Transfer of phosphatidic acid between microsomal and mitochondrial outer and inner membranes

A protein fraction from rat liver cytoplasm, precipitable at 50-95% saturation of ammonium sulphate, binds phosphatidic acid from mitochondrial and microsomal membranes. Protein-bound phosphatidic acid was eluted from Sephadex G-75 in fractions corresponding to a molecular weight of about 10 000. No such binding was observed with mitochondrial soluble proteins, either total or precipitated with ammonium sulphate between 50 and 95% saturation. The transfer of phosphatidic acid from microsomes to mitochondria was increased by liver cytoplasmic proteins precipitable at 50-95% saturation of ammonium sulphate but not with mitochondrial soluble proteins. This increase by cytoplasmic proteins was pronounced in 200 mM sucrose but was negligible in 100 mM KCI where the spontaneous transfer was quite high. Cytoplasmic proteins stimulated the synthesis of cardiolipin and phosphatidylglycerol in mitochondria deprived of the outer membrane but not in intact mitochondria when phosphatidic acid was supplied either by microsomes or liposomes. It is suggested that the transfer of phosphatidic acid from the outer to the inner mitochondrial membrane is not mediated by transfer proteins but occurs either by direct contact of the membranes or as free diffusion through the aqueous phase.     

Integration of porin synthesized in vitro into outer mitochondrial membranes

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell-free in a cell-free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochondria at 30 degrees C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197-202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0 degrees C for 5 min. When the incubation period at 0 degrees C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30 degrees C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).     

Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane

The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Non-protein-mediated transfer of phosphatidic acid between microsomal and mitochondrial membranes

The transfer of phosphatidic acid between rat liver microsomes loaded with [32P]-phosphatidic acid and rat liver mitochondria was studied in the absence of added lipid transfer proteins. It was found that during 1 h at 37 degrees C in the medium containing 100 mM KCl, 20-30% of phosphatidic acid but only 2.5% of phosphatidylcholine were transferred. This spontaneous transfer of phosphatidic acid remained the same after pretreatment of microsomes and mitochondria with 125 mM KCl or microsomes alone with 1 mM Tris, pH 8.6, procedures reported to remove adsorbed lipid transfer proteins. This transfer was insensitive to thiol-blocking reagents. The initial rate of this non-protein-mediated transfer of phosphatidic acid was virtually independent of the concentration of the acceptor membranes (mitochondria), thus indicating that it occurs by diffusion of the phospholipid through the aqueous phase rather than by membrane collision. About 80% of phosphatidic acid synthesized in the outer mitochondrial membrane was recovered in the inner membrane after a 1-h incubation, pointing to a high rate of the intermembrane transfer of this phospholipid within intact mitochondrion.     

Regulation of mitochondrial membrane protein turnover by thyroid hormone(s)

Effects of thyroidectomy on turnover rate of proteins of rat liver mitochondria, mitochondrial membranes and microsomes were examined. Thyroidectomy resulted in a significant increase in the half-lives of whole mitochondria and inner membrane-matrix, the effect being less pronounced on the half-lives of outer mitochondrial membrane and microsomes.     

Optimal assay and subcellular location of phosphatidylglycerol synthesis in lung

Synthesis of phosphatidylglycerol from CDPdiacylglycerol and glycerol 3-phosphate by membranous subcellular fractions of rat lung and liver was optimal when assayed in the presence of bovine serum albumin and Triton X-100. Specific activities of glycerolphosphate phosphatidyltransferase in all membranous subcellular fractions of lung were several times higher than the corresponding fractions from liver. Distribution of this enzyme in subcellular fractions of lung or liver closely parallel the activity of the mitochondrial enzymes monoamine oxidase and succinate cytochrome c reductase. The phosphatidylglycerol-synthesizing activity in microsomes of both lung and liver was a minor fraction of total tissue activity and could be interpreted as due either to contamination with outer mitochondrial membrane or to a small amount of activity innate to microsomes. These results suggest that phosphatidylglycerol, which is believed to be a component of pulmonary surfactant, is synthesized by lung at a rapid rate relative to liver and that the subcellular distribution of its synthesis is similar in both tissues, with mitochondria as the major site.     

