Intracellular maturation and secretion of acid phosphatase of Saccharomyces cerevisiae

To elucidate intracellular maturation and secretion of acid phosphatase of Saccharomyces cerevisiae we prepared a monoclonal antibody that recognizes specifically the protein moiety of this cell surface glycoprotein. With this antibody membranes and soluble fractions of wild-type cells, grown in low-phosphate medium in the presence and absence of tunicamycin, were examined by the immunoblot technique. Similarly, secretory mutants, blocked at distinct steps in the secretory pathway at the restrictive temperature as well as a strain harboring several copies of the structural gene PHO5 for repressible acid phosphatase, were analyzed. The data suggest the following sequence of events in acid phosphatase maturation and secretion: three unglycosylated precursors with molecular masses of 60 kDa, 58 kDa and 56 kDa are synthesized into membranes of the endoplasmic reticulum, where these are core glycosylated in a membrane-bound form. They appear on sodium dodecyl sulfate gels as bands with molecular masses of 76 kDa and 80 kDa. Owing to a rate-limiting maturation step, occurring after core glycosylation, they can accumulate in a membrane-bound form. At the Golgi apparatus outer carbohydrate chains are attached to the core and the enzyme appears in a soluble form, indicating a release of acid phosphatase from the membrane between the endoplasmic reticulum and the Golgi. Pulse-chase experiments suggest that the time for acid phosphatase synthesis and its transport to the Golgi is about 5 min.     

Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulated macrophages

The biosynthesis and processing of cachetin/tumor necrosis-factor (TNF) were examined in the murine macrophage-like cell line RAW 264.7. Lipopolysaccharide-stimulated cells secreted both glycosylated and nonglycosylated 17-kilodalton (kDa) mature cachectin/TNF into the culture medium. Secreted cachectin/TNF was derived from membrane-associated precursors that were precipitated by polyclonal antisera raised against either the mature protein or synthetic peptide fragments of the 79 amino acid cachectin/TNF prohormone sequence. About half of the precursors were N-glycosylated, apparently cotranslationally. The cachectin/TNF precursors were then proteolytically cleaved to release soluble mature cytokine into the medium, while the membrane-bound 14-kDa presequence remained cell associated. During the period of LPS stimulation, the amount of macrophage cell surface cachectin/TNF remained at a low level, suggesting that both nonglycosylated and glycosylated precursors of cachectin/TNF are efficiently cleaved by these cells. These findings suggest the presence of a unique mechanism for the secretion of cachectin/TNF.     

Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes

The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985) J. Cell Biol. 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D.A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986) J. Biol. Chem. 261, 14697-14703].     

Three Forms of α-Glucosidase from Welsh Onion (Allium fistulosum L.)

Tremerogen A-10 is an S-polyisoprenyl peptide mating pheromone secreted by the AB cells of the heterobasidiomycetous yeast Tremella mesenterica. We investigated the feasbility of the use of compactin (ML 236-B), a potent inhibitor of mevalonate synthesis in mammalian cells, for the study of the mating pheromone production. Compactin specifically inhibited mevalonate synthesis of the yeast cells without affecting protein synthesis. The secretion of tremerogen A-10 was effectively blocked by the drug. Accumulation of a large precursor polypeptide (M.W. 28,000) for the mature pheromone (M.W. 1,480) in the membrane fraction of compactin-treated cells was demonstrated by immunoprecipitation of ³⁵-S-labeled proteins. The results suggested that the addition of the nonpolar residue to a polypeptide precursor is important for the production of tremerogen A-10 especially in the intracellular transport and processing of the precursor molecules.     

Product of SEC53 is required for folding and glycosylation of secretory proteins in the lumen of the yeast endoplasmic reticulum

Yeast secretory mutant sec53 cells accumulate inactive secretory glycoprotein precursors that remain associated with the endoplasmic reticulum (ER) at the restrictive temperature (37 degrees C). The possibility that precursor polypeptides fail to penetrate completely into the ER lumen was tested by examining the protease accessibility of accumulated invertase, mating pheromone precursor prepro-alpha-factor and the vacuolar protein precursor procarboxypeptidase Y in cell lysates. In all three cases, the secretory protein precursors are protected from the action of exogenous protease unless the membrane is permeabilized by including Triton X-100 or saponin in the incubation. These results suggest that the sec53 defect allows complete polypeptide translocation. Consistent with this interpretation, the precursor of invertase accumulates in a signal peptide-processed form. In addition, invertase and prepro-alpha-factor precursors contain a small amount of possibly aberrant carbohydrate. In mutant cells or in wild type cells treated with tunicamycin, a 10-kDa fragment of the N terminus of mature invertase assumes a conformation that is resistant to trypsin with or without detergent. This domain may be associated with an ER protein or may simply assume an unusual conformation as a consequence of deficient glycosyl modification.     

