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.     

Lysosomal acid alpha-glucosidase consists of four different peptides processed from a single chain precursor

Pompe's disease is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and N-glycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78-113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204-782) contains the 3.9- and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.     

Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase.

One of the major proteins of the Saccharomyces cerevisiae cell wall, a beta-glucanase (BGL2 gene product), has been isolated and purified to homogeneity under conditions for preserving enzyme activity. The study of enzyme properties of the protein revealed that it is an endo-beta-1,3-glucanase and not an exoglucanase as reported previously (F. Klebl and W. Tanner, J. Bacteriol. 171:6259-6264, 1989). The examination of the glucanase structure showed that the lower apparent molecular mass of the protein (29 kDa) compared with what was calculated from the amino acid sequence of the enzyme (33.5 kDa) is due to anomalous migration in sodium dodecyl sulfate gels and not to posttranslational processing of the polypeptide chain. Of two potential N glycosylation sites at Asn-202 and Asn-284, only the latter site is glycosylated. The overproduction of the beta-glucanase from the high-copy-number plasmid brought about a significant decrease in the growth rate of transformed yeast cells.     

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.     

Peptide amidation: evidence for multiple molecular forms of the amidating enzyme

Amidating enzyme extracted from porcine pituitary was separated into glycosylated and non-glycosylated forms by fractionation on a column of Concanavalin-A Sepharose. The molecular weights of the species present were assessed by HPLC gel exclusion chromatography, which demonstrated that both the glycosylated and the non-glycosylated forms of the enzyme comprise multiple components. The apparent molecular weights of the non-glycosylated forms ranged from approximately 35 kDa to 100 kDa; the glycosylated enzyme contained species with molecular weights ranging from 65 kDa to 135 kDa. Similar proportions of glycosylated to non-glycosylated enzyme (approximately 1:4) were found in the anterior and posterior regions of the pituitary; higher proportions (approximately 1:1) were observed in the thyroid, adrenals and pancreas. The glycosylated forms of the amidating enzyme were shown to exhibit the same mandatory requirement for copper as the non-glycosylated forms, and no differences were seen in respect of their stimulation by dopamine or their pH optima. Both forms catalysed the hydroxylation of glyoxylic acid phenylhydrazone, indicating a common mechanism of action. By these criteria, glycosylation does not affect the activity of the amidating enzyme.     

High yield synthesis and characterization of phosphorylated recombinant human procathepsin D expressed in mammalian cells

We used a vaccinia virus expression system for the production of recombinant human cathepsin D (CD), a lysosomal protease implicated in various patho-physiological processes including cancer, neurodegeneration, and development. The recombinant protein was successfully expressed in various human and non-human cells. It was correctly synthesized as a glycosylated 53 kDa precursor (proCDrec) that reacted with a polyclonal antibody against residues 7-21 of the propeptide sequence. In contrast to the control, in cells infected with the recombinant virus proCDrec was largely secreted into the culture medium, although it contained high-mannose oligosaccharides with uncovered mannose-6-phosphate residues. Intracellular proCDrec was processed into the 48 kDa intermediate single-chain and the 31 plus 13 kDa double-chain forms, however, the processing was slower than in normal cells. A method based on Pepstatin A-affinity chromatography allowed to isolate the recombinant protein from the medium of infected cells. Based on its latency in activity assay at acid pH and on its reactivity with antibodies specific for the N-terminus, the purified protein was judged to be in the inactive precursor form. During incubation at acid pH the purified proCDrec underwent autocatalytic processing and acquired pepstatin A-sensitive enzyme activity, as expected for correctly folded proCD. Antiserum raised in rabbits against proCDrec specifically reacted with human, but not with mouse proCD under non-denaturing conditions. We conclude that our vaccinia virus-directed proCDrec displays structural and functional features resembling those of native human proCD. This system can therefore be exploited for the synthesis of large quantities of human proCD, allowing further studies on the structure and function of this interesting protein.     