Cell-free synthesis of a putative precursor to the rat liver mitochondrial glycerol-3-phosphate dehydrogenase

Antibodies to purified glycerol-3-phosphate dehydrogenase were raised in rabbits and purified from serum by affinity chromatography on enzyme-bound Sepharose columns. RNA from membrane-free polyribosomes, or poly(A)+ RNA (total cellular RNA) of rat liver, was translated in a rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, and the glycerol-3-phosphate dehydrogenase synthesized was isolated by immunoprecipitation using the antibody. The in vitro product moved on sodium dodecyl sulfate-polyacrylamide gels as a polypeptide that was about 5,000 daltons larger than the subunit of the mature enzyme (74,000 daltons). Digestion of both the mature and the in vitro newly synthesized forms of the enzyme yielded respective sets of peptide fragments which had similar patterns upon sodium dodecyl sulfate-gel electrophoresis. When the presumptive precursor that had been synthesized in vitro was incubated with isolated intact rat liver mitochondria, it was converted to "mature" subunits that were no longer susceptible to externally added proteases. Import of the presumptive precursor is dependent upon an electrochemical potential across the inner mitochondrial membranes. The mature form of the protein is assembled in its native location (the outer surface of the inner mitochondrial membrane).     

The in vitro insertion of monoamine oxidase B into mitochondrial outer membranes

Bovine monoamine oxidase (MAO) B has been synthesized in vitro using a reticulocyte lysate translation system directed by bovine liver poly(A)+ RNA. The newly synthesized enzyme apparently lacks a cleavable N-terminal extension, but MAO B is readily incorporated into mitochondria or isolated mitochondrial outer membranes prepared from rat liver. ATP is not required for the binding of the newly synthesized enzyme to the outer membranes, but is necessary for the insertion of MAO B into these membrane vesicles. The ATP is not required to generate a mitochondrial membrane potential as assembly occurs under conditions that preclude either the formation or the maintenance of the potential. MAO B will bind to but not become incorporated into outer membrane vesicles which have been treated with trypsin, suggesting that the insertion of MAO B also depends on protein factors present on the outer membranes.     

Transport of proteins into hepatic and nonhepatic mitochondria: specificity of uptake and processing of precursor forms of carbamoyl-phosphate synthetase I

An in vitro system reconstituted with mouse liver polysome translation products was used to study the nature of polypeptide species imported into mitochondria from different mouse tissues such as liver, kidney, brain, and heart, as well as from Ehrlich ascites, Novikoff hepatoma, and Morris hepatoma 3924A tumor lines. Mouse hepatic mitochondria import a number of proteins including 160-kilodalton (kDa) carbamoyl-phosphate synthetase I (CPS-I). Two other proteins of 63 and 57 kDa of unknown function are also imported as major components by mouse liver mitochondria. Under these in vitro conditions, however, mitochondria from non-CPS-I expressing tissues such as brain, kidney, and heart failed to import and process the precursor forms of CPS-I (pCPS-I). Furthermore, mitochondria from three different tumor lines (Novikoff hepatoma, Morris hepatoma, and Ehrlich ascites) containing negligible CPS-I activity were also unable to import and process pCPS-I to any significant level. Similarly, the 63-kDa protein was selectively transported into liver and kidney mitochondria and also into Ehrlich ascites mitochondria at reduced levels, but not into mitochondria from heart and brain. Nevertheless, the 57-kDa protein and a number of proteins of less than 45 kDa are transported efficiently by all of the mitochondrial types studied. These results provide evidence for tissue- or cell-specific selectivity at the mitochondrial membrane level for the transport of some proteins. The transports of 63- and 57-kDa proteins are differentially inhibited by mouse liver mitochondrial matrix and membrane fractions, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein import into yeast mitochondria is inhibited by antibodies raised against 45-kd proteins of the outer membrane.

Import of several precursor proteins into isolated yeast mitochondria is inhibited by rabbit antiserum raised against the total mitochondrial outer membrane or against electrophoretically purified 45-kd outer membrane proteins. Antisera against other outer membrane proteins are only marginally active or inactive. Inhibition by the antiserum against 45-kd proteins is only weak with untreated mitochondria, but reaches 80-90% with mitochondria that had been pretreated with 0.1 mg/ml trypsin. This trypsin pretreatment by itself inhibits precursor import only slightly (30-50%). Selective inhibition of import does not correlate with binding of the various IgGs to the mitochondrial surface and is also observed with the corresponding Fab fragments. Inhibition by antibodies against 45-kd outer membrane proteins strongly suggests the existence of a mitochondrial surface protein mediating protein import and offers a means of isolating this protein.     

The insertion of monoamine oxidase A into the outer membrane of rat liver mitochondria.

