is isolated and reconstituted with medium-length linear-chain alkyl polyoxyethylene. In the critical step of vesicle formation, the stepwise removal of the detergent by polystyrene beads is applied. Vesicles are generated in the presence of solutes and buffers to be internalized which are then removed by gel filtration. The internal volume is about 4 microliters/mg phospholipid with a vesicle diameter of 100 nm. One vesicle contains, on average, six molecules UCP. The best results are obtained with purified egg yolk phosphatidylcholine. Addition of PtdEtn, PtdSer decreases the vesicle size and, still more, H(+)-transport activity by UCP. Asolectin completely inactivates UCP. K(+)-gradient-driven H+ uptake is 80% inhibited by external GTP and 95% by internal plus external GTP. When H+ transport is recorded externally by a pH electrode and internally by pyranine, the kinetics show no delay resulting from intervening membrane-bound H+ pools. Total H+ uptake after addition of carbonylcyanine m-chlorophenylhydrazone (CCCP) and valinomycin corresponds to the diffusion between H+ and K+ and is unchanged by GTP. The linear correlation of H(+)-transport inhibition to GTP binding demonstrates that all UCP molecules incorporated are equally active. The exchange diffusion between H+ uptake and K+ efflux is demonstrated using a K+ electrode and 86Rb measurements. Recording delta psi using 3,3'-diispropylthiadicarbocyanine shows a rapid generation of delta psi on valinomycin addition, which decreases only slightly with H+ uptake, even after addition of CCCP or gramicidin. The delta psi collapses only after addition of external K+. By demonstrating that valinomycin-induced K+ and H+ fluxes reflect relaxation into the diffusion equilibrium state, the transport rate of UCP can be evaluated as a first-order rate, VH+/CH+, in which the rate, VH+, is related to H(+)-uptake capacity, CH+. This allows quantitative comparison of transport rates independently of the variable CH+. The dependence on delta psi of H+ transport is measured by varying external K+ concentration. A virtually linear relation of the rate to the K(+)-diffusion potential is observed, although the capacity is only slightly changed. The linear VH+/delta psi relationship resembles an open-channel type of transport, but is discussed in terms of a low-activation-barrier type of carrier mechanism, in contrast to the log (VH+/delta psi) relation found for the ADP/ATP carrier with high activation barriers.

Acclimatization↗
PubMed1992

Photoaffinity labeling of the nucleotide-binding site of the uncoupling protein from hamster brown adipose tissue.

The nucleotide binding center of the uncoupling protein from brown adipose tissue (UCP) was probed by photoaffinity labeling with 8-azido-ATP. The isolated dimeric UCP in non-ionic detergent was used. 8-azido-ATP binds to UCP with a Kd = 3 microM, i.e. with an only threefold lower affinity than ATP and a maximum number of binding sites of about 12 mumol/g protein corresponding to about 1 mol/mol dimer UCP. UCP is rapidly degraded by ultraviolet radiation, and therefore only near ultraviolet and visible light can be used for photoaffinity labeling. The total covalent incorporation is shown to be dependent on the concentration of azido-ATP and on competing phospholipids. The specific, i.e. ATP-sensitive incorporation only to the binding site depends on the presence of cysteine. With CNBr cleavage the 8-azido-[gamma-32P]ATP insertion within the primary structure was located by identifying ATP-sensitive labeled peptides in SDS/PAGE. A major specific 8-azido-ATP incorporation was found by autoradiography in the smallest CNBr fragments. Identification of the radioactive peptides was difficult since 8-azido-ATP insertion causes a distinct shift in the gels from the stained peptides. Identification was possible by specific disulfide formation at the C-terminal within the UCP dimer which only removed the CB7 (CB, CNBr fragment) portion of the low-molecular-mass peptides but did not move the radioactive band. This excludes the C-terminal CB7 and identifies the labeled peptide as CB6. Also, limited tryptic cleavage of intact UCP at Lys293 did not remove the radioactivity. Cleavage of tryptophanes support localization of 8-azido-ATP between residues 173-280 which includes CB6. Solid-phase sequencing of the labeled CB6 both after serine lactone and carboxyl coupling suggest incorporation into Thr260. These results indicate that the adenine-binding site is within the third domain of the tripartite UCP structure at a putative hydrophilic channel which can be assessed both from the cytosol and matrix of mitochondria.