Identification of a new active site for autocatalytic processing of penicillin acylase precursor in Escherichia coli ATCC11105

Penicillin acylase (PA) from Escherichia coli ATCC11105 is a periplasmic heterodimer consisting of a 24 kDa small subunit and a 65 kDa large subunit. It is synthesized as a single 96 kDa precursor and then matures to functional PA via a posttranslational processing pathway. The GST-PA fusion protein expression system was established for monitoring the precursor PA processing in vitro. The purified PA precursor was processed into mature PA the same way as in vivo, but pH dependently. From the primary sequence analysis, we identified a putative conserved lysine residue (K299) responsible for the pH dependent processing. The substitution of K299 residue by site-directed mutagenesis affected both the enzyme activity and the precursor PA processing in vivo. Furthermore, it was shown that the processing rates of wild-type and mutant precursor PAs depended on the pKa values of their side chain R group. These results demonstrated that the lysine residue (K299) was involved in the precursor processing of PA together with N-terminal serine residue (S290) of the large subunit.     

Rotaviruses code for two types of glycoprotein precursors

Rotaviruses are nonenveloped viruses that code for two glycoproteins: a structural glycoprotein (VP7) and a nonstructural glycoprotein (NS29). The precursor to VP7 (37K) was shown to contain a 1.5K cleavable signal sequence. The 37K precursor was authentically processed (signal sequence cleaved and the polypeptide "core" glycosylated) when synthesized in a cell-free system supplemented with dog pancreatic microsomes. Similar experiments were performed with the nonstructural glycoprotein precursor (20K); however, the 20K precursor contained an integral (noncleavable) signal sequence. Both precursors were inserted into membranes cotranslationally and both glycosylated products underwent posttranslational oligosaccharide processing. The results suggest a morphogenetic scheme for the simian rotavirus SA11.     

Characterization of the intracellular processing and secretion of hepatic lipase in FU5AH rat hepatoma cells

The processing and secretion of newly synthesized hepatic lipase was characterized in FU5AH rat hepatoma cells. Pulse-chase experiments revealed two immunoreactive species with apparent molecular weights of 55,400 and 57,600. The 55.4 kDa species was detectable only in cell extracts, whereas the 57.6 kDa species was present in both cell extracts and media. Following a 5 min pulse with L-[35S]methionine and a 10 min chase, these two species represented only 0.003% of the total labelled protein. Quantitation of the 55.4 kDa and 57.6 kDa species in a chase time course taken together with their respective sensitivity and resistance to digestion with endo-beta-N-acetylglucosaminidase H indicates that the 55.4 kDa species is a high mannose precursor to the mature 57.6 kDa enzyme which contains only complex N-linked oligosaccharides. From a time course of endo-beta-N-acetylglucosaminidase H digestion, it was determined that hepatic lipase contains a minimum of two N-linked oligosaccharides. Treatment of the 55.4 kDa species with endo-beta-N-acetylglucosaminidase H yields a protein with a kDa value similar to that observed after treatment of the mature secreted enzyme with endo-beta-N-acetylglucosaminidase F or trifluoromethanesulfonic acid. Therefore, processing of N-linked oligosaccharides is probably the only post-translational modification responsible for the observed change in the apparent molecular weight of hepatic lipase. The half-residence times of hepatic lipase in the endoplasmic reticulum-cis Golgi region and in the cell were estimated at 34 min and 57 min, respectively. Newly synthesized hepatic lipase in Fu5AH cells is secreted constitutively and is not stored in an intracellular pool. Finally, little of the newly synthesized enzyme is degraded during the course of a 1 h chase.     

Unique autolytic cleavage of human myeloperoxidase. Implications for the involvement of active site MET409.

