Rat Liver Mitochondrial Matrix Research Articles

Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue.

Increased levels of circulating saturated free fatty acids, such as palmitate, have been implicated in the etiology of type II diabetes and cancer. In addition to being a constituent of glycerolipids and a source of energy, palmitate also covalently attaches to numerous cellular proteins via a process named palmitoylation. Recognized for its roles in membrane tethering, cellular signaling, and protein trafficking, palmitoylation is also emerging as a potential regulator of metabolism. Indeed, we showed previously that the acylation of two mitochondrial proteins at their active site cysteine residues result in their inhibition. Herein, we sought to identify other palmitoylated proteins in mitochondria using a nonradioactive bio-orthogonal azido-palmitate analog that can be selectively derivatized with various tagged triarylphosphines. Our results show that, like palmitate, incorporation of azido-palmitate occurred on mitochondrial proteins via thioester bonds at sites that could be competed out by palmitoyl-CoA. Using this method, we identified 21 putative palmitoylated proteins in the rat liver mitochondrial matrix, a compartment not recognized for its content in palmitoylated proteins, and confirmed the palmitoylation of newly identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. We postulate that covalent modification and perhaps inhibition of various mitochondrial enzymes by palmitoyl-CoA could lead to the metabolic impairments found in obesity-related diseases.

Glycation damage targets glutamate dehydrogenase in the rat liver mitochondrial matrix during aging

Aging is accompanied by gradual cellular dysfunction associated with an accumulation of damaged proteins, particularly via oxidative processes. This cellular dysfunction has been attributed, at least in part, to impairment of mitochondrial function as this organelle is both a major source of oxidants and a target for their damaging effects, which can result in a reduction of energy production, thereby compromising cell function. In the present study, we observed a significant decrease in the respiratory activity of rat liver mitochondria with aging, and an increase in the advanced glycation endproduct-modified protein level in the mitochondrial matrix. Western blot analysis of the glycated protein pattern after 2D electrophoresis revealed that only a restricted set of proteins was modified. Within this set, we identified, by mass spectrometry, proteins connected with the urea cycle, and especially glutamate dehydrogenase, which is markedly modified in older animals. Moreover, mitochondrial matrix extracts exhibited a significant decrease in glutamate dehydrogenase activity and altered allosteric regulation with age. Therefore, the effect of the glycating agent methylglyoxal on glutamate dehydrogenase activity and its allosteric regulation was analyzed. The treated enzyme showed inactivation with time by altering both catalytic properties and allosteric regulation. Altogether, these results showed that advanced glycation endproduct modifications selectively affect mitochondrial matrix proteins, particularly glutamate dehydrogenase, a crucial enzyme at the interface between tricarboxylic acid and urea cycles. Thus, it is proposed that glycated glutamate dehydrogenase could be used as a biomarker of cellular aging. Furthermore, these results suggest a role for such intracellular glycation in age-related dysfunction of mitochondria.

Import of mitochondrial transcription factor A (TFAM) into rat liver mitochondria stimulates transcription of mitochondrial DNA.

Mitochondrial transcription factor A (TFAM) has been shown to stimulate transcription from mitochondrial DNA promoters in vitro. In order to determine whether changes in TFAM levels also regulate RNA synthesis in situ, recombinant human precursor proteins were imported into the matrix of rat liver mitochondria. After uptake of wt-TFAM, incorporation of [alpha-32P]UTP into mitochondrial mRNAs as well as rRNAs was increased 2-fold (P < 0.05), whereas import of truncated TFAM lacking 25 amino acids at the C-terminus had no effect. Import of wt-TFAM into liver mitochondria from hypothyroid rats stimulated RNA synthesis up to 4-fold. We conclude that the rate of transcription is submaximal in freshly isolated rat liver mitochondria and that increasing intra-mitochondrial TFAM levels is sufficient for stimulation. The low transcription rate associated with the hypothyroid state observed in vivo as well as in organello seems to be a result of low TFAM levels, which can be recovered by treating animals with T3 in vivo or by importing TFAM in organello. Thus, this protein meets the criteria for being a key factor in regulating mitochondrial gene expression in vivo.

Subcellular localization of methionine sulphoxide reductase A (MsrA): evidence for mitochondrial and cytosolic isoforms in rat liver cells.

