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.     

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.     

Degradation of ornithine decarboxylase in reticulocyte lysate is ATP-dependent but ubiquitin-independent

Reticulocyte lysate contains all the components of the ubiquitin-dependent proteolytic system. Several proteins are degraded in reticulocyte lysate in a ubiquitin-dependent manner. However, none of the proteins studied has a short intracellular half-life. We have investigated the degradation of ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells. ODC is efficiently degraded in reticulocyte lysate depleted of the ubiquitin activating enzyme, E1, in fraction II of reticulocyte lysate completely lacking ubiquitin, and in fraction II depleted of the entire complex of enzymes responsible for the ligation of ubiquitin to target proteins. The degradation of ODC is ATP dependent. Therefore, our results demonstrate that in addition to the ubiquitin-dependent proteolytic pathway, reticulocyte lysate contains at least one additional ATP-dependent proteolytic pathway. In vitro synthesized ODC served as a substrate in the present degradation study. Its successful utilization establishes a general strategy for investigating the degradation of short-lived proteins (for which a corresponding cDNA is available), that constitute a very small fraction of cellular proteins and for which purification is difficult or impossible. In contrast to ODC synthesized in vitro, that isolated from cells was not degraded by the reticulocyte lysate degradation system, suggesting that post-translational modifications may be involved in regulating ODC degradation.     

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Import of chemically synthesized signal peptides into rat liver mitochondria

The signal peptides of pre-aldehyde dehydrogenase (22-mer) and pre-ornithine transcarbamylase (27-mer) were chemically synthesized and their imports into rat liver mitochondria were studied. Both signal peptides were imported rapidly (within 2 min) in the absence of a membrane potential, exogenous ATP, or rabbit reticulocyte lysate. Signal peptides also were imported into mitochondria treated with a low concentration of trypsin which removed the outer membrane proteins. It was concluded that the chemically synthesized signal peptide could be imported differently than the precursor proteins. The imported signal peptide were found to be associated with both outer and inner membranes. Pulse-chase experiments showed that the import was unidirectional and that the signal peptides associated with inner membranes increased during the chase time. The signal peptides inhibited import of precursor proteins to different extents. Association of signal peptides with inner membrane near or at translocator sites might result in inhibition of precursor import.     

Oxygenation of mitochondrial membranes by the reticulocyte lipoxygenase. Action on monoamine oxidase activities A and B

Incubation of isolated rat liver mitochondria with the pure rabbit reticulocyte lipoxygenase caused a time-dependent inactivation of the monoamine oxidase activities A and B. Furthermore, a conversion of the monoamine oxidase into a diamine oxidase was observed. The inactivation kinetics for both monoamine oxidase activities A and B showed a biphasic behaviour; a reversible short-term inhibition during the first 5 min of incubation was followed by an irreversible inactivation of the enzyme. The kinetic studies suggest that the slow irreversible inactivation of the monoamine oxidase activities is due to secondary reactions subsequent to the initial attack of the lipoxygenase on the mitochondrial outer membrane. During the interaction of the lipoxygenase with the mitochondria, only about 1.5% of the polyenoic fatty acids present in the mitochondrial membranes were oxygenated. The predominant products formed during the interaction of the lipoxygenase with the mitochondrial membranes are (13S)-hydro(pero)xy-9Z,11E-octadecadienoic acid and (15S)-hydro(pero)xy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid.     

The chloroplast 32 kDa protein is synthesized on thylakoid-bound ribosomes in Chlamydomonas reinhardtii

A cloned cpDNA fragment containing a portion of the gene for the 32–36 kDa thylakoid protein of Chlamydomonas (polypeptide D-1) was isolated. Hybridization probing of RNA from soluble and membrane fractions of Chlamydomonas showed that the mRNA for D-1 is bound to thylakoid membranes. Run-off translation of thylakoid-bound polysomes (rough thylakoids) with [³⁵S]-methionine yields polypeptide D-1 as the major product. Peptide mapping with S. aureus V-8 protease of D-1 synthesized (1) in vivo, (2) in vitro by rough thylakoids and (3) in the reticulocyte lysate directed by non-polyadenylated RNA showed that D-1 is synthesized as a precursor in the reticulocyte lysate but as the mature polypeptide by rough thylakoids.     

