Distance estimate of the active center of D-beta-hydroxybutyrate dehydrogenase from the membrane surface

D-beta-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) is a membrane-bound, lipid-requiring enzyme which has a reactive sulfhydryl in the vicinity of the active center. The spin-probe-spin-label technique has been used to estimate the distance of separation of the reactive sulfhydryl of D-beta-hydroxybutyrate dehydrogenase from the bilayer surface. The reactive sulfhydryl of the enzyme was derivatized with the maleimide spin-label reagent 4-maleimido-2,2,6,6-tetramethylpiperidinyl-1-oxy in the presence of the cofactor NAD+. The derivatized enzyme, inserted (inlaid orientation) into phospholipid vesicles, was titrated with spin probes, either Mn2+ or Gd3+, until the spin-label EPR spectrum was reduced in amplitude to its residual (limiting) value. From this limiting amplitude, the dipolar interaction coefficient was obtained, which is related to the reciprocal of the distance to the sixth power. The radial distances of closest approach of the paramagnetic Mn2+ and Gd3+ ions to the spin-label nitroxide on the enzyme were found to be 18 and 16 A, respectively. These calculated distances were in accord with those determined by comparison with a phosphatidylcholine calibration system having 2,2-dimethyloxazolidinyl-1-oxy spin-labels located at selected positions along the sn-2 fatty acyl chain. Since the distal nitroxide moiety of the maleimide spin-label (17 A from the bilayer surface) is 8 A from the sulfhydryl addition site, the two limiting distances of immersion of the reactive sulfhydryl within the bilayer are 9 and 25 A. The shorter distance is considered more compatible with facile access of the coenzyme to the active site of the enzyme.     

Reversible association of ox liver glutamate dehydrogenase with the inner mitochondrial membrane

A comparative study on the catalytic and allosteric properties of particulate and soluble forms of ox liver glutamate dehydrogenase has been carried out. The response of the bound enzyme to release by various effectors was investigated. The particulate enzyme was found to have catalytic activities similar to the free enzyme in contrast to its behaviour when bound to pure anionic phospholipids. Possible reasons for such outstanding differences are discussed.     

The organization of NADH dehydrogenase polypeptides in the inner mitochondrial membrane.

The organization of the constituent polypeptides of mitochondrial NADH dehydrogenase was studied by using two membrane-impermeable probes, diazobenzene[35S]sulphonate and lactoperoxidase-catalysed radioiodination. The incorporation of label into the subunits of the isolated enzyme was compared with that obtained with enzyme immunoprecipitated from labelled mitochondria or inverted submitochondrial particles. On the basis of accessibility to these two labels, we divide the polypeptides of Complex I into five groups: those that are apparently buried in the enzyme, those that are accessible to labelling in the isolated enzyme but not in the membrane, those that are exposed on the cytoplasmic face of the membrane, those that are exposed on the matrix face and finally those that are exposed on both faces and are therefore transmembranous. We conclude that NADH dehydrogenase is asymmetrically organized across the inner mitochondrial membrane.     

Lipid-protein interactions in biomembranes studied through the phospholipid specificity of D-beta-hydroxybutyrate dehydrogenase

Since the biological membranes are fundamental units in the living cells, the studies of lipid-protein interactions are crucial for the understanding of their structure, functions and properties. Beside hydrophobic interactions between fatty acids chain of phospholipids and intrinsic membrane proteins, the interactions between charged groups of the protein with the polar heads of phospholipids generally confer the specificity which may be absolute or preferential. This paper reports essential results obtained these last few years with D-beta-hydroxybutyrate dehydrogenase (BDH) from inner mitochondrial membrane, one of the most interesting and best documented examples of a lipid-requiring enzyme. This is a review of the molecular basis--knowledge and strategy of study--of the lipid specificity for membrane protein functions.     

Dual specificity phosphatases 18 and 21 target to opposing sides of the mitochondrial inner membrane

Although large-scale approaches have identified numerous mitochondrial phosphoproteins, little is known about the mitochondrial kinases and phosphatases that regulate these phosphoproteins. Here, we identify two members of the atypical dual specificity phosphatases (DSP), DSP18 and DSP21, that are localized in mitochondria. Although DSP18 is widely expressed in several mammalian tissues, DSP21 is selectively expressed in the testes. We demonstrate that DSP18 and DSP21 are targeted to mitochondria by cryptic internal localization signals. Subfractionation of mitochondria demonstrated that DSP18 is located in the intermembrane space as a peripheral membrane protein of the inner membrane. In contrast, subfractionation of rat testis mitochondria revealed DSP21 is localized to the matrix as a peripheral membrane protein of the inner membrane. Moreover, we demonstrate that a previously reported substrate for DSP18, the stress-activated protein kinase, does not localize to mitochondria in several different tissues, making it an unlikely substrate for DSP18. Finally, we show that induction of apoptosis by treatment with staurosporine causes translocation of DSP18 from the intermembrane space into the cytosol similar to other apoptogenic factors such as cytochrome c. This work rigorously demonstrates the unique location of two highly similar DSPs on opposing sides of the mitochondrial inner membrane.     

