Biosynthesis of nonspecific lipid transfer protein (sterol carrier protein 2) on free polyribosomes as a larger precursor in rat liver

The biosynthesis of nonspecific lipid transfer protein (nsLTP) was investigated. Total RNA of rat liver was translated in a rabbit reticulocyte lysate cell-free protein-synthesizing system with [35S]methionine as label. The immunoprecipitation of translation products with affinity-purified anti-nsLTP antibody yielded 14.5- and 60-kDa [35S]polypeptides. The molecular mass of the former polypeptide was approximately 1.5 kDa larger than that of the purified mature nsLTP (13 kDa). The site of synthesis of nsLTP was studied by in vitro translation of free and membrane-bound polyribosomal RNAs followed by immunoprecipitation. mRNA for both the 14.5- and 60-kDa polypeptides were found predominantly in the free polyribosomal fraction in both normal and clofibrate-treated rats. Clofibrate, a hypolipidemic drug that proliferates peroxisomes, did not increase the relative amount of nsLTP mRNA in rat liver. Pulse-chase experiments in rat hepatoma H-35 cells suggested that nsLTP was synthesized as a larger precursor of 14.5 kDa and converted to a mature form of 13 kDa. We have recently shown that nsLTP is highly concentrated in peroxisomes in rat hepatocytes [Tsuneoka et al. (1988) J. Biochem. 104, 560-564]. Taken together, these results suggest that nsLTP is synthesized as a larger precursor of 14.5 kDa on cytoplasmic free polyribosomes, then post-translationally transported to peroxisomes, where the precursor is presumably proteolytically processed to its mature form of 13 kDa. The relationship between the 13-kDa nsLTP and the 60-kDa polypeptide is also discussed.     

A purified precursor polypeptide requires a cytosolic protein fraction for import into mitochondria.

The beta-subunit of mitochondrial ATPase is coded by a nuclear gene, synthesized outside the mitochondria as a larger precursor and imported into mitochondria. The beta-subunit precursor was purified from yeast, both as a homogeneous, unlabeled polypeptide and in radiochemically pure form. Both precursor preparations were cleaved to the mature beta-subunit by partially purified processing protease from the mitochondrial matrix. However, import of the radiochemically pure precursor into isolated yeast mitochondria required a cytosolic fraction from yeast or reticulocytes. The cytosolic factor was non-dialyzable and trypsin-sensitive; its apparent mol. wt. was approximately 40 000 as judged by gel filtration. Import of some proteins into mitochondria thus requires proteins of the 'soluble' cytoplasm.     

Posttranslational processing of polysaccharide lyase: maturation route for gellan lyase in Bacillus sp. GL1

Cells of Bacillus sp. GL1 extracellularly secrete a gellan lyase with a molecular mass of 130 kDa responsible for the depolymerization of a heteropolysaccharide (gellan), although the gene is capable of encoding a huge protein with a molecular mass of 263 kDa. A maturation route for gellan lyase in the bacterium was determined using anti-gellan lyase antibodies. The fluid of the bacterial exponentially growing cultures on gellan contained two proteins with molecular masses of 260 and 130 kDa, both of which reacted with the antibodies. The 260 kDa protein was purified from the cultured fluid and characterized. The protein exhibited gellan lyase activity and showed similar enzyme properties, such as optimal pH and temperature, thermal stability, and substrate specificity, to those of the 130 kDa gellan lyase. The N-terminal amino acid sequences of the 260 and 130 kDa enzymes were found to be identical. Determination of the C-terminal amino acid of the 130 kDa enzyme indicated that the 260 kDa enzyme is cleaved between the 1205Gly and 1206Leu residues to yield the mature form (130 kDa) of the gellan lyase. Therefore, the mature enzyme consists of 1170 amino acids (36Ala-1205Gly) with a molecular weight of 125,345, which is in good agreement with that calculated from SDS-PAGE analysis. Judging from these results, gellan lyase is first synthesized as a preproform (263 kDa) and then secreted as a precursor (260 kDa) into the medium through cleavage of the signal peptide. Finally, the precursor is post-translationally processed into the N-terminal half domain of 130 kDa as the mature form, the function of C-terminal half domain being unclear.     