Proteins are sensitive to reactive oxygen species, and the accumulation of oxidized proteins has been implicated in the aging process and in other age-related pathologies. In proteins, methionine residues are especially sensitive to oxidation, leading to S - and R -methionine sulphoxide diastereoisomers, the reversion of which is achieved by the peptide methionine sulphoxide reductases MsrA and MsrB respectively. The MsrA enzyme, in addition to its role in repair, forms part of the reactive oxygen species scavenging systems that are important in cellular antioxidant defence. MsrA is present in most living organisms, and the mammalian enzyme has been detected in all tissues investigated. In the present study, we investigated the subcellular distribution of MsrA in rat liver cells. Since it seemed likely that MsrA may be localized in areas where reactive oxygen species are produced, rat liver mitochondrial matrix and cytosolic extracts were prepared. The presence of MsrA was assayed in these subcellular compartments by monitoring peptide methionine sulphoxide reductase enzymic activity, by Western blotting and by in situ immunolocalization by electron microscopy using a specific antibody. Moreover, MsrA was identified by MS in a partially purified cytosolic fraction and in a mitochondrial matrix crude extract. Rat MsrA isoforms are encoded by a single gene, and it is suggested that the precursor of the mitochondrial form contains an N-terminal cleavable signal sequence that localizes the MsrA to this organelle. Finally, two-dimensional gel electrophoresis followed by Western-blot analysis of partially purified MsrA from the cytosol and mitochondria, and comparison with the two-dimensional patterns of oxidized recombinant MsrA, revealed oxidative modifications of cysteine residues.

Anti-mitochondrial antibodies in patients with dilated cardiomyopathy (anti-M7) are directed against flavoenzymes with covalently bound FAD.

Anti-mitochondrial antibodies (anti-M7) in sera from patients with dilated cardiomyopathy and myocarditis recognize, besides mitochondrial antigens, bacterial sarcosine dehydrogenase. The common target antigen was identified as the covalently bound FAD of mitochondrial and bacterial flavoenzymes. Thus, anti-M7-positive serum reacted on Western blots exclusively with covalently flavinylated enzymes. The antigenic specificity of anti-M7 sera was reproduced by an antiserum raised in rabbits with 6-hydroxy-D-nicotine oxidase. The heart mitochondrial membrane antigen recognized by anti-M7 serum was identified as the flavoprotein subunit of succinate dehydrogenase, the antigens in rat liver mitochondrial matrix as the flavoenzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase. Anti-M7 serum contained a specific anti-flavoenzyme antibody fraction. Nanomolar concentrations of FAD and riboflavin inhibited the immune reaction on Western blots and in ELISA, and incubation with FAD-agarose depleted the anti-M7 activity of the serum. N-terminally deleted dimethylglycine dehydrogenase proteins were only immunoprecipitated by anti-M7 sera when the FAD was covalently incorporated. An affinity constant (KD) of 10(-8) M was established for the anti-flavoenzyme antibodies by competitive ELISA. Of patients with cardiomyopathy and myocarditis, 36% and 25%, respectively, were anti-flavoenzyme-positive by Western blot and ELISA, but only two of 15 patients with other heart diseases and none of 50 healthy controls.

Betaine aldehyde dehydrogenase from rat liver mitochondrial matrix

An NAD-linked aldehyde dehydrogenase which in addition to aliphatic and aromatic aldehydes, metabolizes aminoaldehydes and betaine aldehyde, has been purified to homogeneity from male Sprague–Dawley rat liver mitochondria. The properties of the rat mitochondrial enzyme are similar to those of a rat liver cytoplasmic betaine aldehyde dehydrognase and the human cytoplasmic E3 isozyme. The primary structure. of four tryptic peptides were also similar; only one difference in primary structure was observed. The close similarity of properties of the cytoplasmic with the mitochondrial form suggest that the cytoplasmic and mitochondrial betaine aldehyde dehydrogenase may be coded for by the same nuclear gene. Investigation of the mitochondrial form by isoelectric focusing resulted in visualization of multiple forms, different from those seen in the cytoplasm suggesting that the enzyme may be processed in the mitochondria.

A 45 kDa protein related to PPARγ2, induced by peroxisome proliferators, is located in the mitochondrial matrix

Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARγ2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance.

A protein factor of rat liver mitochondrial matrix involved in flavinylation of dimethylglycine dehydrogenase.

