Phaseolus vulgaris cytoplasmic leucyl-tRNA synthetase. Purification and comparison of its catalytic, structural, and immunological properties with those of the chloroplastic enzyme

The cytoplasmic leucyl-tRNA synthetase was purified from bean (Phaseolus vulgaris) leaves. After ammonium sulfate fractionation and chromatography on Sephadex G-50, DEAE-cellulose, hydroxylapatite, and phosphocellulose, complete purification was achieved by blue Sepharose CL-6B chromatography using specific elution with pure yeast tRNALeu1. The enzyme was purified 1050-fold and had a specific activity of 940 nmol of leucyl-tRNA formed/min/mg of protein. Polyacrylamide gel electrophoresis of the native enzyme showed one band, but the denatured enzyme showed two bands. These two protein bands are structurally related. The smallest protein appears to be a cleavage product from the largest one, suggesting the presence of a sensitive cleavage site in the cytoplasmic leucyl-tRNA synthetase. The cytoplasmic enzyme is a monomer (Mr = 130,000), larger than its chloroplastic counterpart (Mr = 120,000). The two enzymes differ in their substrate (tRNA) specificity, tryptic peptide map, and amino acid composition. Antibodies were raised against the cytoplasmic enzyme and against the chloroplastic enzyme and no cross-immunological reaction was detected, showing that the two enzymes do not share any antigenic determinant. Taken together, these results suggest that P. vulgaris cytoplasmic and chloroplastic leucyl-tRNA synthetases are coded for by different genes.     

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases from Euglena gracilis. Comparative study of their structural properties

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126,000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.     

Chloroplastic and cytoplasmic valyl- and leucyl- tRNA synthetases from Euglena gracilis: Comparative study of their structural properties

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126 000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.     

The biogenesis of the cyanellae of Cyanophora paradoxa. III. In vitro synthesis of cyanellar polypeptides using separated cytoplasmic and cyanellar RNA

RNA from Cyanophora paradoxa was separated into cytoplasmic and cyanellar fractions by using a combination of subcellular fractionation and oligo-dT chromatography. In vitro translation of the separated cytoplasmic and cyanellar RNAs in a rabbit reticulocyte lysate system in the presence of [35S]methionine resulted in the incorporation of radiolabel into electrophoretically distinct sets of polypeptides. Monospecific and polyspecific antibodies that react with cyanellar polypeptides were used to probe the in vitro translation products by indirect immunoprecipitation by using Staphylococcus protein A conjugated to Sepharose beads. The results indicate that linker polypeptide L1 of the phycobilisome, the gamma subunit of coupling factor CF1, and subunit II of PS I are synthesized in the cytoplasm as precursor molecules that are 5-8 kDa larger than their mature sizes. Antibodies directed against the psbA gene product (the D1 protein) precipitated a polypeptide found in the translation products of the cyanellar RNA-directed reactions, which is about 1.5 kDa larger than the mature protein.     

Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.     

Biochemical comparison of the Neurospora crassa wild type and the temperature-sensitive and leucine-auxotroph mutant leu-5. Purification of the cytoplasmic and mitochondrial leucyl-tRNA synthetases and comparison of the enzymatic activities and the degradation patterns

The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 degrees C) and mutant (grown at 28 degrees C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28 degrees C and transferred then to 37 degrees C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37 degrees C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish and difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 degrees C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of approximately 1:600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme.     

The C-terminal appended domain of human cytosolic leucyl-tRNA synthetase is indispensable in its interaction with arginyl-tRNA synthetase in the multi-tRNA synthetase complex