Adenosine Triphosphate↗
PubMed1991

Molecular species of cardiolipin in relation to other mitochondrial phospholipids. Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier?

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment.

Animals↗
PubMed1990

Lipid-protein interactions in ADP-ATP carrier/egg phosphatidylcholine recombinants studied by spin-label ESR spectroscopy.

The stoichiometry and specificity of lipid-protein interaction, as well as the lipid exchange rates at the protein interface, have been determined from the electron spin resonance spectra of spin-labeled lipids in reconstituted complexes of the mitochondrial ADP-ATP carrier with egg phosphatidylcholine. With the exception of cardiolipin and phosphatidic acid, the lipids studied are found to compete for approximately 50 sites at the intramembranous surface of the protein dimer. This number of first-shell lipid sites is unusually large for a protein of this size. The specificity for the protein is in the order stearic acid approximately phosphatidic acid approximately cardiolipin greater than phosphatidylserine greater than phosphatidylglycerol approximately phosphatidylcholine, with the maximum association constant relative to phosphatidylcholine being approximately 4. The selectivity for anionic lipids was partially screened with increasing ionic strength, but to a lesser extent for cardiolipin and phosphatidic acid than for stearic acid. Only in the case of phosphatidylserine was the selectivity reduced at high ionic strength to a level close to that for phosphatidylcholine. The off rates for lipid exchange at the protein surface were independent of lipid/protein ratio and correlated in a reciprocal fashion with the different lipid selectivities, varying from 5 x 10(6) s-1 for stearic acid at low ionic strength to 2 x 10(7) s-1 for phosphatidylcholine and phosphatidylglycerol. The off rates for cardiolipin were unusually low in comparison with the observed selectivity, and indicated the existence of a special population of sites (ca. 30% of the total) for cardiolipin, at which the exchange rate was very low.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Structure-function studies of adenine nucleotide transport in mitochondria. I. Construction and genetic analysis of yeast mutants encoding the ADP/ATP carrier protein of mitochondria.

The gene encoding the major ADP/ATP carrier in yeast AAC2 (pet9; Lawson, J., and Douglas, M. (1988) J. Biol. Chem. 263, 14812-14818) has been disrupted (delta AAC2) by itself and in combination with a disruption of a second translocator gene AAC1 (delta AAC1). Disruption of AAC2 like the pet9 mutation renders yeast unable to grow on a nonfermentable carbon source. The AAC1 AAC2 double disruption exhibits a phenotype identical to the AAC2. This provides the host strain for the analysis of point mutations in the AAC protein. We have initiated this structure-function analysis by characterizing and confirming that the pet9 mutation is a G to A transition resulting in an arginine to histidine change at position 96. Site-directed replacements at Arg96 confirm its essential function for growth on a nonfermentable carbon source. These data also suggest that in the absence of functional AAC1 and AAC2 gene products, adenine nucleotide transport across the mitochondrial inner membrane must occur by an as yet unidentified translocator or translocation mechanism or that within these cells separate intra- and extramitochondrial adenine nucleotide pools can exist to support growth.

Amino Acid Sequence↗
PubMed1990

Structure-function studies of adenine nucleotide transport in mitochondria. II. Biochemical analysis of distinct AAC1 and AAC2 proteins in yeast.

