Heart mitochondrial creatine kinase revisited: the outer mitochondrial membrane is not important for coupling of phosphocreatine production to oxidative phosphorylation

The state of mitochondrial creatine kinase (CKmi-mi) in intact dog heart mitochondria and mitoplasts and the mechanism of its functional coupling with the oxidative phosphorylation system have been reinvestigated under different osmotic conditions and ionic compositions of the medium. It has been established that in a medium which mimics the cardiac cell cytoplasma, dissociation of CKmi-mi from the membrane of mitoplasts increases when the mitoplasts are swollen due to hypoosmotic treatment. It was shown by EPR that hypoosmotic treatment results in the enhancement of the mobility of phospholipids in the membrane bilayer. It has been also shown that when CKmi-mi is detached from the inner membrane in intact mitochondria in isotonic KCl solution, the effects of the coupling between CKmi-mi and oxidative phosphorylation via ATP/ADP translocase disappear in spite of the presence of CKmi-mi in the intermembrane space and intactness of the outer mitochondrial membrane. Therefore, this coupling cannot be explained by the "compartmented coupling" mechanism or "dynamic adenine nucleotide compartmentation" in the intermembrane space due to diffusion limitation for adenine nucleotides through the outer mitochondrial membrane, as has been supposed by several authors (F.N. Gellerich et al. (1987) Biochim. Biophys. Acta 890, 117-126; S.P.J. Brooks and C.H. Suelter (1987) Arch. Biochem. Biophys. 253, 122-132). The data obtained show that the displacement of the enzyme from the membrane results in significantly increased sensitivity of the coupled processes of aerobic phosphocreatine synthesis to inhibition by the product, phosphocreatine. Thus, all results show that under physiological osmotic and ionic conditions CKmi-mi remains firmly attached to the inner mitochondrial membrane and effectively coupled with ATP/ADP translocase due to intimate dynamic interaction between those proteins.     

Association of chicken mitochondrial creatine kinase with the inner mitochondrial membrane

The stoichiometry and dissociation constant for the binding of homogeneous chicken heart mitochondrial creatine kinase (MiMi-CK) to mitoplasts was examined under a variety of conditions. Salts and substrates release MiMi-CK from mitoplasts in a manner that suggests an ionic interaction. The binding of MiMi-CK to mitoplasts is competitively inhibited by Adriamycin, suggesting that they compete for the same binding site. Fluorescence measurements also show that Adriamycin binds to MiMi-CK so that the effect of Adriamycin on the binding of MiMi-CK to mitoplasts is not simple. Titrating mitoplasts with homogeneous MiMi-CK at different pH values shows a pH-dependent equilibrium involving a group(s) on either the membrane or the enzyme with a pKa = 6. Extrapolating these titrations to infinite MiMi-CK concentration gives 14.6 IU bound/nmol cytochrome aa3 corresponding to 1.12 mol MiMi-CK/mol cytochrome aa3. Chicken heart mitochondria contain, after isolation, 2.86 +/- 0.42 IU/nmol cytochrome aa3. Titrating respiring mitoplasts with carboxyatractyloside gives at saturation 3.3 mol ADP/ATP translocase/mol cytochrome aa3. Therefore, chicken heart mitoplasts can maximally bind about 1 mol of MiMi-CK per 3 mol translocase; in normal chicken heart mitochondria about 1 mol of MiMi-CK is present per 13 mol translocase.     

Localization of the NADH kinase in the inner membrane of yeast mitochondria

The bulk of NADH kinase of Saccharomyces cerevisiae was recovered in the mitochondrial fraction prepared from spheroplasts. Most of the NADH kinase was localized in the inner membrane fraction, which was separated from other mitochondrial components by the combined swelling, shrinking, and sonication procedure. Treatment of mitoplasts with antiserum against the NADH kinase caused inactivation of the enzyme. On the contrary, no influence was observed upon the same treatment of intact mitochondria. p-Chloromercuribenzoate and eosin-5-maleimide inactivated the enzyme without affecting the matrix ATPase. The NADH kinase was enzymatically iodinated in mitoplasts, but not in the intact mitochondria. These results support the conclusion that NADH kinase is localized and functions at the intermembrane space side of the mitochondrial inner membrane. It is evident that the NADH kinase is encoded by nuclear gene(s) because it is synthesized in the presence of chloramphenicol or acriflavine, and a significant amount of the enzyme was detected in mitochondrial DNA-deficient mutants.     

