Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of inner membrane.     

A membrane-associated isozyme of invertase in yeast precursor of the external glycoprotein

An enzymatic test is described which allows the localization of yeast invertase activity directly on sodium dodecyl sulfate gels. When crude membrane fractions are prepared from Saccharomyces cerevisiae cells which are actively synthesizing external invertase, these membranes show an activity band on sodium dodecyl sulfate gels additional to the external and the internal invertase. In the soluble fraction this form was completely absent. It has a molecular weight of approximately 190 000 and was 50 000 smaller than the external invertase. It showed kinetic characteristics of a precursor of the external enzyme. Thus it appeared transiently, when yeast cells were shifted from a condition of non-synthesizing external invertase to one where the enzyme was synthesized. When the increase in the external enzyme slowed down after some time, the membrane-associated form almost completely disappeared.The addition of tunicamycin to yeast cells synthesizing external invertase inhibited further synthesis of the enzyme by 97%; also the formation of the membrane-associated form was strongly inhibited. The amount of it present before the addition of tunicamycin completely disappeared in the presence of the antibiotic. The precursor form, therefore, seems to possess already those carbohydrate parts which contain N-acetylglucosamine and are transferred via dolichyl phosphate-bound intermediates. The membrane-associated precursor amounts to less than 5% of the total invertase activity of a yeast cell.     

Transport of proteins across the mitochondrial outer membrane. A precursor form of the cytoplasmically made intermembrane enzyme cytochrome c peroxidase.

Cytochrome c peroxidase, a cytoplasmically made enzyme located between the inner and outer membrane of yeast mitochondria, is synthesized as larger precursor in a reticulocyte cell-free lysate as well as in pulsed yeast spheroplasts. When the pulsed spheroplasts are chased, the precursor is converted to the mature apoprotein. When the in vitro synthesized precursor is incubated with isolated yeast mitochondria in the absence of protein synthesis, it is cleaved to the mature form; the mature form co-sediments with the mitochondria and is resistant to externally added proteases. These results, in conjunction with those reported earlier (Maccecchini, M.-L., Rudin, Y., Blobel, G., and Schatz, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 343-347) suggest that the mechanism of protein transport into the mitochondrial intermembrane space is quite similar to that of protein transport into the matrix or the inner membrane.     

Synthesis of glutamic oxaloacetic transaminase isozymes in rat liver cells

The synthesis of glutamic oxaloacetic transaminase isozymes in rat liver explants was studied using specific antisera against the cytosolic and mitochondrial isozymes. The pulse-labeled cytosolic isozyme was detected in the cytosolic fraction and remained there in pulse-chase experiments. On the other hand, the pulse-labeled mitochondrial isozyme was detected as a larger precursor in the cytosolic fraction. During chase, the amount of pulse-labeled precursor of the mitochondrial isozyme decreased and labeled mature mitochondrial isozyme appeared in the mitochondrial fraction.     

In vitro synthesis of a putative precursor of serine: pyruvate aminotransferase of rat liver mitochondria

Serine:pyruvate aminotransferase [EC 2.6.1.51] of rat liver, an enzyme induced by glucagon in mitochondria, was synthesized in cell-free protein synthesizing systems derived from nuclease-treated rabbit reticulocyte lysate and wheat germ extract as a putative precursor which was approximately 2,000 daltons larger than the subunit of mature enzyme. The hepatic level of translatable messenger RNA coding for the putative precursor was approximately 40 times higher in rats received a glucagon administration 3.5 h before sacrifice than in control animals.     