AAC1 and AAC2 genes in yeast each encode functional ADP/ATP carrier (AAC) proteins of the mitochondrial inner membrane. In the present study, mitochondria harboring distinct AAC proteins and the pet9 Arg96 to HIS mutant (Lawson, J., Gawaz, M., Klingenberg, M., and Douglas, M. G. (1990) J. Biol. Chem. 265, 14195-14201) protein have been characterized. In addition, properties of the different AAC proteins have been defined following reconstitution into proteoliposomes. Deletion of AAC2 but not AAC1 causes a major reduction in the mitochondrial cytochrome content and respiration, and this level remains low even when the level of AAC1 protein is increased to 20% that of the AAC2 gene product. In reconstitution studies, the rate of nucleotide transport by isolated AAC1 protein is approximately 40% that of the AAC2 protein. Thus, the lack of mitochondrial-dependent growth supported by the AAC1 gene product alone may be due to the combination of low abundance and reduced activity. Surprisingly, analysis of the Arg96 to His mutant protein revealed binding and transport activities similar to the functional AAC1 and AAC2 gene products. These observations are discussed in relation to a molecular analysis of this highly conserved small transporter and its function in conjunction with other proteins in the mitochondrial membrane.

Adenosine Triphosphate↗
PubMed1990

Mechanism and evolution of the uncoupling protein of brown adipose tissue.

The uncoupling protein found in mitochondria from thermogenic brown adipose tissue is structurally very similar to two other mitochondrial carrier proteins transporting ADP/ATP and phosphate, respectively. Similarities are also seen with the mechanism of these carriers, which are part of a family of H+/OH(-)-substrate anion co-transporters, further strengthening the evidence that the uncoupling protein has evolved from this family of mitochondrial carrier proteins.

Adipose Tissue, Brown↗
PubMed1989

The uncoupling protein dimer can form a disulfide cross-link between the mobile C-terminal SH groups.

Isolated uncoupling protein (UCP) can be cross-linked, by various disulfide-forming reagents, to dimers. The best cross-linking is achieved with Cu2+-phenanthroline oxidation. Because cross-linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross-linking is prevented by SH reagent and reversed by SH-reducing reagents. In mitochondria, cross-linking of UCP with disulfide-forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide-bridge formation does not inhibit GTP-binding to UCP. Cross-linked UCP re-incorporated in proteoliposomes either before or after cross-linking fully retains the H1-transport function. Rapid cross-linking by membrane impermeant reagents indicates a surface localization of the C-terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide-bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross-linked UCP was cleaved by BrCN. The CB-7 C-terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys-292, removed cross-linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C-terminal peptide of 11 residues contains only cystein-304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S-S bridge formation. Obviously, the C-terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross-linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane.

Animals↗
PubMed1989

The ADP/ATP carrier from yeast (AAC-2) is uniquely suited for the assignment of the binding center by photoaffinity labeling.

The ADP/ATP carrier from yeast was photoaffinity-labeled in mitochondria with 2-azido-[alpha-32P]ATP in a binding-center-specific, i.e. carboxyatractylate-sensitive, manner. After isolation, fragmentation possibilities unique for the yeast AAC-2 could be exploited to assign the insertion to a narrow range of the sequence. The CNBr fragment 115-210 contained all the incorporated label which corresponds to the second domain within the triple-domain primary structure of the AAC. With hydroxylamine cleavage directed to the Asn 171-Gly 172 site, all the label was found in the C-terminal 16 kDa fragment. Thus the 2-azido-ATP incorporation is clearly delimited to the 172-210 segment. 8-Azido-[alpha-32P]ATP could be site-specifically incorporated only in isolated AAC since it has a much lower affinity for AAC than 2-azido-ATP. The label was also exclusively found in the 172-210 region. With both forms no incorporation into the C-terminal region was found, as claimed for bovine AAC. The labeled segment contains Lys 179 and 182 which are homologous to bovine Lys 162 and 165 and which have been proposed to be in the translocation path.

Adenosine Triphosphate↗
PubMed1989

The isolation and reconstitution of the ADP/ATP carrier from wild-type Saccharomyces cerevisiae. Identification of primarily one type (AAC-2).