Human cytosolic leucyl-tRNA synthetase is one component of a macromolecular aminoacyl-tRNA synthetase complex. This is unlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes. There is little known about it, since the purified enzyme has been unavailable. Herein, human cytosolic leucyl-tRNA synthetase was heterologously expressed in a baculovirus system and purified to homogeneity. The molecular mass (135 kDa) of the enzyme is close to the theoretical value derived from its cDNA. The kinetic constants of the enzyme for ATP, leucine, and tRNA(Leu) in the ATP-PP(i) exchange and tRNA leucylation reactions were determined, and the results showed that it is quite active as a free enzyme. Human cytosolic leucyl-tRNA synthetase expressed in human 293 T cells localizes predominantly to the cytosol. Additionally, it is found to have a long C-terminal extension that is absent from bacterial and yeast LeuRSs. A C-terminal 89-amino acid truncated human cytosolic leucyl-tRNA synthetase was constructed and purified, and the catalytic activities, thermal stability, and subcellular location were found to be almost identical to native enzyme. In vivo and in vitro experiments, however, show that the C-terminal extension of human cytosolic leucyl-tRNA synthetase is indispensable for its interaction with the N-terminal of human cytosolic arginyl-tRNA synthetase in the macromolecular complex. Our results also indicate that the two molecules interact with each other only through their appended domains.     

The interaction between biologically inactive tRNA conformers and leucyl-tRNA synthetase from rabbit liver

The interaction between tRNA conformers inactive in aminoacylation and leucyl-tRNA synthetase has been investigated. Heat inactivation of the enzyme in the presence of inactive tRNA conformers is shown to lead to a marked increase of inactivation rate while active tRNA conformers, on the other hand, reveal a protecting effect. To study the properties of the enzyme complexed with different tRNA conformers limited proteolysis has been used. Active tRNA conformers are found to protect leucyl-tRNA synthetase against hydrolysis while inactive ones tend to intensify it. Inactive tRNA conformers are also shown to inhibit the aminoacylation of native tRNA in vitro. On the basis of these data biologically inactive conformers of animal tRNA are assumed to form an unproductive complex with leucyl-tRNA synthetase and the structure of the enzyme involved in such interaction is supposed to be more labile and 'extended' than that in complex with active tRNA conformers.     

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.     

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.     

In vitro translation of intestinal sucrase-isomaltase and glucoamylase

It is has been proposed that both sucrase-isomaltase and glucoamylase are initially synthesized as large single-chain precursors which are then processed to heterodimers. We have tested this hypothesis by in vitro translation of their mRNAs. The primary translation product of sucrase-isomaltase mRNA was a single polypeptide of Mr = 190,000. Similar experiments using antiserum against glucoamylase revealed a single polypeptide of Mr = 145,000. These results are consistent with the single chain precursor hypothesis for sucrase-isomaltase. However, the glucoamylase product (145 kDa) is too small to be processed to a heterodimer of Mr = 230,000. Moreover, the mature subunits (Mr = 135,000 and 125,000) probably are derived from the 145 kDa precursor by proteolytic trimming and must include and share most of the precursor protein.     

Further studies of glycosylation and intracellular transport of lactase-phlorizin hydrolase in rat small intestine.

Previous studies [Büller, Montgomery, Sasak & Grand (1987) J. Biol. Chem. 262, 17206-17211] have demonstrated that lactase-phlorizin hydrolase is inserted into the microvillus membrane (MVM) as a large precursor of approx. 220 kDa, which then undergoes two proteolytic cleavage steps to become the 130 kDa mature MVM protein. In order to assess the role of glycosylation in intracellular transport, the processing of this enzyme has been studied in the presence of castanospermine, an inhibitor of N-linked oligosaccharide modification and subsequent treatment with two endoglycosidases, endo-beta-N-acetyl-glucosaminidase (endo-H) and peptide:N-glycosidase-F (N-glycanase). We now show that the intracellular precursor (205 kDa) undergoes carbohydrate processing (220 kDa) and transport to the MVM where its further proteolytic cleavage is as described. Treatment of the intracellular 205 kDa precursor with either endo-H which cleaves only high-mannose N-linked oligosaccharides, or with N-glycanase, which cleaves both high-mannose and complex N-linked oligosaccharides, results in the conversion of the 205 kDa protein band to one of 195 kDa. These data suggest that the 205 kDa precursor contains only high-mannose N-linked carbohydrates, and that the unglycosylated nascent protein is 195 kDa. In the presence of castanospermine, an intracellular precursor of approx. 210 kDa is observed. When treated with endo-H or N-glycanase, this form also produces a protein of 195 kDa. The transport of the intracellular precursor to the MVM and further proteolytic processing is not blocked by the inhibitor. However, all MVM forms of lactase-phlorizin hydrolase show an increase of approx. 5 kDa. Treatment of these three MVM forms with endo-H indicates the increased presence of high mannose oligosaccharides in comparison with non-castanospermine-treated forms. The susceptibility to endo-H of the 130 kDa MVM band synthesized in the absence of castanospermine implies the presence of high-mannose N-linked oligosaccharides in the mature form of lactase-phlorizin hydrolase. Incubation of these MVM forms with N-glycanase further reduces their electrophoretic mobility, indicating the presence of complex N-linked oligosaccharides in the MVM forms, in contrast with the intracellular precursor. Altered glycosylation reduces but does not abolish intracellular transport of lactase-phlorizin hydrolase to the MVM.     