Myeloperoxidase (MPO) is a functionally important component of the normal human neutrophil host defense system. This enzyme possesses a dimeric structure composed of two heavy subunit (55-63 kDa)/light subunit (10-15 kDa) protomers, each of which is associated with a heme-like prosthetic group. In addition, MPO species of approximately 38 and 22 kDa have been reported by many different investigators, but their nature and mode of origin are not understood. In the present study, we demonstrate that when MPO is heated under nonreducing, denaturing conditions, these two species are produced via a novel autolytic cleavage of the heavy subunit. The 38-kDa species was isolated by fast-protein liquid chromatography and identified by sequencing as the carboxyl-terminal portion of the heavy subunit, and the cleavage was shown to occur exclusively between Met409 and Pro410. In order to further characterize this unusual cleavage reaction, the 22-kDa species was digested with endoproteinase Asp-N, and the peptide corresponding to its carboxyl terminus was isolated and analyzed by sequencing and mass spectrometry. These data indicated that during cleavage of the heavy subunit, Met409 was converted to homoserine lactone. Thus, the cleavage appeared to formally resemble the cyanogen bromide-dependent cleavage of Met-X peptide bonds. Recent x-ray crystallographic data for canine MPO have indicated that Met409 is in close proximity to the heme-like prosthetic group of MPO. Our studies suggest that interaction of Met409 with this group leads to the formation of a methionyl sulfonium derivative which undergoes intramolecular rearrangement with subsequent peptide cleavage under nonreducing conditions. This derivative may be, at least in part, responsible for the unusual spectral characteristics and enzymatic properties of the enzyme. The primary structure of the 22-kDa MPO species is also reported, and direct evidence is provided for asparagine-linked oligosaccharide moieties at two of the three predicted glycosylation sites.     

Synthesis and processing of lysosomal alpha-fucosidase in cultured human fibroblasts

The lysosomal enzyme alpha-L-fucosidase from human skin fibroblasts is synthesized as a 53 kDa glycosylated precursor which is then proteolytically processed to a 50 kDa mature form. This was confirmed by pulse-chase labeling studies with chase times up to 72 h. In fibroblasts treated with 1-deoxymannojirimycin to prevent trimming of high mannose oligosaccharides, endoglycosidase H (endo H) treatment completely deglycosylated and reduced the size of immunoprecipitated alpha-fucosidase by 4-5 kDa, suggesting the presence of two oligosaccharide units. Endoglycosidase H and endo F studies on untreated alpha-fucosidase suggested the presence of one complex-type and one high mannose-type unit, and that the final processing from 53 to 50 kDa did not involve the removal of carbohydrate. Processing was inhibited by the thiol proteinase inhibitor Ep-459, but not by Ep-475 or leupeptin. Since Ep-459 treatment increased both alpha-fucosidase activity (3-fold) and the amount of immunoprecipitable alpha-fucosidase protein in normal human skin fibroblasts, this suggests a role for cysteine-like proteinases either directly or indirectly in lysosomal hydrolase processing and turnover. Subcellular fractionation studies revealed that the proteolytic processing of the 53 kDa precursor to the 50 kDa mature form occurred in the lysosome, or some other dense organelle.     

Conformational and thermal stability of mature dimeric human myeloperoxidase and a recombinant monomeric form from CHO cells

Myeloperoxidase (MPO) is a lysosomal heme enzyme present in the azurophilic granules of human neutrophils and monocytes. It is a critical element of the human innate immune system by exerting antimicrobial effects. It is a disulfide bridged dimer with each monomer containing a light and a heavy polypeptide and its biosynthesis and intracellular transport includes several posttranslational processing steps. By contrast, MPO recombinantly produced in Chinese hamster ovary cell lines is monomeric, partially unprocessed and contains a N-terminal propeptide (proMPO). It mirrors a second form of MPO constitutively secreted from normal bone marrow myeloid precursors. In order to clarify the impact of posttranslational modifications on the structural integrity and enzymology of these two forms of human myeloperoxidase, we have undertaken an investigation on the conformational and thermal stability of leukocyte MPO and recombinant proMPO by using complementary biophysical techniques including UV-Vis, circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Mature leucocyte MPO exhibits a peculiar high chemical and thermal stability under oxidizing conditions but is significantly destabilized by addition of dithiothreitol. Unfolding of secondary and tertiary structure occurs concomitantly with denaturation of the heme cavity, reflecting the role of three MPO-typical heme to protein linkages and of six intra-chain disulfides for structural integrity by bridging N- and C-terminal regions of the protein. Recombinant monomeric proMPO follows a similar unfolding pattern but has a lower conformational and thermal stability. Spectroscopic and thermodynamic data of unfolding are discussed with respect to the known three-dimensional structure of leukocyte MPO as well as to known physiological roles.     