Ubiquitin-proteasome-dependent degradation of apolipoprotein B100 in vitro

Apolipoprotein B100 (apoB) is a large secretory protein that forms very low density lipoprotein in liver. An in vitro degradation assay was developed using rabbit reticulocyte (RR) lysate in order to investigate the mechanism of intracellular degradation of newly synthesized apoB by the ubiquitin-proteasome pathway. [³H]apoB, isolated from [³H]leucine pulsed/chased Hep G2 cells, was degraded 51% when incubated for 2 h at 37°C in an assay mixture that included RR lysate (source of the ubiquitin conjugation system and proteasome) and an exogenous ATP regenerating system. ApoB degradation was ATP-dependent and degradation fragments were not observed suggesting that the very large apoB molecule was extensively degraded. ApoB degradation was decreased to 50% when potent proteasome inhibitors, clasto-lactacystin β-lactone (10 μM) or MG-132 (50 μM), were added to the reaction mixture, but was not affected by the cysteine protease inhibitor, E-64, or the serine protease inhibitor, phenylmethylsulfonyl fluoride. ApoB degradation was inhibited by the mutant ubiquitin protein K48R and by ubiquitin aldehyde, an inhibitor of ubiquitin-protein isopeptidases. During incubation ubiquitination of apoB increased even as apoB was being degraded. These results suggest that in vitro degradation of apoB, a large secretory protein that is normally found in the endoplasmic reticulum (ER) lumen or associated with the ER membrane, was proteasome-dependent and involved both ubiquitination and deubiquitination steps.     

Ubiquitin carboxyl-terminal peptides. Substrates for ubiquitin activating enzyme

The carboxyl terminus of ubiquitin is activated in the presence of ATP to enter the ubiquitin cycle in cells. Peptides corresponding to the COOH-terminal region of ubiquitin were synthesized to investigate their effects on the ATP/ubiquitin-dependent proteolytic pathway. Their activities in the PPi exchange assay with ubiquitin activating enzyme (E1) were proportional to their length. The hexapeptide Ac-Leu-Arg-Leu-Arg-Gly-Gly reacted with ATP to form an enzyme-adenylate-hexapeptide complex and at high concentrations was 20-25% as active as human ubiquitin in the PPi exchange assay with E1. However, the hexapeptide was not transferred to the sulfhydryl "thiol" site on E1. In addition, the COOH-terminal peptides did not support the degradation of 125I-bovine serum albumin in the reticulocyte lysate system. A nonhomologous peptide of equivalent length was inactive in all assays. Thus, synthetic COOH-terminal peptide(s) of ubiquitin can partially substitute for ubiquitin in its reactions with E1 but do not support subsequent steps of the energy-dependent proteolytic pathway. These results show that it may be possible to design small molecules that either serve as substrates or inhibitors for other specific steps in ubiquitin-dependent pathways.     

Multi-ubiquitination of a nascent membrane protein produced in a rabbit reticulocyte lysate.

During a large-scale in vitro translation analysis of a human full-length cDNA bank, we found many clones producing in vitro translation products showing ladder bands on a fluorogram with the equidistance of about 9 kDa at the position larger than the molecular mass expected from the open reading frame. We have analyzed a clone showing a typical pattern of the ladder bands. This clone encoded a 188-amino acid polypeptide containing a putative transmembrane domain. A green fluorescent protein-tagged polypeptide expressed in COS7 cells was localized in the endoplasmic reticulum and the Golgi apparatus. The ladder bands were observed in a rabbit reticulocyte lysate system, but not in a wheat germ extract system. Addition of the glutathione S-transferase-fused ubiquitin into the lysate caused upward shifts of the ladder bands. Addition of microsomal membranes prevented the formation of the ladder bands. Time course experiments demonstrated that the in vitro translation products increased in the presence of microsomal membranes, but were gradually degraded in their absence. These results suggest that the ladder formation resulted from the ubiquitination of misfolded polypeptide that failed to translocate to its proper position, and that an exclusion mechanism of misfolded membrane protein works in the rabbit reticulocyte lysate system.     

THE SUBMITOCHONDRIAL LOCALIZATION OF MONOAMINE OXIDASE : An Enzymatic Marker for the Outer Membrane of Rat Liver Mitochondria

Controlled osmotic lysis (water-washing) of rat liver mitochondria results in a mixed population of small vesicles derived mainly from the outer mitochondrial membrane and of larger bodies containing a few cristae derived from the inner membrane. These elements have been separated on Ficoll and sucrose gradients. The small vesicles were rich in monoamine oxidase, and the large bodies were rich in cytochrome oxidase. Separation of the inner and outer membranes has also been accomplished by treating mitochondria with digitonin in an isotonic medium and fractionating the treated mitochondria by differential centrifugation. Treatment with low digitonin concentrations released monoamine oxidase activity from low speed mitochondrial pellets, and this release of enzymatic activity was correlated with the loss of the outer membrane as seen in the electron microscope. The low speed mitochondrial pellet contained most of the cytochrome oxidase and malate dehydrogenase activities of the intact mitochondria, while the monoamine oxidase activity could be recovered in the form of small vesicles by high speed centrifugation of the low speed supernatant. The results indicate that monoamine oxidase is found only in the outer mitochondrial membrane and that cytochrome oxidase is found only in the inner membrane. Digitonin treatment released more monoamine oxidase than cytochrome oxidase from sonic particles, thus indicating that digitonin preferentially degrades the outer mitochondrial membrane.     