The involvement of rat liver mitochondria in the flavinylation of the mitochondrial matrix flavoenzyme dimethylglycine dehydrogenase (Me2GlyDH) has been investigated. Me2GlyDH was synthesized as an apoenzyme in the rabbit reticulocyte lysate (RL) transcription/translation system and its flavinylation was monitored by virtue of the trypsin resistance of the holoenzyme. The rate of holoenzyme formation in the presence of FAD was stimulated with increasing efficiency by the addition of solubilized mitoplasts, mitochondrial matrix and DEAE-purified matrix fraction. Apo-Me2GlyDH was also converted into holoenzyme when the solubilized mitoplasts were supplemented with FMN and ATP. This observation is consistent with the existence of a mitochondrial FAD synthetase generating the FAD needed for holoenzyme formation from its precursors. Holoenzyme formation in the presence of FAD increased linearly with the concentration of matrix protein in the assay, and depended on the amount of externally added Me2GlyDH with saturation characteristics. These findings suggest the presence of a protein factor in the mitochondrial matrix which stimulates Me2GlyDH flavinylation. This factor was different from both mitochondrial heat shock protein (Hsp)70, as shown by immunodepletion experiments, and mitochondrial Hsp60, as demonstrated by the capability of a DEAE-purified matrix fraction devoid of Hsp60 to accelerate flavinylation of both RL translated and purified Me2GlyDH.

Effect of extra- and intra-mitochondrial calcium on citrulline synthesis

A quantitative relationship between rat liver mitochondrial matrix free Ca2+ ([Ca2+]m) and citrullinogenesis has been observed. Maximum citrulline synthesis occurred at 100 to 200nM [Ca2+]m; higher [Ca2+]m caused inhibition. When [Ca2+]m was decreased to below 50nM, by addition of A23187 and EGTA, inhibition also occurred. By incubating mitochondria with ruthenium red ([Ca2+]m = 200nM) prior to addition of extramitochondrial free Ca2+ ([Ca2+]o) it was found that high external Ca2+ (800nM) did not inhibit citrulline synthesis thus demonstrating that [Ca2+]m, not [Ca2+]o was controlling citrullinogenesis.

Identification, purification and partial characterization of a carboxypeptidase from the matrix of rat liver mitochondria: a novel metalloenzyme.

A novel carboxypeptidase has been purified to apparent homogeneity from the matrix fraction of rat liver mitochondria by using a procedure mainly based on immobilized-metal-affinity chromatography (IMAC). This carboxypeptidase has been named mCP-III, since it represents the third major peak of carboxypeptidase activity after the IMAC step of purification. mCP-III hydrolyses a number of N-blocked dipeptides, with preference for Cbz-Phe-Ala, and shows no degrading activity towards 125I-casein. The optimal pH of its activity is 7.6, the apparent Km for Cbz-Phe-Ala is 0.12 mM and the specific activity is 145.5 mumol/min per mg of protein. The enzyme is a typical metalloproteinase, is inhibited by 1,10-phenanthroline and carboxypeptidase inhibitor and re-activated by added Zn2+ and Co2+. The molecular mass estimated by molecular-sieve h.p.l.c. was approx. 115 kDa with two protein bands of 61 and 50 kDa shown by SDS/PAGE analysis, indicating that the enzyme is active as a dimer. This is the first clearly identified carboxypeptidase within mitochondria.

Rat liver mitochondrial intermediate peptidase (MIP): purification and initial characterization.

A number of nuclearly encoded mitochondrial protein precursors that are transported into the matrix and inner membrane are cleaved in two sequential steps by two distinct matrix peptidases, mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). We have isolated and purified MIP from rat liver mitochondrial matrix. The enzyme, purified 2250-fold, is a monomer of 75 kDa and cleaves all tested mitochondrial intermediate proteins to their mature forms. About 20% of the final MIP preparation consists of equimolar amounts of two peptides of 47 kDa and 28 kDa, which are apparently the products of a single cleavage of the 75 kDa protein. These peptides are not separable from the 75 kDa protein, nor from each other, under any conditions used in the purification. The peptidase has a broad pH optimum between pH 6.6 and 8.9 and is inactivated by N-ethylmaleimide (NEM) and other sulfhydryl group reagents. The processing activity is divalent cation-dependent; it is stimulated by manganese, magnesium or calcium ions and reversibly inhibited by EDTA. Zinc, cobalt and iron strongly inhibit MIP activity. This pattern of cation dependence and inhibition is not clearly consistent with that of any known family of proteases.

Purification and N-terminal sequencing of peptidyl-prolyl cis-trans-isomerase from rat liver mitochondrial matrix reveals the existence of a distinct mitochondrial cyclophilin.