The submitochondrial localization adenylate kinase: an enzymatic marker for the inner surface of the outer membrane of lung mitochondria in guinea pig

Guinea pig lung mitochondrial adenylate kinase activity was measured under isotonic and hypotonic conditions. The activity differed in sensitivity to trypsin. Under isotonic conditions, the enzyme resisted the action of trypsin, where as the enzyme was destroyed substantially by trypsin under hypotonic conditions.     

Palmitoylation is required for membrane association of activated but not inactive invertebrate visual Gqalpha

The invertebrate visual G protein, iGqα plays a central role in invertebrate phototransduction by relaying signals from rhodopsin to phospholipase C leading to membrane depolarization. Previous studies have shown reversible association of iGqα with rhabdomeric membranes regulated by light. To address the mechanism of membrane association we cloned iGqα from a Loligo pealei photoreceptor cDNA library and expressed it in HEK293T cells. Mutations were introduced to eliminate putative sites for palmitoylation at cysteines in positions 3 and 4. Membrane and soluble fractions were prepared from cells where iGqα was either activated or maintained in the GDP-bound form, followed by identification of iGqα through immunoblot analysis. The wild-type iGqα was entirely membrane-bound and shown to be post-translationally modified by palmitoylation. The mutant iGqα (C3,4A) was not palmitoylated yet it was found to be membrane-associated in the inactive state, however, approximately half of the protein became soluble when activated. These results suggest that palmitoylation is not required for membrane association of iGqα in the inactive state but is important in maintaining the stable membrane association of activated iGqα–GTP. The mechanism by which iGqα moves away from the membrane into the cytosol in response to prolonged light-stimulation in the native squid eye appears, therefore, to involve both activation and depalmitoylation processes.     

Myristoylation of flagellar creatine kinase in the sperm phosphocreatine shuttle is linked to its membrane association properties.

TCK, the flagellar creatine kinase (ATP:creatine N-phosphotransferase) of sperm from the sea urchin Strongylocentrotus purpuratus is a membrane-associated lipophilic protein involved in energy transport. The cDNA derived protein sequence contains a consensus site sufficient for the covalent attachment of myristate. To examine whether TCK was myristoylated, mouse fibroblast Swiss 3T3 and baby hamster kidney cell lines were transfected with a cDNA encoding the entire TCK protein linked to a metallothionein promotor. TCK expression was induced by zinc and paralleled by incorporation of [3H]myristic acid derived label into the protein. 3H Label incorporated into TCK was resistant to hydroxylamine treatment. The 3H-labeled material released from TCK by acid methanolysis eluted from a C18 reverse phase high pressure liquid chromatography column at the positions of myristic acid and methylmyristate. Thus, TCK expressed in transfected mammalian cell lines contains authentic myristic acid, covalently attached through amide linkage. [3H]Myristoyl TCK comigrated on two-dimensional gels with the purified lipophilic isoform TCK II from sea urchins. Furthermore, like TCK II, [3H]myristoyl TCK associated with phospholipid liposomes, suggesting that myristoylation may mediate the observed membrane association of TCK. Myristoylation of sea urchin sperm flagellar creatine kinase may play a role in confining this enzyme to the flagellum during spermatogenesis.     