The cytosolic factor required for import of precursors of mitochondrial proteins into mitochondria

The mechanism of import of proteins into mitochondria was studied by using the peptide of the presequence of ornithine aminotransferase (the extrapeptide), which was chemically synthesized and is composed of 34 amino acids. When the extrapeptide was incubated with isolated mitochondria in the presence of a rabbit reticulocyte lysate at 25 degrees C, it was imported into the mitochondrial matrix, and the import depended on the inner membrane potential, but not added ATP. The import of several precursors of mitochondrial proteins was competitively inhibited by the presence of excess extrapeptide in the reaction system, indicating that the extrapeptide and mitochondrial proteins were imported by the same machinery. Import of the extrapeptide was significantly stimulated by addition of a rabbit reticulocyte lysate, and a component of the lysate (the cytosolic factor) stimulating import of the extrapeptide was purified about 20,000 times by successive column chromatography on DEAE-cellulose and aminopentyl-Sepharose 4B. The binding of the extrapeptide to liposomes composed of egg lecithin and partially purified receptor of the precursor of mitochondrial protein (Ono, H., and Tuboi, S., (1985) Biochem. Int. 10, 351-357) required the cytosolic factor when the concentration of the peptide was less than 1.5 X 10(-8) M, suggesting that the physiological binding of the precursors of mitochondrial proteins to the receptor is dependent on the cytosolic factor. The extrapeptide and the cytosolic factor were shown to form a complex. From these results, the mechanism of binding of the extrapeptide to the receptor of the mitochondrial outer membrane is suggested to be as follows: the peptide (the precursor of mitochondrial protein) and the cytosolic factor form a complex, and then the complex is recognized by and bound to the receptor.     

In vitro biosynthesis of the beta-subunit of the Na+/K+-ATPase in developing brine shrimp: glycosylation and membrane insertion

We demonstrate here translation, glycosylation, and membrane insertion of the beta-subunit of the Na+/K+-ATPase of the developing brine shrimp, Artemia, in a reticulocyte lysate translation system. The apparent molecular weight of the primary translation product as determined by SDS-PAGE is 33,000 +/- 1000 (n = 7). When microsomal membranes are present during the entire translation period, a new band with an apparent molecular weight of 37,000 +/- 1000 (n = 7) appears. This change in apparent molecular weight is due to the addition of about two N-linked oligosaccharides. The temporal relationship between protein synthesis and glycosylation have also been examined. Glycosylation and membrane insertion could be achieved if membranes were added after completion of about 70% of the peptide chain. However, glycosylation did not occur if membranes were added after the completion of translation of the beta-subunit. The beta-subunit was synthesized on membrane-bound polysomes, where about two N-linked oligosaccharides were added to the growing polypeptide chain. These studies demonstrate that in vitro translation systems will be useful for studying the biosynthesis of the beta-subunit of the brine shrimp, which is a good model system to examine the developmental regulation of the Na+/K+-ATPase.     

Differential post-translational modification of human type I keratins synthesized in a rabbit reticulocyte cell-free system

The translation products synthesized in a rabbit reticulocyte lysate system, from total cellular mRNA from the human epithelial cell-line ME-180, have been examined. Keratin proteins are prominent among these translation products, and they precisely coelectrophorese in sodium dodecyl sulfate-polyacrylamide gels with keratins purified from the cells. Type-I, acidic, keratins which are acetylated in vivo, are also acetylated by the reticulocyte lysate. Examination by two-dimensional electrophoresis, of two acidic keratins known to be phosphorylated in vivo reveals that only one of these proteins is phosphorylated in the lysate system. Phosphorylation of this protein occurs after release of the completed polypeptide chain from the ribosome. The protein phosphorylated by the lysate is known to be the only ME-180 phosphokeratin modulated by cyclic AMP, reflecting in vitro the differential modification of ME-180 keratins in vivo.     