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.     

Expression and activation of the vacuolar processing enzyme in Saccharomyces cerevisiae

Vacuolar processing enzymes (VPEs) are cysteine proteinases responsible for maturation of various vacuolar proteins in plants. A larger precursor to VPE synthesized on rough endoplasmic reticulum is converted to an active enzyme in the vacuoles. In this study, a precursor to castor bean VPE was expressed in a pep4 strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE. Two VPE proteins of 59 and 46 kDa were detected in the vacuoles of the transformant. They were glycosylated in the yeast cells, although VPE is not glycosylated in plant cells in spite of the presence of two N-linked glycosylation sites. During the growth of the transformant, the level of the 59 kDa VPE increased slightly until a rapid decrease occurred after 9 h. By contrast, the 46 kDa VPE appeared simultaneously with the disappearance of the 59 kDa VPE. Vacuolar processing activity increased with the accumulation of the 46 kDa VPE, but not of the 59 kDa VPE. The specific activity of the 46 kDa VPE was at a similar level to that of VPE in plant cells. The 46 kDa VPE instead of proteinase A mediated the conversion of procarboxypeptidase Y to the mature form. This indicates that proteinase A responsible for maturation of yeast vacuolar proteins can be replaced functionally by plant VPE. These findings suggest that an inactive VPE precursor synthesized on the endoplasmic reticulum is transported to the vacuoles in the yeast cells and then processed to make an active VPE by self-catalytic proteolysis within the vacuoles.     

Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105

The two constituent subunits of the enzyme penicillin acylase from Escherichia coli strain ATCC 11105 are derived from a single precursor polypeptide by post-translational processing. Mutant penicillin acylase precursors were constructed carrying insertions and deletions in various domains and they were analysed for their processing behaviour. It was found that an endopeptide region of appropriate size and an intact C-terminus were absolutely necessary for the maturation process. Internal deletions within the beta-subunit domain also prevented post-translational cleavage. Processing competence, therefore, was not merely determined by the amino acid sequence in the vicinity of the processing sites but relied on a correct overall conformation of the protein. The processing pathway in vivo proceeds via an intermediate comprising the alpha subunits plus endopeptide and is thus identical to the pathway which has been determined previously by in vitro analysis. The post-translational modification of the precursor is probably not carried out by a specific processing enzyme(s) as the heterologous expression of the penicillin acylase (pac) structural gene yielded processed and active enzyme in different enterobacteria and in a Pseudomonas species.     

The Alzheimer amyloid precursor protein. Identification of a stable intermediate in the biosynthetic/degradative pathway

The amyloid forming beta-peptide of Alzheimer's disease is synthesized as part of a larger integral membrane precursor protein (beta APP) of which three alternatively spliced versions of 695, 751, and 770 amino acids have been described. A fourth beta APP form of 563 amino acids does not contain the beta-peptide region. Recent experiments using transient expression in HeLa cells (Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L., and Beyreuther, K. (1989) Cell 57, 115-126) indicate that the beta APP undergoes several posttranslational modifications including the cleavage and secretion of a large portion of its extracellular domain. The nature and fate of the fragment that remains cell-associated following this cleavage has not heretofore been described. The metabolism of this fragment may have particular significance in Alzheimer's disease since it must contain at least part of the beta-peptide. To study the metabolic fate of this fragment, we have established cell lines overexpressing the 695- and 751-amino acid versions of beta APP. Pulse-chase studies show that this system is similar to the HeLa cell system in that both proteins are synthesized first as membrane-bound proteins of approximately 98 and 108 kDa carrying asparagine-linked sugar side chains and are subsequently processed into higher molecular mass forms by the attachment of sulfate, phosphate, and further sugar groups including sialic acid, adding approximately 20 kDa in apparent molecular mass. The mature form of beta APP is cleaved and rapidly secreted, leaving an 11.5-kDa fragment with the transmembrane region and the cytoplasmic domain behind in the cell. This fragment is stable with a half-life of at least 4 h.     