Functional studies with the octameric and dimeric form of mitochondrial creatine kinase. Differential pH-dependent association of the two oligomeric forms with the inner mitochondrial membrane

Phosphate extraction of mitochondrial creatine kinase (Mi-CK, EC 2.7.3.2) from freshly isolated intact mitochondria of chicken cardiac muscle, after short swelling in hypotonic medium, yielded more than 90% of octameric and only small amounts of dimeric Mi-CK as judged by fast protein liquid chromatography-gel permeation analysis of the supernatants immediately after extraction of the enzyme. In extraction buffer, octameric Mi-CK displayed a tendency to dissociate, albeit at a slow rate with a half-life of approximately 3-5 days, into stable dimers. Experiments with purified Mi-CK octamers or dimers, or defined mixtures thereof, incubated under identical conditions with Mi-CK-depleted mitoplasts revealed that both oligomeric forms of Mi-CK can rebind to mitoplasts. However, the association of Mi-CK was strongly pH-dependent and, in addition, octameric and dimeric Mi-CK showed different pH dependences of rebinding. Therefore, it was possible under certain pH conditions to rebind either both oligomeric forms or selectively the octamers only. Furthermore, evidence is presented that Mi-CK dimers partially form octamers upon rebinding to the inner membrane. The differential association of the two oligomeric Mi-CK forms with the inner mitochondrial membrane together with the dynamic equilibrium between octameric and dimeric Mi-CK (Schlegel, J., Zurbriggen, B., Wegmann, G., Wyss, M., Eppenberger, H.M., and Wallimann, T. (1988) J. Biol. Chem., 263, 16942-16953) suggest that both oligomeric forms are physiologically relevant. A change in the octamer to dimer ratio may influence the association behavior of Mi-CK in general and thus modulate mitochondrial energy flux as discussed in the phosphoryl creatine circuit model (Wallimann, T., Schnyder, T., Schlegel, J., Wyss, M., Wegmann, G., Rossi, A.-M., Hemmer, W., Eppenberger, H.M., and Quest, A.F.G. (1989) Prog. Clin. Biol. Res. 315, 159-176.     

Osa protein encoded by plasmid pSa is located at the inner membrane but does not inhibit membrane association of VirB and VirD virulence proteins in Agrobacterium tumefaciens

Abstract The osa gene of IncW plasmid pSa encodes a 21-kDa protein that completely abolishes the oncogenic activity encoded by virulence genes in Agrobacterium tumefaciens. osa is the last gene of a four-gene operon in pSa, the expression of which appears to be highly regulated since the Osa protein is absent when either pSa or the osa operon is present in the Agrobacterium cell. When the osa gene alone or together with upstream genes within the operon are expressed under the control of a constitutive promoter, Osa protein is produced, enabling us to determine its subcellular location. Immunoblot analyses located Osa protein at the inner membrane of both A. tumefaciens and Escherichia coli . Because Osa inhibits oncogenicity of A. tumefaciens , and because alterations of the products of the virB and virD genes affect oncogenicity, studies were conducted to determine if there are changes in their specific association with the membranes in the presence Osa. Immunoblot analyses of VirB2, VirB3, VirB4, VirB9, and VirD4 in the presence and absence of Osa revealed no differences between the two treatments in these Vir protein associations with the membranes. These results indicate that both virB and virD gene products are produced in the presence of Osa; that they appear unaffected in their association with the membranes; and that Osa is associated with the inner membrane, where VirB2, VirB4, and VirD4 proteins are also located.     

Human preproparathyroid hormone synthesized in Escherichia coli is transported to the surface of the bacterial inner membrane but not processed to the mature hormone

cDNA encoding human preproPTH (hpreproPTH) was expressed in Escherichia coli to study the processing of the precursor to hPTH and its secretion by the bacterial secretory apparatus. We first constructed hybrid genes that differed randomly in the distance between the E. coli lac promoter's ribosomal binding site and DNA encoding a fusion protein with beta-galactosidase activity and the prepro sequence of hpreproPTH on the aminoterminus. Starting with clones identified as efficient producers of beta-galactosidase on indicator agar plates, the coding sequence for hpreproPTH was reconstituted intact. In a different construction we placed the hpreproPTH coding sequence downstream from the lac promoter at a distance of 12 base pairs from the ribosomal binding site. PTH immunoreactive proteins from multiple clones were identified by protein gel electrophoresis and by protein microsequencing. PTH-related proteins encoded by different plasmids were shown to be hpreproPTH with amino-terminal extensions of either two or four amino acids and as authentic hpreproPTH. Two hPTH fragments, hPTH(3-84) and hPTH(8-84), were also observed. The trypsin accessibility of hpreproPTH and of the two hPTH fragments in pulse-chase, cell-fractionation experiments using intact and lysed spheroplasts lets us conclude that the mammalian signal sequence directs hpreproPTH to the surface of the spheroplast membrane but is not appropriately cleaved by the signal peptidase.     