Human monoamine oxidase A that had been synthesized in a reticulocyte lysate translation system was capable of binding to and inserting into either rat liver mitochondria or isolated mitochondrial outer membranes. The inserted form was as resistant to proteinase K as endogenous mitochondrial monoamine oxidase A. The insertion, but not the binding, of monoamine oxidase A was prevented by depleting the reaction mixture of either ATP (with apyrase) or ubiquitin (with purified antibodies against this polypeptide). Addition of ATP or ubiquitin, respectively, to these depleted mixtures restored the insertion of the enzyme. In the absence of mitochondria, in vitro synthesized monoamine oxidase A did not catalyze its own alkylation by the mechanism-based inhibitor, [3H]clorgyline. However, both monoamine oxidase A that had been membrane-inserted in vitro and monoamine oxidase A that had been bound to the mitochondria under conditions of ATP depletion catalyzed adduct formation. Furthermore, reaction of either clorgyline or another mechanism-based inhibitor, pargyline, with the membrane-bound enzyme during ATP depletion inhibited the insertion of monoamine oxidase A when ATP was restored. These observations indicate that monoamine oxidase A acquired a catalytically active conformation on interaction with the mitochondrial outer membranes prior to its ATP and ubiquitin-dependent insertion into the membrane.     

Microtubule-associated proteins bind to 30 kDa and 60 kDa proteins of rat brain mitochondria: visualization by ligand blotting

Axonal transport of mitochondria is a microtubule-associated movement. Microtubule-mitochondria interactions were studied in vitro using organelles isolated from rat brain. Thanks to the ligand blotting method we were able to show two mitochondrial membrane proteins with apparent molecular masses of 30 kDa and 60 kDa that bind microtubule-associated proteins. The binding of the 30 kDa protein has an apparent Kd of 8 x 10(-8) M. Digitonin fractionation of mitochondria reveals a bimodal localization of the 30 kDa and the 60 kDa proteins within the outer membrane. The data suggest that these polypeptides could participate to the interactions observed in situ between microtubules and mitochondria.     

Subcellular distribution of rat liver porin

The subcellular distribution of rat liver porin was investigated using the immunoblotting technique and monospecific antisera against the protein isolated from the outer membrane of rat liver mitochondria. Subfractionation of mitochondria into inner membranes, outer membranes and matrix fractions revealed the presence of porin only in the outer membranes. Porin was also not detected in highly purified subcellular fractions, including plasma membranes, nuclear membranes, Golgi I and Golgi II, microsomes and lysosomes. Thus, liver porin is located exclusively in the outer mitochondrial membrane.     

Import of rat liver mitochondrial malate dehydrogenase. Binding of the precursor to mitochondria, an intermediate step in import

We have previously reported that the precursor of rat liver mitochondrial malate dehydrogenase, synthesized in vitro, is about 1,500 to 2,000 Mr larger than the mature enzyme and can be processed to the mature size by isolated mitochondria from Chinese hamster ovary cells (Chien, S.-M. and Freeman, K. B. (1984) J. Biol. Chem. 259, 3337-3342). Furthermore, binding, but not processing, was observed in the presence of an uncoupler. Binding was insensitive to temperature and was completed within 2.5 min at 0 degrees C. The role of binding in the overall process of import of the precursor is now further characterized. The precursor form, bound either in the presence of an uncoupler or at 0 degrees C, was sensitive to trypsin suggesting that binding occurs on the mitochondrial outer membrane. Saturation of binding was observed with a limited amount of mitochondria and an excess of in vitro translated rat liver proteins indicating that there is a finite number of binding sites. Furthermore, when the precursor was prebound to mitochondria at 0 degrees C for 5 min, the precursor was processed to the mature size and the rate of processing was independent of the volume of reaction mixture. In contrast, the rate of processing of unbound precursor was dependent on reaction volume. These results strongly suggest that binding of the precursor of malate dehydrogenase to the mitochondrial outer membrane is an intermediate step in its import.     

A receptor for protein import into potato mitochondria

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.     

Phosphatidic acid synthesis in mitochondria. Topography of formation and transmembrane migration.

The topography of formation and migration of phosphatidic acid (PA) in the transverse plane of rat liver mitochondrial outer membrane (MOM) were investigated. Isolated mitochondria and microsomes, incubated with sn-glycerol 3-phosphate and an immobilized substrate palmitoyl-CoA-agarose, synthesized both lyso-PA and PA. The mitochondrial and microsomal acylation of glycerophosphate with palmitoyl-CoA-agarose was 80-100% of the values obtained in the presence of free palmitoyl-CoA. In another series of experiments, both free polymyxin B and polymyxin B-agarose stimulated mitochondrial glycerophosphate acyltransferase activity approximately 2-fold. When PA loaded mitochondria were treated with liver fatty acid binding protein, a fifth of the phospholipid left the mitochondria. The amount of exportable PA reduced with the increase in the time of incubation. In another approach, PA-loaded mitochondria were treated with phospholipase A(2). The amount of phospholipase A(2)-sensitive PA reduced when the incubation time was increased. Taken together, the results suggest that lysophosphatidic acid (LPA) and PA are synthesized on the outer surface of the MOM and that PA moves to the inner membrane presumably for cardiolipin formation.