Ornithine transcarbamylase in liver mitochondria

Summary Ornithine transcarbamylase (ornithine carbamoyltransferase, EC 2.1.3.3), the second enzyme of urea synthesis, is localized in the matrix of liver mitochondria of ureotelic animals. The enzyme is encoded by a nuclear gene, synthesized outside the mitochondria, and must then be transported into the organelle. The rat liver enzyme is initially synthesized on membrane-free polysomes in the form of a larger precursor with an amino-terminal extension of 3 400–4 000 daltons. In rat liver slices and isolated rat hepatocytes, the pulse-labeled precursor is first released into the cytosol and is then transported with a half life of 1 2 min into the mitochondria where it is proteolytically processed to the mature form of the enzyme. The precursor synthesized in vitro exists in a highly aggregated form and has a conformation different from that of the mature enzyme. The precursor has an isoelectric point (pI = 7.9) higher than that of the mature enzyme (pI = 7.2).The precursor synthesized in vitro can be taken up and processed to the mature enzyme by isolated rat liver mitochondria. The mitochondrial transport and processing system requires membrane potential and a high integrity of the mitochondria. The transport and processing activities are conserved between mammals and birds or amphibians and is presumably common to more than one precursor. Potassium ion, magnesium ion, and probably a cytosolic protein(s), in addition to the transcarbamylase precursor and the mitochondria, are required for the maximal transport and processing of the precursor.A mitochondrial matrix protease which converts the precursor to a product intermediate in size between the precursor and the mature subunit has been highly purified. The protease has an estimated molecular weight of 108 000 and an optimal pH of 7.5–8.0, and appears to be a metal protease. The protease does not cleave several of the protein and peptide substrates tested. The role of this protease in the precursor processing remains to be elucidated.Rats subjected to different levels of protein intake and to fasting show significant changes in the level of enzyme protein and activity of ornithine transcarbamylase. The dietary-dependent changes in the enzyme level are due mainly to an altered level of functional mRNA for the enzyme. In contrast, during fasting, the increase in the enzyme level is associated with a decreased level of translatable mRNA forthe enzyme.Pathological aspects of ornithine transcarbamylase including the enzyme deficiency and reduced activities of the enzyme in Reye's syndrome are also described. A possibility that impaired transport of the enzyme precursor into the mitochondria leads to a reduced enzyme activity, is proposed.Abbreviation pOTC precursor of ornithine transcarbamylase     

Adenylate cyclase system of human skeletal muscle: Subcellular distribution and general properties

The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na⁺ + K⁺)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low K_m phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent K_m for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg²⁺, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg²⁺ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle.     

In vitro translation of canine mitochondrial creatine kinase messenger RNA

The cell-free translation products of mRNA from canine myocardium were immunoprecipitated using antiserum specific for either the MM or mitochondrial creatine kinase subunit. The two subunits were shown to be encoded by the nuclear genome and translated from separate mRNAs. The mitochondrial subunit was translated as a polypeptide with a molecular weight approximately 6,000 greater than the mature form of the enzyme. In contrast, the M-subunit was translated as a polypeptide having a molecular weight identical to that of the mature cytosolic M-subunit. It is assumed that the mitochondrial subunit precursor must be proteolytically processed during translocation from the cytoplasm into mitochondria.     

Import of rat liver mitochondrial malate dehydrogenase. Synthesis, transport, and processing in vitro of its precursor

Rat liver total RNA was translated in a reticulocyte lysate, and the precursor of rat liver mitochondrial malate dehydrogenase was identified by a monospecific antibody against the denatured mature enzyme. The precursor is about Mr = 1500 to 2000 larger than the monomeric form of the mature protein. The major spots of the two-dimensional peptide map of the two proteins were identical. The precursor was synthesized on free polysomes, but not membrane-bound polysomes. Upon fractionation by molecular sieve chromatography on Sephadex G-100, the size of the precursor was slightly larger than the dimeric form of the mature protein. Incubation of the precursor with isolated mitochondria from Chinese hamster ovary cells resulted in uptake and processing of the precursor to the mature size. The processed form was resistant to trypsin indicating that it was translocated into mitochondria. Processing was complete in 10 to 30 min at 30 degrees C. Rapid binding of the precursor to mitochondria was also observed at 0 or 30 degrees C. Processing but not binding was inhibited by an uncoupler.     

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).     