Glycosylation is important for cell surface expression of the water channel aquaporin-2 but is not essential for tetramerization in the endoplasmic reticulum

Aquaporin-2 (AQP2) is a pore-forming protein that is required for regulated reabsorption of water from urine. Mutations in AQP2 lead to nephrogenic diabetes insipidus, a disorder in which functional AQP2 is not expressed on the apical cell surface of kidney collecting duct principal cells. The mechanisms and pathways directing AQP2 from the endoplasmic reticulum to the Golgi complex and beyond have not been defined. We found that approximately 25% of newly synthesized AQP2 is glycosylated. Nonglycosylated and complex-glycosylated wild-type AQP2 are stable proteins with a half-life of 6-12 h and are both detectable on the cell surface. We show that AQP2 forms tetramers in the endoplasmic reticulum during or very early after synthesis and reaches the Golgi complex in 1-1.5 h. We also report that glycosylation is neither essential for tetramerization nor for transport from the endoplasmic reticulum to the Golgi complex. Instead, the N-linked glycan is important for exit from the Golgi complex and sorting of AQP2 to the plasma membrane. These results are important for understanding the molecular mechanisms responsible for the intracellular retention of AQP2 in nephrogenic diabetes insipidus.     

Role of quaternary structure in muscle creatine kinase stability: Tryptophan 210 is important for dimer cohesion

A mutant of the dimeric rabbit muscle creatine kinase (MM-CK) in which tryptophan 210 was replaced has been studied to assess the role of this residue in dimer cohesion and the importance of the dimeric state for the native enzyme stability. Wild-type protein equilibrium unfolding induced by guanidine hydrochloride occurs through intermediate states with formation of a molten globule and a premolten globule. Unlike the wild-type enzyme, the mutant inactivates at lower denaturant concentration and the loss of enzymatic activity is accompanied by the dissociation of the dimer into two apparently compact monomers. However, the Stokes radius of the monomer increases with denaturant concentration as determined by size exclusion chromatography, indicating that, upon monomerization, the protein structure is destabilized. Binding of 8-anilinonaphthalene-1-sulfonate shows that the dissociated monomer exposes hydrophobic patches at its surface, suggesting that it could be a molten globule. At higher denaturant concentrations, both wild-type and mutant follow similar denaturation pathways with formation of a premolten globule around 1.5-M guanidine, indicating that tryptophan 210 does not contribute to a large extent to the monomer conformational stability, which may be ensured in the dimeric state through quaternary interactions. Proteins 32:43–51, 1998. © 1998 Wiley-Liss, Inc.     

Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria

Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O₂ min^?1mg^?1, which was reduced to 98 nmol O₂ min^?1 (mg mitochondrial protein)^?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O₂ min^?1mg^?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent K_m of 7 μM for NADH, a V_max of 80 nmol NADH min^?1mg^?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a K_m of 80 μM and a V_max of 131 nmol NADH min^?1mg^?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria.     

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1‐Δglf strain did not produce the galactofuranose‐containing glycans as shown by immunoblots and immuno‐electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also ‘leaking’ as revealed by a β‐galactosidase‐based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose‐containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.     

Tic40 is important for reinsertion of proteins from the chloroplast stroma into the inner membrane

Chloroplast inner-membrane proteins Tic40 and Tic110 are first imported from the cytosol into the chloroplast stroma, and subsequently reinserted from the stroma into the inner membrane. However, the mechanism of reinsertion remains unclear. Here we show that Tic40 itself is involved in this reinsertion process. When precursors of either Tic40 or a Tic110 C-terminal truncate, tpTic110-Tic110N, were imported into chloroplasts isolated from a tic40-null mutant, soluble Tic40 and Tic110N intermediates accumulated in the stroma of tic40-mutant chloroplasts, due to a slower rate of reinsertion. We further show that a larger quantity of soluble Tic21 intermediates also accumulated in the stroma of tic40-mutant chloroplasts. In contrast, inner-membrane insertion of the triose-phosphate/phosphate translocator was not affected by the tic40 mutation. Our data suggest that multiple pathways exist for the insertion of chloroplast inner-membrane proteins.     