Precursor of mitochondrial glutamic oxaloacetic transaminase isozyme exists as a dimer

Mitochondrial glutamic oxaloacetic transaminase is synthesized in the cytoplasm as a larger precursor and then imported into mitochondria in association with its processing to mature form. The precursor corresponded to about 0.05-0.1% of the total proteins synthesized on membrane-free polysomes. Gel permeation chromatography showed that the molecular weight of the precursor was about 100,000 daltons, which was slightly larger than that of mature enzyme (homodimer: 45,000 x 2). On the other hand, sodium dodecylsulfate treated precursor had the molecular size (about 50,000 daltons) slightly larger than that of the subunit of mature one by the gel permeation chromatography. These results suggest that the precursor of mitochondrial glutamic oxaloacetic transaminase exists as a dimer.     

Characterization of different forms of yeast acid trehalase in the secretory pathway

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec 18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.     

Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii

Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum. A 42-kDa polypeptide with isoelectric point (pI) of 7.1-7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pI of 6.1-6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.     

Biosynthesis of mitochondrial porin and insertion into the outer mitochondrial membrane of Neurospora crassa

Mitochondrial porin, the major protein of the outer mitochondrial membrane is synthesized by free cytoplasmic polysomes. The apparent molecular weight of the porin synthesized in homologous or heterologous cell-free systems is the same as that of the mature porin. Transfer in vitro of mitochondrial porin from the cytosolic fraction into the outer membrane of mitochondria could be demonstrated. Before membrane insertion, mitochondrial porin is highly sensitive to added proteinase; afterwards it is strongly protected. Binding of the precursor form to mitochondria occurs at 4 degrees C and appears to precede insertion into the membrane. Unlike transfer of many precursor proteins into or across the inner mitochondrial membrane, assembly of the porin is not dependent on an electrical potential across the inner membrane.     

Glycosylation of P-glycoprotein in a multidrug-resistant KB cell line, and in the human tissues.

P-glycoprotein (P-gp) is thought to transport anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. Immunohistochemistry reveals that P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, and the capillary endothelium of the brain and testis. However, little is known about the structural and functional variations of P-gp in these tissues. With immunoblotting and photoaffinity labeling, we found that the molecular mass of P-gp in these tissues varied between 130-140 kDa. To clarify the post-translational modification of P-gp, we studied the biosynthesis of P-gp in a human multidrug-resistant cell line (KB-C2). We found that P-gp was produced in KB-C2 cells as a 125 kDa precursor and was slowly processed (t1/2 = 45-60 min) to the mature form of 140 kDa. In the presence of tunicamycin, a 120 kDa form of P-gp was synthesized and this form was no longer processed. Treating the 125 kDa precursor form with endo-beta-N-acetylglucosaminidase H (Endo H) and the 140 kDa mature form with N-glycanase diminished the molecular size of P-gp to that of the tunicamycin-treated form. N-Glycanase almost completely removed [3H]glucosamine labeling from P-gp. These data indicate that the major modification of P-gp is N-linked glycosylation. P-gps from KB-C2 cells, kidney and adrenal gland had a different lectin-binding capacity. There seems to be a variety of N-linked glycosylations in tissue and tumor P-gps.     

Identification of the amino terminal subunit of the glycoprotein of Borna disease virus

The only surface membrane glycoprotein of Borna disease virus (BDV) is synthesized as a polypeptide with a molecular mass of 57 kDa and N-glycosylated to a precursor glycoprotein (GP) of about 94 kDa. It is processed by the cellular protease furin into the C-terminal membrane-anchored subunit GP-C, also known as gp43, and a presumptive N-terminal subunit GP-N, that is highly glycosylated and has a molecular mass of about 51 kDa. However, up to now the latter remained undetected in BDV-infected material. We describe a novel approach to identify glycan masked linear antigenic epitopes. In the present study, GP-N was identified in BDV-infected cells by a combination of lectin precipitation, enzymatic deglycosylation on blot and immunochemistry using an N-terminal specific antiserum. The GP-N has an apparent molecular mass of 45-50 kDa in its glycosylated form and 27 kDa in its deglycosylated form. N-glycan analysis revealed that the precursor GP contains only mannose-rich N-glycans, whereas GP-N and GP-C contain mannose-rich and complex-type N-glycans.     