In vitro import into mitochondria of the precursor of mitochondrial aspartate aminotransferase

The import of the precursor of mitochondrial aspartate aminotransferase was reconstituted in vitro with isolated mitochondria thus corroborating the earlier conclusion of a post-translational uptake. The higher Mr precursor was synthesized in a reticulocyte lysate programmed with free polysomes from chicken liver. After incubation with intact mitochondria from chicken heart about 50% of the precursor was converted to the mature form in a time-dependent process, its rate being a function of the amount of mitochondria added. The same amount of precursor was processed to the mature form on addition of a mitochondrial extract. No conversion to the mature enzyme took place when the precursor was incubated with intact mitochondria in the presence of the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone or of the chelator o-phenanthroline which penetrates the mitochondrial inner membrane. In contrast, the chelator bathophenanthroline disulfonate which does not diffuse into the mitochondrial matrix did not inhibit the appearance of the mature form. The results indicate that that precursor must pass through an energized inner mitochondrial membrane before it is processed by a chelator-sensitive protease in the mitochondrial matrix. Excess mature mitochondrial aspartate aminotransferase did not compete with the precursor for its uptake into mitochondria. Mature mitochondrial aspartate aminotransferase is an alpha 2-dimer with Mr = 2 X 45,000. Both the precursor synthesized in a rabbit reticulocyte lysate and the precursor accumulated in the cytosol of carbonyl cyanide m-chlorophenylhydrazone-treated chicken embryo fibroblasts were found to exist as homodimer or hetero-oligomer and high Mr complexes (Mr greater than 300,000).     

The mechanism of replication of phi x174 DNA. XVI. Evidence that the phi x174 viral strand is synthesized discontinuously

Chymostatin is a naturally occurring inhibitor of serine proteases that have chymotryptic-like specificity. This tetrapeptide inhibitor is produced by various species of Streptomyces bacteria. Chymostatin reacts with the serine enzyme Streptomyces griseus protease A in the crystalline state to produce an adduct, the structure of which is in agreement with hemiacetal formation between the C-terminal l-phenylalaninal residue of the inhibitor and the O^γ atom of the active Ser195 residue of S. griseus protease A. The 2.8 Å difference electron density map of the complex is also consistent with the novel structural features previously deduced spectroscopically for chymostatin; i.e. an essential (for inhibition) aldehyde function in the C-terminal l-phenylalaninal residue, an unusual arnino acid, 2-(2-iminohexahydro-(4 S)-pyrimidyl)-(S)-glycine as the third residue from the C terminus and an N-terminal amino group blocked by a (1S)-carboxyphenylethyl-carbamoyl group. There is no significant movement of the active site residues of S. griseus protease A upon complexation with chymostatin.     

The synthesis and processing of β-glucuronidase in normal and egasyn deficient mouse kidney

The presence of a precursor form of β-glucuronidase, with a subunit molecular weight of 75,000 was demonstrated in mouse kidney. This was later processed to the mature form, with subunit molecular weight of 71,500. Tissue fractionation revealed that the precursor was associated with the microsomes whereas the mature form was associated with the lysosomes. In mice lacking egasyn both forms of β-glucuronidase were present, but the rate of processing was elevated compared to normal.     

Expression and function of heterologous forms of malate dehydrogenase in yeast.

The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.     

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins

The precursor of cytochrome b₂ (a cytoplasmically-synthesized mitochondrial protein) binds to isolated mitochondria or to isolated outer membrane vesicles. Binding does not require an energized inner membrane, is diminished by trypsin treatment of the membranes and is not observed with the partially processed (intermediate) form of the cytochrome b₂ precursor or with non-mitochondrial proteins. Upon energization of the mitochondria, the bound precursor is imported and cleaved to the mature form. Similar results were obtained with the precursor of citrate synthase. This receptor-like binding activity was present in isolated outer, but not inner membrane. It was solubilized from outer membrane with non-ionic detergent and reconstituted into liposomes.     