Circular pancreatic trypsin inhibitor. A novel substrate for studies on intracellular proteolysis

Several recent studies indicate that substrates for ubiquitin-dependent proteolysis must possess unblocked alpha-amino termini. To examine further the importance of free amino groups for proteolytic susceptibility we selected pancreatic trypsin inhibitor (PTI) as a test substrate. PTI can be circularized to form cPTI, a molecule that lacks alpha-amino groups in the absence of an endoproteolytic cleavage. We compared the breakdown of radioiodinated PTI and cPTI in rabbit reticulocyte lysate and found that cPTI was not stabilized relative to PTI. In addition, proteolysis of PTI or cPTI was not inhibited upon conversion of their lysine residues to homoarginine. However, neither degradation of PTI nor cPTI required ATP, and ubiquitin conjugation to either molecule was minor relative to known substrates of the ubiquitin pathway. Thus, PTI and cPTI are cleaved by an ATP-independent endoprotease(s) that does not require the substrate to be ubiquitinated. Such an activity was identified in low salt fractions obtained upon DEAE chromatography of reticulocyte lysate. The ubiquitin/ATP-dependent protease and another large multisubunit protease, both of which elute from DEAE-Fractogel at higher salt concentrations, do not degrade PTI or cPTI. Although monomeric PTI was rapidly degraded in reticulocyte lysate, cross-linked PTI molecules were stable both in reticulocyte lysate and following introduction into cultured cells using red blood cell-mediated microinjection. Thus, increased rates of turnover do not necessarily correlate with greater molecular mass of the substrate.     

In vitro synthesis and assembly of a 68 kDa outer mitochondrial membrane protein from rat liver

Outer mitochondrial membrane was purified from rat liver. Its constituent proteins were analyzed by SDS-polyacrylamide gel electrophoresis and by electrophoretic immunoblotting employing antibodies raised against total outer mitochondrial membrane. Anti-outer mitochondrial membrane antiserum reacted with only one polypeptide (15 kDa) in rough microsomes, whereas no immunological cross-reactivity was observed with other mitochondrial compartments (intermembrane space, inner membrane, or matrix) or with lysosomes or total cytosol. The antiserum was employed to characterize precursors of outer mitochondrial membrane proteins synthesized in vitro in a rabbit reticulocyte cell-free system. One product (a 68 kDa polypeptide designated OMM-68) bound efficiently to mitochondria in vitro but did not interact with either dog pancreas or rat liver microsomes, either co-translationally or post-translationally. OMM-68 was synthesized exclusively by the membrane-free class of polyribosomes. Attachment of precursor OMM-68 to mitochondria was not accompanied by processing of the polypeptide to a different size.     

Import of precursor proteins into mitochondria: site of polypeptide unfolding

The matrix-targeting signal of mitochondrial preornithine carbamyl transferase has been fused to either murine dihydrofolate reductase (pODHFR) or bacterial chloramphenicol acetyltransferase (pOCAT). Loosening of the tightly folded "native" structure of the two proteins following their synthesis in a rabbit reticulocyte lysate was assayed by the acquisition of protease sensitivity (pODHFR and pOCAT) or by the loss of enzyme activity (pOCAT). By these criteria, the bulk population of both precursor proteins was tightly folded following release from the ribosome, even in the presence of ATP and excess reticulocyte lysate. Neither protein unfolded as a consequence of binding to the surfaces of anionic liposomes or intact mitochondria. However, a non-native form of full-length pOCAT, exhibiting a loss of enzymatic activity and an enhanced protease sensitivity, was detected in association with a submitochondrial fraction that banded between the inner and outer mitochondrial membrane fractions on sucrose density gradients. Delivery of the precursor molecule to this position required ATP and a proteinaceous component on the surface of the organelle.     

Membrane perturbing factor in reticulocyte lysate, which is transiently activated by proteases.

Proteases have been used to examine the topology of proteins on various membranes. We reexamined the conditions of protease treatment for rough microsomal membranes and found that proteinase K degraded the lumenal proteins in the presence of reticulocyte lysate. The lysate treated with either heat or N-ethylmaleimide no longer promoted the degradation. The reticulocyte dependent degradation was also observed with papain, trypsin, and elastase. This activity was transiently generated by treating reticulocyte lysate short-term with trypsin. We thus concluded that a membrane perturbing factor(s) must exist in reticulocyte which is transiently activated by protease treatment.     

Distinct steps in the import of ADP/ATP carrier into mitochondria

Transport of the precursor to the ADP/ATP carrier from the cytosol into the mitochondrial inner membrane was resolved into several consecutive steps. The precursor protein was trapped at distinct stages of the import pathway and subsequently chased to the mature form. In a first reaction, the precursor interacts with a protease-sensitive component on the mitochondrial surface. It then reaches intermediate sites in the outer membrane which are saturable and where it is protected against proteases. This translocation intermediate can be extracted at alkaline pH. We suggest that it is anchored to the membrane by a so far unknown proteinaceous component. The membrane potential delta psi-dependent entrance of the ADP/ATP carrier into the inner membrane takes place at contact sites between outer and inner membranes. Completion of translocation into the inner membrane can occur in the absence of delta psi. A cytosolic component which is present in reticulocyte lysate and which interacts with isolated mitochondria is required for the specific binding of the precursor to mitochondria.