Methods for isolation of the ADP/ATP carrier (AAC) from yeast (Saccharomyces cerevisiae) are described which allow separation of the carrier from the initially copurified porin which poses a specific problem in yeast. The procedure varies according to whether one wishes to obtain a stable CAT-AAC complex, the free and active AAC for reconstitution, or the SDS-denatured pure AAC peptide. CNBr cleavage of AAC enabled us to differentiate clearly between isogenes AAC-1 and AAC-2 recently found in yeast, due to the exclusive occurrence of a methionine (M-115) residue at the end of the first domain in AAC-2. Thus the AAC isolated from wild-type yeast is primarily or exclusively AAC-2. The isolated AAC is active in ADP/ATP exchange in reconstituted liposomes with a Vmax of 1100 mumol/min per g protein and Km = 15 microM for ADP, and a Vmax of 900 mumol/min per g protein and Km = 9 microM for ATP.

Atractyloside↗
PubMed1989

Rotational diffusion of mitochondrial ADP/ATP carrier studied by saturation-transfer electron spin resonance.

The rotational mobility of the mitochondrial ADP/ATP carrier has been studied solubilized in Triton micelles, reincorporated in phospholipid liposomes, and in mitochondria. Spin-labeled analogues of the noncovalent inhibitors carboxyatractyloside and atractyloside were found to be strongly immobilized when bound to the carrier [Munding, A., Beyer, K., & Klingenberg, M. (1983) Biochemistry 22, 1941-1947], such that saturation-transfer electron spin resonance spectroscopy could be used to study the rotational motion of the protein. Spin-labeled maleimide covalently bound to the carrier was found to have independent segmental motion and hence to be unsuitable for studies of protein rotation. The ESR spectra of the carboxyatractyloside and atractyloside spin labels were found to contain a second component from label in the lipid or detergent, necessitating the use of the saturation-transfer ESR integral method [Horváth, L. I., & Marsh, D. (1983) J. Magn. Reson. 54, 363-373] to analyze the rotational motion of the label component bound to the protein. Effective rotational correlation times obtained from integration of the high-field region of the spectrum were lower than those obtained from the total spectral integral, indicating strongly anisotropic rotational diffusion of the carrier in the vesicular and membrane systems, with the spin-label z axis oriented preferentially perpendicular to the rotation axis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Molecular aspects of the adenine nucleotide carrier from mitochondria.

The ADP/ATP carrier (AAC) of mitochondria is a functionally central and characteristic component of the eukaryotic cell. By linking the thermodynamically divergent metabolites in the intra- and extramitochondrial compartments, it had to evolve with the emergence of the eukaryotic cell. Because of a number of unique properties, the AAC provided advanced insight into the molecular basis of solute transport through biomembrane carriers. With highly specific and unusually large substrates, ADP and ATP, and with high-affinity inhibitors binding selectively either from the inside or the outside, the first molecular demonstration of the single-binding-center gated pore mechanism was made. This framework can only partially be interpreted with the available yet rapidly increasing structural information on the AAC. The primary structure, first established for the AAC from beef heart mitochondria, showed a relatively wide distribution of hydrophilic residues which permits assignment of only two hydrophobic transmembrane stretches. However, a striking tripartition of the primary structure into about three 100-residue-long domains allows a more significant assignment of transmembrane elements. With alignment of these three domains for maximum conservation of structurally critical residues, each domain can be assigned to have two transmembrane alpha elements between 18 and 22 residues long. The interdomain homology between these alpha regions is low. The central regions flanked by these helices contain most of the polar residues and are significantly interdomain conserved. With lysine probes the central regions are assigned to the matrix side (m-side) and the two connecting regions as well as C and N termini to the cytosolic side (c-side). Out of the central regions a loop is assumed to protrude through the membrane, probably for lining the translocation channel. This localization of a major protein mass within the membrane agrees with hydrodynamic evidence, the carrier being an oblate ellipsoid with only about 50 A along the short axis. In accordance, the loops of domains 2 and 3 are affinity labeled by azido-ADP or azido-atractylate. Primary structures of AAC from other sources (fungi, plants) also exhibit the tripartition. The interdomain conserved residues are also interspecies conserved, thus showing that they are essential. These repeat domains have probably evolved from a common gene coding for about 100 residues. Isoforms of the AAC exist, as shown by primary structure analysis of human cDNA libraries from different organs. Three different isoforms are identified in human organs.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