1. Rat liver mitochondrial matrix peptidyl-prolyl cis-trans-isomerase (PPIase) has been purified. The major form of the enzyme has a molecular mass of 18.6 kDa, with a minor active component of 17.6 kDa. 2. The second-order rate constant for cyclosporin A binding to the enzyme was determined from the time-dependence of the inhibition of PPIase by low concentrations of cyclosporin A and found to be 0.9 microM-1.s-1 at 10 degrees C. 3. The Ki for cyclosporin A inhibition of the enzyme was 3.6 nM, and the half-life for dissociation of the enzyme-inhibitor complex was 3.6 min. 4. From the specific activity of the pure enzyme it can be calculated that isolated liver mitochondria contain approx. 45 pmol of enzyme per mg of total mitochondrial protein. Higher values estimated previously [Halestrap & Davidson (1990) Biochem. J. 268, 153-160] are explained by the use of a short (30 s) preincubation period of the enzyme with cyclosporin, which is insufficient to allow full equilibration of the binding of the inhibitor to the PPIase. 5. N-Terminal sequencing of the 18.6 and 17.5 kDa forms of PPIase show the presence of mitochondrial presequences of 13 and three amino acids respectively, with the remaining sequence having a strong sequence similarity to other cyclophilins. 6. Parallel purification and N-terminal sequencing of rat cytosolic PPIase showed the two proteins to have significant differences, implying that they are probably products of separate genes.

Electrophoretic polyamine transport in rat liver mitochondria

Naturally occurring polyamines (spermidine, putrescine, cadaverine), as the well studied spermine, are transported into rat liver mitochondrial matrix provided that mitochondria are energized and the electrical membrane potential has a value of about 180 mV. This condition is achieved by the presence of inorganic phosphate, or acetate, or nigericin in the incubation medium. Valinomycin plus K(+) almost completely blocks polyamine transport.The obtained results clearly show that all naturally occurring polyamines are transported by an electrophoretic mechanism in responce to a high negative inner electrical potential.The distribution ratio of polyamines across the mitochondrial membrane is far from the thermodynamic equilibrium by many orders of magnitude. This result might suggest the existence of a different pathway for polyamine efflux.

Dynamics, structure, and function are coupled in the mitochondrial matrix.

The coupling between molecular diffusion and the structure and function of the rat liver mitochondrial matrix was explored using fluorescence anisotropy techniques and electron microscopy. The results confirm that matrix ultrastructure and the concentration of matrix protein are influenced by the respiratory state of mitochondria and the osmolarity of the external medium. At physiological osmolarity, a fluorescent metabolite-sized probe was found to diffuse slowly in the mitochondrial matrix but not to be completely immobile. In addition, significant differences in diffusion rates were found to exist between different mitochondrial respiratory states, with the slowest diffusion occurring in states with the highest matrix protein concentration. These data support the concept of a matrix structure in which diffusion is considerably hindered due to limited probe-accessible water and further suggest that volume-dependent regulation of matrix protein packing may modulate metabolite diffusion and, in turn, mitochondrial metabolism.

A novel glutathione transferase (13–13) isolated from the matrix of rat liver mitochondria having structural similarity to class theta enzymes

A rat liver mitochondrial-matrix fraction was prepared and shown to have 1-chloro-2,4-dinitrobenzene(CDNB)-metabolizing glutathione transferase (GST) activity. Further fractionation by sequential gel filtration, isoelectric focusing or chromatofocusing and hydroxyapatite chromatography yielded three GSTs of pI 9.3, 8.9 and 7.5, none of which bound to a GSH-agarose affinity matrix. Most of the activity was associated with the pI-9.3 form, which was selected for further study. Its activity was tested with the following potential substrates in addition to CDNB: 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, trans-4-phenylbut-3-en-2-one, 1,2-epoxy-3-(p-nitrophenoxy)propane, ethacrynic acid, menaphthyl sulphate, cumene hydroperoxide, linoleic acid hydroperoxide and 4-hydroxynon-2-enal. Appreciable activity was obtained only with CDNB and ethacrynic acid (82 and 26 mumol/min per mg of protein respectively). The apparent Km for GSH, using 1 mM-CDNB, was 1.9 mM. The enzyme is a dimer of subunit Mr 26,500. It has a free N-terminus, which has enabled the first 33 amino acids to be sequenced. This portion of primary structure has a sequence in common with members of the Theta class of GSTs (eg. 36% identity with subunit 12) and also a sequence which might function as a mitochondrial import signal. It is novel and has been named 'GST 13-13'.