The Pediocin AcH Precursor Is Biologically Active

The properties of the pediocin AcH precursor, prepediocin AcH, have been studied to gain insight into how producer cells may protect themselves from the activity of intracellular prebacteriocins. The native 62-amino-acid precursor and the 44-amino-acid mature species were expressed in Escherichia coli host strains that lack the leader peptide processing enzyme, PapD. Both forms inhibited the growth of the test bacterium Listeria innocua Lin11, indicating that the native precursor is biologically active. The two species also were synthesized in the context of maltose-binding protein chimeric proteins to facilitate the measurement of their relative specific activities. The chimeric form of the precursor was ~80% as active as the chimeric mature species. Of relevance to cell protection and pediocin AcH production, it was determined that the precursor is strongly susceptible to inactivation by reducing agents and to degradation by chymotrypsin and endogenous E. coli proteases. Taken together, the results indicate that the activity of prepediocin AcH may have to be controlled prior to secretion to prevent toxicity to the host. Perhaps producer cells avoid membrane damage by maintaining the precursor in a reduced inactive state or by degrading molecules whose secretion is delayed.     

Structure of phage P22 coat protein aggregates formed in the absence of the scaffolding protein.

Previous studies have shown that the assembly of the precursor shell (prohead) of bacteriophage P22 requires the copolymerization of the gene 5 coat protein with the gene 8 scaffolding protein. Removal of the scaffolding protein by mutation prevents efficient coat protein assembly, but some aberrant particles do form. We have now isolated these structures and characterized them with respect to morphology, protein composition, and small-angle X-ray scattering properties.The aberrant particles fall into three morphological classes, i.e. complex spirals and closed shells of two sizes. Small-angle X-ray scattering studies confirm that the larger particles are hollow shells with the radius of proheads ($\text{r = 260}\text{A}\text{?}$), and not of the mature virus ($\text{r = 285}\text{A}\text{?}$). These structures lack the inner shell of scaffolding protein found in proheads. The small particles have a radius of 195 Å, smaller than proheads, and appear to contain material, not scaffolding protein, within the outer shell.The aberrant particles contain two minor protein species, the gene 9 tail-spike protein, and an unidentified 67,000 molecular weight polypeptide, probably from the host. Neither is found in normal proheads. Removal of gene.9 product by mutation did not affect the formation of the aggregates. Fractionation of the morphological classes of particles revealed that the 67,000 molecular weight band was associated with the closed shells. It may be serving as a pseudo-initiator.Earlier studies had shown that treatment of proheads with sodium dodecyl sulfate in vitro resulted in loss of the scaffolding protein, and expansion of the shell to the mature radius of 285 Å. When the 8^? prohead-sized shells were treated similarly, they also expanded to the mature-sized shell. These results support the idea that there are at least two stable states of the coat protein, one of which, the prohead form, is an obligatory precursor of the mature form.     

The conversion of the precursor form of γ-glutamyltranspeptidase to its subunit form takes place in brush border membranes

The subcellular distributions of the precursor form and mature form of γ-glutamyltranspeptidase of rat kidney were studied by labeling the enzyme with [³H] fucose in vivo. In trans Golgi elements and basolateral membranes, γ-glutamyltranspeptidase was present as a precursor form with a single polypeptide chain. However, the brush border membranes contained the heavy and light subunits as well as precursor. These results suggest that the precursor is converted to the mature form after its transport to the brush border membranes.     

Expression and transport into mitochondria of bovine cytochrome P-450(SCC) in insect cells using the baculovirus expression system.

Bovine cytochrome P-450(SCC) introduced with the baculovirus host vector system was found to be expressed in Spodoptera frugiperda cells. Cell fractionation analysis indicated that the P-450(SCC) expressed as the precursor form was transported into mitochondria and converted to a mature form. However, this form did not exhibit definite activity for cholesterol side chain cleavage. These findings suggest that most of the P-450(SCC) expressed by this system is an inactive protein within mitochondria that is not folded to the conformation of the active enzyme and/or does not incorporate heme appropriately.     

Characterization of a latent cysteine proteinase from ascitic fluid as a high molecular weight form of cathepsin B

The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of M_r 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of M_r 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.