In vitro synthesis of bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases. Characterization and processing of a precursor polypeptide for the chloroplast enzyme

Bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases differ in their structural and catalytic properties and do not share common antigenic determinants. Polyadenylated mRNAs, prepared from young bean leaves, have been translated in vitro in a rabbit reticulocyte lysate cell-free system. The newly synthesized polypeptides have been submitted to immunoadsorption on protein A-Sepharose in the presence of the antibodies raised against the chloroplastic or the cytoplasmic leucyl-tRNA synthetase. The specificity of the immunoadsorption has been checked by competition experiments involving the pure enzymes. Bean chloroplastic leucyl-tRNA synthetase is synthesized in vitro from a polyadenylated mRNA as a precursor polypeptide of 130 kDa, which is somewhat larger than the mature enzyme of 120 kDa. Bean cytoplasmic leucyl-tRNA synthetase is synthesized in vitro as a polypeptide which has the size of the mature monomer (130 kDa). Processing of the precursor polypeptide of the chloroplastic leucyl-tRNA synthetase, yielding the mature enzyme, has been obtained by performing the in vitro translation in the presence of canine pancreatic microsomal membranes. These results suggest that in vivo bean chloroplastic leucyl-tRNA synthetase could be synthesized in the cytoplasm as a precursor which would be transported into the chloroplasts.     

Processing and Secretion of Lysosomal Acid α-Glucosidase In Tetrahymena Wild Type and Secretion-Deficient Mutant Cells

ABSTRACT. The proteolytic processing and secretion of a lysosomal enzyme, acid α-glucosidase, was studied by pulse-chase labeling with [³⁵S]methionine in Tetrahymena thermophila CU-399 cells treated with ammonium chloride. This cell secreted a large amount of acid α-glucosidase into the cultured medium during starvation. the secretion was found to be repressed by addition of ammonium chloride (NH₄Cl). Acid α-glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min. This mature enzyme was secreted into the media within 2-3 h after chase, whereas the precursor form was not secreted by either control cells or NH₄Cl-treated cells. NH₄Cl did not affect the processing of the precursor acid α-glucosidase. Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS-1 defective in lysosomal enzyme secretion. Furthermore, the purified extracellular (CU-399) and intracellular (MS-1) acid a-glucosidases were the same in molecular mass (105 kDa) and enzymatic properties. They contained no mannose 6-phosphate residues in N-linked oligosaccharides. These results suggested that unlike mammalian cells, Tetrahymena acid α-glucosidase may be transferred to lysosomes by a mannose 6-phosphate receptor-independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.     

In vitro translation and processing of rat kidney gamma-glutamyl transpeptidase

Rat kidney gamma-glutamyl transpeptidase is composed of two nonidentical glycosylated subunits. The enzyme is localized on the lumenal surface of the brush-border membranes of proximal tubule epithelial cells; it is attached to the membranes via an NH2-terminal segment of the larger of the two subunits. Tissue-labeling experiments followed by immunoprecipitation with antibodies directed against the enzyme and its two subunits demonstrate that a glycosylated single chain precursor (Mr = 78,000), containing the elements of both the subunits, is initially synthesized. Pulse-chase studies in the presence of pactamycin, and inhibitor of protein synthesis initiation, indicate that the larger of the two subunits is located at the NH2 terminus of the Mr = 78,000 precursor. The initial events in the biosynthesis and processing of gamma-glutamyl transpeptidase were investigated by in vitro translation of rat kidney mRNA. Such translation results in the synthesis of a Mr = 63,000 unglycosylated polypeptide which has been shown immunologically to contain the domains for both subunits. The Mr = 63,000 species is processed to a Mr = 78,000 core-glycosylated polypeptide when translation of mRNA is carried out in the presence of dog pancreas microsomes. This processing does not appear to be associated with cleavage of an NH2-terminal leader sequence. The Mr = 78,000 polypeptide is integrated into the microsomal membranes with an orientation that is analogous to that found on the brush-border membranes. Glycosylation and membrane integration of transpeptidase are cotranslational events. Upon longer incubation, the Mr = 78,000 species sequestered within the microsomal vesicles is cleaved to species corresponding in size to the two subunits of the kidney enzyme.