The carboxyl-terminal third of the dicarboxylate carrier is crucial for productive association with the inner membrane twin-pore translocase

The carrier proteins of the mitochondrial inner membrane consist of three structurally related tandem repeats (modules). Several different, and in some cases contradictory, views exist on the role individual modules play in carrier transport across the mitochondrial membranes and how they promote protein insertion into the inner membrane. Thus, by use of specific translocation intermediates, we performed a detailed analysis of carrier biogenesis and assessed the physical association of carrier modules with the inner membrane translocation machinery. Here we have reported that each module of the dicarboxylate carrier contains sufficient targeting information for its transport across the outer mitochondrial membrane. The carboxyl-terminal module possesses major targeting information to facilitate the direct binding of the carrier protein to the inner membrane twin-pore translocase and subsequent insertion into the inner membrane in a membrane potential-dependent manner. We concluded that, in this case, a single structural repeat can drive inner membrane insertion, whereas all three related units contribute targeting information for outer membrane translocation.     

Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria

The immunosuppressive peptide cyclosporin A is a powerful inhibitor of the Ca2+-dependent permeability transition in rat liver mitochondria. When swelling is used to monitor the transition, the inhibitor is effective regardless of whether N-ethylmaleimide, Hg2+, WY-14643, t-butyl hydroperoxide, oxalacetate, rhein, phosphate, phosphoenolpyruvate, or ruthenium red plus uncoupler is used as the inducing agent. Twenty-five to fifty pmol/mg protein of cyclosporin A reduces the swelling response by 50% with complete inhibition obtained at about 150 pmol/mg protein. The compound, which does not inhibit Ca2+ uptake or mitochondrial phospholipase A2, is effective when added before or after the transition promoting agent. These findings, together with the shape of the inhibition dose-response curve, suggest that cyclosporin A essentially titrates a mitochondrial component which is present at 80-90 pmol/mg protein. It is proposed that this component is a solute unselective, regulated pore or a factor involved in controlling such a structure.     

Evidence for the existence of an inner membrane anion channel in mitochondria

Mitochondria normally exhibit very low electrophoretic permeabilities to physiologically important anions such as chloride, bicarbonate, phosphate, succinate, citrate, etc. Nevertheless, considerable evidence has accumulated which suggests that heart and liver mitochondria contain a specific anion-conducting channel. In this review, a postulated inner membrane anion channel is discussed in the context of other known pathways for anion transport in mitochondria. This anion channel exhibits the following properties. It is anion-selective and inhibited physiologically by protons and magnesium ions. It is inhibited reversibly by quinine and irreversibly by dicyclohexylcarbodiimide. We propose that the inner membrane anion channel is formed by inner membrane proteins and that this pathway is normally latent due to regulation by matrix Mg2+. The physiological role of the anion channel is unknown; however, this pathway is well designed to enable mitochondria to restore their normal volume following pathological swelling. In addition, the inner membrane anion channel provides a potential futile cycle for regulated non-shivering thermogenesis and may be important in controlled energy dissipation.     

Phosphorylation by cyclic AMP-dependent protein kinase does not affect the association of ATP citrate-lyase with isolated mitochondria

ATP citrate-lyase is known to be a substrate for various protein kinases, but the functional role, if any, of kinase-directed phosphorylation of this enzyme has not been identified. Recently, Strålfors [(1987) J. Biol. Chem. 262, 11486–11489] has suggested that effects on the association of this enzyme with mitochondria may account for the observed ability of isoproteronol or insulin to promote immobilization of ATP citrate-lyase in permeabilized cells. Here we report studies involving phosphorylation of the pure enzyme in vitro using cyclic AMP-dependent protein kinase. We show that phosphorylation has no significant effect on the fraction of the enzyme that may be bound to isolated mitochondria.