Evidence for an in vivo modification of mitochondrial proteins by coenzyme A

Following denaturation of mitochondrial proteins by sodium dodecyl sulfate, a [1- 14C]pantothenic acid-derived radioactivity proved to be acid precipitable in the outer membrane, the intermembrane space, the inner membrane and in the matrix of rat liver mitochondria, where it had the highest specific radioactivity of 541 ± 29 cpm/100 μg protein. This tightly and/or covalently bound protein radioactivity could be released by incubation in the presence of dithioerythreitol; it was identified as [ 14C]coenzyme A by its HPLC retention time, its absorption spectrum and its radioactivity. This acid-stable and thiol-labile coenzyme A-binding apparently refers to specific protein binding sites. With the purified, homogeneous mitochondrial matrix enzymes acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) (EC 2.3.1.9, acetyl-CoA; acetyl-CoA C-acetyltransferase) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16) coenzyme A was found exclusively, e.g., in the modified, partially-active forms A1 und A2 of acetyl-CoA acetyltransferase and not in the unmodified fully-active enzyme. Thus it is evident that this coenzyme A modification is transient. We suggest that coenzyme A-modification is a signal involved in the assembly or the degradation processes of distinct mitochondrial matrix proteins.

Purification and characterization of aldehyde dehydrogenase from rat liver mitochondrial matrix

Aldehyde dehydrogenase (EC 1.2.1.3) has been purified to homogeneity from Sprague-Dawley rat liver mitochondrial matrix; its specific activity with propionaldehyde (1 mM at pH 9.0) is 1.4 mumol/min/mg. It has a native molecular weight of ca. 260,000 daltons, a subunit weight of 54,000 daltons and separates into two bands on isoelectric focusing (pI, 5.15 and 5.30); its extinction coefficient at 280 nm for 1 mg/ml solution is 1.2 and 280/260 nm ratio is 1.6. The enzyme prefers NAD over NADP as the coenzyme; the Km for NADP (67,000 microM) is three orders of magnitude greater than that for NAD (61 microM); the Km for acetaldehyde is 2 microM and for propionaldehyde is 0.8 microM at pH 7.0. The enzyme is reversibly inhibited by chloral (Ki = 3 microM) but is resistant to disulfiram inhibition. In addition another aldehyde dehydrogenase, first observed in the mitochondrial matrix during isoelectric focusing, has been partially purified. It has a Km for propionaldehyde of 0.7 mM and an isoelectric point of 6.4. Its activity with glutamic-gamma-semialdehyde (Km = 0.13 mM) is ca. 20 times higher than with propionaldehyde identifying the enzyme as glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12).

Phosphorus-31 NMR visibility and characterization of rat liver mitochondrial matrix adenine nucleotides

Compartmentation and NMR visibility of mitochondrial adenine nucleotides were quantitated in isolated rat liver mitochondria respiring on succinate and glutamate in vitro at 8 and 25 degrees C. Intra- and extramitochondrial nucleotides were discriminated by adding the chelator trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA). T1 values of about 0.2-0.3 s for magnesium-bound matrix nucleotides were determined. Adenine nucleotide T1 values were influenced by the ionic environment; only magnesium-free ATP T1's were affected by temperature. Intra- and extramitochondrial adenine nucleotide ratios were varied in ATP-loaded mitochondria with added ATP and phosphate using the mitochondrial inhibitors oligomycin and carboxyatractyloside, and adenine nucleotides were quantitated by using NMR and enzymatic analysis. There was good agreement between matrix ATP concentrations (magnesium-bound ATP) calculated by using NMR and standard biochemical techniques. Although matrix ADP could be detected by NMR, it was difficult to quantitate accurately by NMR. The data indicate that mitochondrial ATP is NMR-visible in isolated mitochondria in vitro.

Import and processing of hybrid proteins by mammalian mitochondria in vitro.

Normal and hybrid proteins were synthesized following transcription of pSP64 recombinant plasmids with SP6 polymerase and translation of resultant mRNAs in a rabbit reticulocyte lysate. The precursor to the rat liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = approximately 40,000), was efficiently imported by rat heart mitochondria in vitro and processed to mature protein. Import of the precursor to a second matrix enzyme, carbamyl-phosphate synthetase I (Mr = approximately 165,000), could not be demonstrated. However, a 33-kDa hybrid protein, bearing the precarbamyl-phosphate synthetase I signal sequence (38 amino acids) and 55 amino acids from the amino terminus of the mature enzyme, followed by the carboxyl-terminal 209 amino acids of ornithine carbamyltransferase, was imported and processed under identical conditions. The topogenic function of the preornithine carbamyltransferase signal sequence (32 amino acids) was confirmed by constructing a hybrid protein bearing the signal sequence and five amino acids of mature enzyme, followed by 250 amino acids of the cytosolic enzyme of Escherichia coli, asparagine synthetase; the hybrid was transported to the matrix compartment of heart mitochondria where processing, albeit incorrect, took place. A hybrid asparagine synthetase bearing the pre-ornithine carbamyltransferase signal sequence plus 28 amino acids of mature ornithine carbamyltransferase, however, was imported and processed with apparent fidelity.

Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase.

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc1 complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.