Biosynthesis and processing of lysosomal acid phosphatase in cultured human cells

The biosynthesis of lysosomal acid phosphatase was studied in a normal human embryonic lung cell line, WI-38. Cells were labeled with radioactive leucine under a variety of conditions, the enzyme was immunoprecipitated using a monospecific antiserum raised against human liver lysosomal acid phosphatase, and the products were separated by electrophoresis and were visualized by fluorography. Lysosomal acid phosphatase constitutes 60% of the total tartrate-inhibitable acid phosphatase in WI-38. It is initially synthesized as a high-molecular-weight precursor polypeptide of 69 kDa. The precursor polypeptide is rapidly glycosylated and processed to a mature enzyme of 53-45 kDa via intermediates of 65 and 60 kDa in WI-38 cells. The 69-kDa precursor polypeptide is also converted to larger precursor polypeptides of 74 and 80 kDa. The multiplicity of precursor polypeptides is due at least in part to differences in the glycosylation and phosphorylation of the polypeptides. Sensitivity of phosphorylated oligosaccharide chains from precursor, mature and small polypeptides to endo-beta-hexosaminidase H-catalyzed cleavage suggests the presence of high-mannose phosphorylated oligosaccharide chains similar to those present on many other lysosomal enzymes. The effects of tunicamycin and ammonium chloride were also studied. In contrast to the effect of ammonium chloride on arylsulfatase A secretion, the lysosomal acid phosphatase in WI-38 cells was not secreted in the presence of NH4Cl. This is consistent with the existence of an alternate route for the transfer of lysosomal acid phosphatase into lysosomes. This alternate route may be the reason that I-cell fibroblasts contain a normal level of lysosomal acid phosphatase.     

Activation of human mitochondrial lysyl-tRNA synthetase upon maturation of its premitochondrial precursor

The cytoplasmic and mitochondrial species of human lysyl-tRNA synthetase are encoded by a single gene by means of alternative splicing of the KARS1 gene. The cytosolic enzyme possesses a eukaryote-specific N-terminal polypeptide extension that confers on the native enzyme potent tRNA binding properties required for the vectorial transfer of tRNA from the synthetase to elongation factor EF1A within the eukaryotic translation machinery. The mitochondrial enzyme matures from its precursor upon being targeted to that organelle. To understand how the cytosolic and mitochondrial enzymes are adapted to participate in two distinct translation machineries, of eukaryotic or bacterial origin, we characterized the mitochondrial LysRS species. Here we report that cleavage of the precursor of mitochondrial LysRS leads to a mature enzyme with reduced tRNA binding properties compared to those of the cytoplasmic counterpart. This adaptation mechanism may prevent inhibition of translation through sequestration of lysyl-tRNA on the synthetase in a compartment where the bacterial-like elongation factor EF-Tu could not assist in its dissociation from the synthetase. We also observed that the RxxxKRxxK tRNA-binding motif of mitochondrial LysRS is not functional in the precursor form of that enzyme and becomes operational after cleavage of the mitochondrial targeting sequence. The finding that maturation of the precursor is needed to reveal the potent tRNA binding properties of this enzyme has strong implications for the spatiotemporal regulation of its activities and is consistent with previous studies suggesting that the only LysRS species able to promote packaging of tRNA(Lys) into HIV-1 viral particles is the mature form of the mitochondrial enzyme.     

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.     

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.     

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.     

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).