Transport and metabolism of 5'-nucleotidase in a rat hepatoma cell line

The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72-kDa form in 15-60 min (t1/2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-beta-N-acetylglycosaminidase H; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20-30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium.     

Cytosolic L-alanine:4,5-dioxovalerate transaminase differs from the mitochondrial form

L-Alanine:4,5-dioxovalerate transaminase was detected in the kidney cytosolic fraction with a lower specific activity than the mitochondrial enzyme. The enzyme was purified from the cytosol to homogeneity with a yield of 32%, and comparative analysis with the mitochondrial form was performed. Both forms of the enzyme have identical pH and temperature optima and also share common antigenic determinants. However, differences in their molecular properties exist. The molecular mass of the native cytoplasmic enzyme is 260 kDa, whereas that of the mitochondrial enzyme is 210 kDa. In addition, the cytoplasmic L-alanine: 4,5-dioxovalerate transaminase had a homopolymeric subunit molecular mass of 67 kDa compared to a subunit molecular mass of 50 kDa for the mitochondrial L-alanine:4,5-dioxovalerate transaminase. This is the first report of two forms of L-alanine:4,5-dioxovalerate transaminase. The different responses of cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminases to hemin supplementation both in vitro and in vivo was demonstrated. Maximum inhibition of mitochondrial L-alanine:4,5-dioxovalerate transaminase activity was demonstrated with hemin injected at a dose of 1.2 mg/kg body mass, whereas the same dose of hemin stimulated the cytosolic enzyme to 150% of the control. A one-dimensional peptide map of partially digested cytosolic and mitochondrial L-alanine:4,5-dioxovalerate transaminase shows that the two forms of the enzymes are structurally related. Partial digestion of the cytosolic form of the enzyme with papain generated a fragment of 50 kDa which was identical to that of the undigested mitochondrial form (50 kDa). Moreover, papain digestion resulted in a threefold increase in cytosolic enzyme activity over the native enzyme, and such enhancement was comparable to the activity of the mitochondrial form of the enzyme. Therefore, we conclude that the cytosolic form of L-alanine: 4,5-dioxovalerate transaminase is different from the mitochondrial enzyme. Furthermore, immunoblot analysis indicated that the mitochondrial enzyme has antigenic similarity to the cytosolic enzyme as well as to the papain-digested cytosolic enzyme 50-kDa fragment.     

Mitochondrial precursor protein. Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation, and import competence

A hybrid precursor protein constructed by fusing the mitochondrial matrix-targeting signal of rat preornithine carbamyl transferase to murine cytosolic dihydrofolate reductase (designated pO-DHFR) was expressed in Escherichia coli. Following purification under denaturing conditions, pO-DHFR was capable of membrane translocation when diluted directly into import medium containing purified mitochondria but lacking cytosolic extracts. This import competence was lost with time, however, when the precursor was diluted and preincubated in medium lacking mitochondria, unless cytosolic proteins (provided by rabbit reticulocyte lysate) were present. Identical results were obtained for purified precursor made by in vitro translation. The ability of the cytosolic proteins to maintain the purified precursor in an import-competent state was sensitive to protease, N-ethylmaleimide (NEM), and was heat labile. Further, this activity appeared to be signal sequence dependent. ATP was not required for the maintenance of pO-DHFR competence, nor did purified 70-kDa heat shock protein (the constitutive form of Hsp70) substitute for this activity. Interestingly, however, purified Hsp70 prevented aggregation of the precursor in an ATP-dependent manner and, as well, retarded the apparent rate and extent of pO-DHFR folding. Partial purification of reticulocyte lysate proteins indicated that competence activity resides within a large mass protein fraction (200-250 kDa) that contains Hsp70. Sucrose density gradient analysis revealed that pO-DHFR reversibly interacts with components of this fraction. Pretreatment of the fraction with NEM, however, significantly stabilized the subsequent formation of a complex with the precursor. The results indicate that Hsp70 can retard precursor polypeptide folding and prevent precursor aggregation; however, by itself, Hsp70 cannot confer import competence to pO-DHFR. Maintenance of import competence correlates with interactions between the precursor and an NEM-sensitive cytosolic protein fraction. Efficient dissociation of the precursor from this complex appears to require a reactive thiol moiety on the cytosolic protein(s).     