Electrophysiology of the inner mitochondrial membrane

The application of electrophysiological techniques to mitochondrial membranes has allowed the observation and partial characterization of several ion channels, including an ATP-sensitive K⁺-selective one, a high-conductance megachannel, a 107 pS anionic channel and three others studied at alkaline pH's. A reliable correlation with the results of non-electrophysiological studies has been obtained so far only for the first two cases. Activities presumed to be associated with the Ca²⁺ uniporter and with the adenine nucleotide translocator, as well as the presence of various other conductances have also been reported. The review summarizes the main properties of these pores and their possible relationship to permeation pathways identified in biochemical studies.     

Involvement of tyrosyl residues in the structure-function relationships of D-beta-hydroxybutyrate dehydrogenase: a phospholipid-requiring enzyme

The involvement of tyrosyl residues in the function of D-beta-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme, has been investigated by using several tyrosyl modifying reagents, i.e., N-acetylimidazole, a hydrophilic reagent, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and tetranitromethane, two hydrophobic reagents. Modification of the tyrosyl residues highly inactivates the derived enzyme: Treatment of the enzyme with 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole leads to an absorbance at 380 nm and to an incorporation of about 1 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole per polypeptide chain for complete inactivation. Inactivation by N-acetylimidazole induces a decrease in absorbance at 280 nm which can be reversed by hydroxylamine treatment. On the other hand, the ligands of the active site, such as methylmalonate, a pseudosubstrate, and NAD+ (or NADH), do not protect the enzyme against inactivation. In contrast, the presence of phospholipids strongly protects the enzyme against hydrophobic reagents. Finally, previous modification of the enzyme with N-acetylimidazole does not affect the incorporation of 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole while modification with tetranitromethane does. These results indicate the existence of two classes of tyrosyl residues which are essential for enzymatic activity, and demonstrate their location outside of the active site. One of these residues appears to be located close to the enzyme-phospholipid interacting sites. These essential residues may also be essential for maintenance of the correct active conformation.     

Dynamic subcompartmentalization of the mitochondrial inner membrane

The inner membrane of mitochondria is organized in two morphologically distinct domains, the inner boundary membrane (IBM) and the cristae membrane (CM), which are connected by narrow, tubular cristae junctions. The protein composition of these domains, their dynamics, and their biogenesis and maintenance are poorly understood at the molecular level. We have used quantitative immunoelectron microscopy to determine the distribution of a collection of representative proteins in yeast mitochondria belonging to seven major processes: oxidative phosphorylation, protein translocation, metabolite exchange, mitochondrial morphology, protein translation, iron-sulfur biogenesis, and protein degradation. We show that proteins are distributed in an uneven, yet not exclusive, manner between IBM and CM. The individual distributions reflect the physiological functions of proteins. Moreover, proteins can redistribute between the domains upon changes of the physiological state of the cell. Impairing assembly of complex III affects the distribution of partially assembled subunits. We propose a model for the generation of this dynamic subcompartmentalization of the mitochondrial inner membrane.     

Metabolic control of the expression of mitochondrial D-beta-hydroxybutyrate dehydrogenase, a ketone body converting enzyme

The effects of various metabolic conditions inducing an overproduction of ketone bodies in the rat were studied at different levels of D-beta-hydroxybutyrate dehydrogenase expression, i.e., enzymatic activity and protein content in purified mitochondria, and translational activity of isolated free cytosolic polysomes. The strongest variations were obtained in diabetes mellitus where the D-beta-hydroxybutyrate dehydrogenase expression is largely decreased. Insulin can reverse this strong effect. Modulation of liver enzyme activity and of enzyme content was observed under the other conditions tested, i.e., a decrease and an increase in starvation and hyperlipidemic conditions, respectively. A comparative study was carried out on the enzyme of extrahepatic tissues, i.e., heart, kidney and brain. The results indicate that the D-beta-hydroxybutyrate dehydrogenase function appears to be controlled, at least at the translational, post-translational and catalytic levels.     

Comparison of D-beta-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria

The properties of D-beta-hydroxybutyrate dehydrogenase (BDH) from rat liver and brain mitochondria were compared to determine if isozymes of this enzyme exist in these tissues. The BDHs from these tissues behaved similarly during the purification process. The enzymes were indistinguishable by sodium dodecyl sulfate-polyacrylamide or acid-urea-polyacrylamide gel electrophoresis and they had identical isoelectric points. The BDHs from rat liver and brain were also quite similar in functional parameters determined by kinetic analysis and phospholipid activation of apo-BDH (i.e., the lipid-free enzyme). Antiserum against rat liver BDH inhibited both enzymes to an equivalent extent in a titration assay. The enzymes had similar patterns of peptide mapping by partial digestion with Staphylococcus aureus V8 protease, followed by immunoblotting using antiserum against the liver enzyme. These results suggest that the BDHs in rat liver and brain are very similar and possibly identical.     

MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.     

Selective solubilization of nicotinamide nucleotide transhydrogenase from the mitochondrial inner membrane

Nicotinamide nucleotide transhydrogenase was solubilized from beef heart submitochondrial particles employing Triton X-100 or lysolecithin. Lysolecithin was considerably more efficient and selective and released over 80 % of the transhydrogenase acdtivity from the membrane together with succinate dehydrogenase. Solubilization of NADH dehydrogenase and cytochrome oxidase was more efficiently accomplished with Triton than with lysolecithin. Both detergents released ATPase to various extents. Transhydrogenase remaining bound to particles after treatment with lysolecithin still catalyzed energy-linked transhydrogenation.     

ChChd3, an inner mitochondrial membrane protein, is essential for maintaining crista integrity and mitochondrial function

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952-14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.