In vitro synthesis of a putative precursor to the mitochondrial enzyme, carbamyl phosphate synthetase.

A simple and rapid procedure is described for purification of carbamyl phosphate synthetase from the matrix fraction of rat liver mitochondria. Antibodies to the enzyme were raised in sheep and purified from antiserum by affinity chromatography on enzyme-bound Sepharose columns. When membrane-free polyribosomes, isolated from a cytosolic fraction of rat liver, were incubated in a messenger-dependent rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, the purified antibody precipitated a product of translation representing 0.2% of total trichloroacetic acid-insoluble radioactivity. It demonstrated mobility characteristics in sodium dodecyl sulfate-polyacrylamide gels expected for a polypeptide of molecular mass approximately 5500 daltons larger than the mature mitochondrial form of the enzyme (160,000 daltons). Proteolysis of both the mature and presumptive in vitro precursor forms of the enzyme yielded respective sets of peptide fragments which gave similar patterns upon gel electrophoresis. Excess mitochondrial enzyme effectively competed with the in vitro product for interaction with anti-carbamyl phosphate synthetase antibody.     

Regulation of steroid hormone biosynthesis. Identification of precursors of a phosphoprotein targeted to the mitochondrion in stimulated rat adrenal cortex cells.

Two short-lived precursor proteins, pp37 and pp32, of the mitochondrial phosphoprotein pp30 (formerly denoted as ib) have been detected in Bt2cAMP-stimulated rat adrenal cortex cells, incubated at 25 degrees C or with 1,10-ortho-phenanthroline at 37 degrees C. Subsequently, these two precursor proteins were also identified in cells incubated at 37 degrees C, where they are present only at low levels due to their short half-life. pp30 is produced in several steroidogenic tissues in response to trophic hormone or second messenger analogue. pp37 and pp32 are also phosphoproteins located in the mitochondrion that are produced in response to cAMP analogue and give rise to proteolytic peptide maps similar to that of pp30. As for pp30, inhibition of cytosolic translation prevents the production of pp37 and pp32. The larger precursor protein pp37 has an apparent molecular mass of 37 kDa, an isoelectric point of approximately 7.1, and a half-life at 37 degrees C of 3-4 min. Pulse-chase studies indicate that this protein is processed into the smaller protein, pp32, which has an apparent molecular mass of 32 kDa, an isoelectric point of approximately 6.4, and a half-life at 37 degrees C of 3-4 min. This latter protein is the immediate precursor of pp30. Since ortho-phenanthroline inhibits the mitochondrial processing protease, while the lower incubation temperature slows both protein import and protease processing, the experimental conditions necessary to detect these proteins are consistent with pp37 being a precursor protein that contains two cleavable presequences and is imported into the mitochondrion. The sequential removal of these sequences produces the mature protein pp30.     

Biosynthesis pathway of gp90MEL-14, the mouse lymph node-specific homing receptor

The mouse lymph node specific homing receptor gp90MEL-14 is a 95-kDa molecular mass ubiquitinated cell surface molecule involved in the binding of lymphocytes to high endothelial venules in peripheral lymph nodes. The molecule is thought to consist of a core protein to which ubiquitin side chains are covalently bound. Recently we cloned the cDNA encoding the core protein; this cDNA clone encodes for a polypeptide with an estimated molecular mass of 37 kDa. We have studied the biosynthesis of gp90MEL-14 in an effort to explain the difference in molecular mass between the core protein and the 95-kDa mature molecule. Pulse labeling experiments show a rapid synthesis of a 70-kDa precursor form that contains high-mannose N-linked oligosaccharides. On processing of the high-mannose oligosaccharides into complex N-linked oligosaccharides, the precursor matures in a single step into the 95-kDa form. Experiments using deglycosylating enzymes and inhibitors of N-linked glycosylation demonstrate that the molecular mass of deglycosylated gp90MEL-14 is 45 kDa; extensive N-linked glycosylation is responsible for the difference in molecular mass with the mature 95-kDa form. The core protein molecular weight of in vitro transcribed and translated gp90MEL-14 cDNA is consistent with the estimated molecular mass of 37 kDa, calculated from the cDNA sequence of the core protein, and 8 to 10 kDa less than the protein molecular mass of gp90MEL-14 translated in vivo in the presence of tunicamycin (45 kDa). Inasmuch as we have ruled out glycosylation as accounting for this discrepancy, this is consistent with the addition of one ubiquitin moiety to the core protein during biosynthesis. Limited proteolysis confirms the similarity between in vitro transcribed gp90MEL-14 cDNA and the tunicamycin form of gp90MEL-14.     

COOH-terminal processing of polypeptide D1 of the photosystem II reaction center of Scenedesmus obliquus is necessary for the assembly of the oxygen-evolving complex

Mutant LF-1 of the green alga Scenedesmus obliquus has been described by Metz and co-workers (Metz, J. G., Pakrasi, H., Seibert, M., and Arntzen, C. J. (1986) FEBS Lett. 205, 269-274) to be inactive for light-driven oxygen evolution, despite a functional Photo-system II reaction center. A polypeptide, D1, implicated in the ligation of the primary photoreactants of photosystem II, was shown to migrate with an apparent higher molecular mass on LDS-PAGE in the mutant than in the wild-type (WT) strain. We show here that polypeptide D1 is synthesized in a precursor form in Scenedesmus WT. Following synthesis and insertion into the thylakoid membrane, a 1.5-2-kDa oligopeptide is clipped off with a half-time of 1-2 min, yielding the mature 34-kDa form of the polypeptide. No processing of polypeptide D1 from mutant LF-1 was observed to take place. We show here that polypeptide D1 of LF-1 displays an identical proteolytic fingerprint pattern to the precursor D1 polypeptide of the wild-type strain. These both have molecular masses about 1.5-2 kDa higher than that of the mature WT polypeptide. A polyclonal antibody elicited by a synthetic oligopeptide (14-mer), predicted from the psbA gene nucleotide sequence to be homologous to the COOH terminus of the precursor D1 of spinach, cross-reacts only with D1 of mutant LF-1 and not with mature D1 of spinach, Chlamydomonas, or of Scenedesmus WT. This observation demonstrates that the greater molecular mass of polypeptide D1 from mutant LF-1 and of Scenedesmus WT precursor D1 is derived from a COOH-terminal extension. We conclude that the LF-1 mutant lacks the appropriate nuclear-encoded protease which processes polypeptide D1 at its COOH terminus from the precursor to the mature form. Such processing would appear to be a necessary step toward the stable incorporation of manganese into the oxygen-evolving site.     

The type I and type II bovine scavenger receptors expressed in Chinese hamster ovary cells are trimeric proteins with collagenous triple helical domains comprising noncovalently associated monomers and Cys83-disulfide-linked dimers.

Scavenger receptors have been implicated in the development of atherosclerosis and other macrophage-associated functions. The structures and processing of type I and type II bovine macrophage scavenger receptors were examined using polyclonal anti-receptor antibodies. Pulse/chase metabolic labeling experiments showed that both types of scavenger receptors expressed in Chinese hamster ovary (CHO) cells behaved as typical cell surface membrane glycoproteins. They were synthesized as endoglycosidase H-sensitive precursors which were converted to endoglycosidase H-resistant mature forms expressed on the cell surface. The reduced precursor and mature forms were doublets on sodium dodecyl sulfate-gel electrophoresis, primarily because of heterogeneous N-glycosylation. The approximate molecular sizes were: type I precursor, 65/63 kDa; type I mature, 82/76 kDa; type II precursor, 57/53 kDa; and type II mature, 72/65 kDa. During post-translational processing, the cysteine-rich C terminus (SRCR domain) of some of the type I receptors was proteolytically removed to form a relatively stable, approximately 69-kDa degradation product. Type II receptors differ from type I receptors in that they do not have SRCR domains and an analogous proteolytic cleavage was not observed. Several experiments provided strong evidence that the Gly-X-Y-repeat domains in the scavenger receptors oligomerize into collagenous triple helices. For example, alpha,alpha'-dipyridyl, an inhibitor of the collagen-modifying enzymes prolyl and lysyl hydroxylases, interfered with both the kinetics and nature of post-translational receptor processing, and both precursor and mature forms of the receptors in intact cells could be cross-linked with difluorodinitrobenzene into reduction-resistant trimers. In intact cells, precursor receptor trimers (type I, 198 kDa; type II, 176 kDa) were assembled in the endoplasmic reticulum by the noncovalent association of monomers and Cys83-disulfide-linked dimers (type I, 129 kDa; type II, 119 kDa). When cells were lysed in the absence of the sulfhydryl trapping agent iodoacetamide, oxidation of the side chain of Cys17 in the cytoplasmic domain leads to the artifactual formation of reduction-sensitive covalently linked trimers. The approximate masses of the mature dimer and trimer forms were 162 and 237 kDa for type I receptors and 147 and 219 kDa for type II receptors. Cys83-disulfide-linked dimer formation was not required for function because mutant receptors (Cys83----Gly83) assembled into trimers of noncovalently associated monomers and exhibited normal receptor activity. Treatment of cells with difluorodinitrobenzene cross-linked some of the receptors into complexes larger than trimers, raising the possibility that the trimers may assemble into higher order oligomers.     

Translocation of proteins into rat liver mitochondria. Existence of two different precursor polypeptides of liver fumarase and import of the precursor into mitochondria

Two different putative precursor polypeptides of rat liver fumarase were synthesized when RNA prepared from rat liver were translated in vitro using the rabbit reticulocyte lysate system. One of these putative precursor polypeptides (P1) was synthesized as a larger molecular mass than the mature subunit of fumarase (45,000 daltons) by about 5,000 daltons and the other (P2) had the same molecular mass as the mature enzyme. When the 35S-labeled cell-free translation products were incubated with rat liver mitochondria at 30 degrees C, P1 and the 35S-labeled mature size fumarase were associated with the mitochondria. Of these, the 35S-labeled mature size fumarase was resistant to externally added protease, but P1 was not, indicating that the 35S-labeled mature size fumarase was located in the mitochondrial matrix. The following observations strongly suggested that the 35S-labeled mature size fumarase in mitochondria was derived from P1, which was energy-dependently imported and concomitantly processed to the mature size. 1) The amount of the 35S-labeled mature size fumarase recovered from the mitochondria increased proportionally to the duration of incubation, while the amount of P1 recovered from the post-mitochondrial and mitochondrial fractions decreased with the duration of the incubation. 2) Only P1 could bind with the mitochondrial outer membrane at 0 degrees C even in the presence of an uncoupler of the oxidative phosphorylation but P2 did not. 3) P1 bound to the mitochondrial outer membrane was imported into the matrix, when the mitochondria binding only P1 at 0 degrees C was reisolated and incubated at 30 degrees C in the presence of an energy-generating system. The specific receptor was involved in the binding of P1 to mitochondria, since a high concentration of NaCl did not interfere with the binding of P1 to the membrane and did not discharge P1 bound onto the membrane. It was shown that P1 formed an aggregate composed of 6 to 8 molecules and P2 was a dimer in the cell-free translation mixture and that P1 and P2 were enzymatically inactive. These results suggest that the precursor for the mitochondrial enzyme has a larger molecular weight than that of the mature enzyme, whereas the precursor for the cytosolic enzyme has the same molecular weight as the mature enzyme.     

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.