Biosynthesis and secretion of M- and Z-type alpha 1-proteinase inhibitor by human monocytes. Effect of inhibitors of glycosylation and of oligosaccharide processing on secretion and function

The biosynthesis and secretion of M-type and Z-type alpha 1-antitrypsin was studied in human monocytes. In monocytes of PiMM individuals alpha 1-antitrypsin represented 0.08% of the newly synthesized proteins and 0.44% of the secreted proteins. Two molecular forms of alpha 1-antitrypsin could be identified: a 51-kDa intracellular form, susceptible to endoglucosaminidase H, thus representing the high-mannose type precursor form and a 56-kDa form resistant to endoglucosaminidase H which was secreted into the medium. Inhibition of de novo glycosylation by tunicamycin impaired the secretion of M-type alpha 1-antitrypsin by about 75% whereas inhibition of oligosaccharide processing by the mannosidase II inhibitor swainsonine did not alter the secretion of M-type alpha 1-antitrypsin. alpha 1-Antitrypsin secreted by human monocytes was functionally active as measured by complex formation with porcine pancreatic elastase. Even unglycosylated alpha 1-antitrypsin secreted by human monocytes treated with tunicamycin formed a complex with elastase. In monocytes of PiZZ individuals the secretion of alpha 1-antitrypsin was decreased. 72% of newly synthesized M-type alpha 1-antitrypsin, but only 35% of newly synthesized Z-type alpha 1-antitrypsin were secreted during a labeling period of 3 h with [35S]methionine. The 51-kDa form of Z-type alpha 1-antitrypsin accumulated intracellularly, whereas the 56-kDa form was secreted. Inhibition of oligosaccharide processing by swainsonine did not alter the decreased secretion of Z-type alpha 1-antitrypsin, whereas inhibition of de novo glycosylation by tunicamycin blocked the secretion of Z-type alpha 1-antitrypsin completely.     

Phosphorylation of caldesmon prevents its interaction with smooth muscle myosin

Caldesmon is known to bind to smooth muscle myosin. Ca2+/calmodulin-dependent phosphorylation of caldesmon completely blocks its interaction with myosin. Cleavage of caldesmon at its 2 cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) occurs initially at one site to yield 108-kDa and 21.2-kDa peptides and subsequently at the second site within the 108-kDa peptide to yield 85-kDa and 23.5-kDa fragments. The 23.5-kDa peptide retains the ability to bind to myosin. The N-terminal (95 kDa) and C-terminal (42 kDa) chymotryptic peptides of caldesmon were isolated and digested with NTCB: the C-terminal actin- and calmodulin-binding peptide was not cleaved, indicating that it does not contain either of the cysteine residues, whereas the 95-kDa N-terminal peptide was cleaved at two sites to yield 56-kDa, 23.5-kDa, and 21.2-kDa fragments. The arrangement of NTCB fragments in caldesmon is, therefore: 21.2 kDa/23.5 kDa/85 kDa from N to C terminus. Digestion of phosphorylated caldesmon with NTCB suggested a single phosphorylation site in the 21.2-kDa peptide and three sites in the 23.5-kDa peptide. These results lead to the development of a model whereby caldesmon may cross-link actin to myosin and such cross-linking is blocked by phosphorylation of caldesmon. This mechanism may explain the formation of reversible "latch bridges" which permit force maintenance at low levels of myosin phosphorylation in intact smooth muscles.     

Immobilization of soluble fibrin on factor XIIIa-coated polystyrene beads mediated by N-terminal fibronectin fragments. II. Demonstration of covalent adducts of fibrin peptide chains and fibronectin fragments.

Previous experiments had shown that the free N-terminal fibronectin 30-kDa-domain mediates binding of soluble 125I-fibrin to transamidase-coated polystyrene beads (H?rmann et al., Biol. Chem. Hoppe-Seyler 368, 669-674, 1987). Now, the formation of covalent adducts of the N-terminal fragment with fibrin peptide chains is demonstrated. Binding of soluble 125I-fibrin was performed in presence of N-terminal fibronectin 30-kDa or 70-kDa fragments. The material adsorbed was removed from the beads under reducing conditions and analysed by dodecylsulfate gel electrophoresis followed by autoradiography. The 30-kDa fragment gave rise to bands of 80 kDa and 180-200 kDa which were lacking in the products of the 70-kDa compound. Instead, they showed bands at 120 kDa and ca. 280 kDa. Evidently, those bands represented covalent adducts of fibrin peptide chains or their dimers with the 30-kDa or the 70-kDa fragment, respectively. In addition, dimeric gamma-chains and alpha-chain polymers of fibrin were present indicating partial polymerization of bead-attached fibrin.     

A 45-kDa human T-cell membrane glycoprotein functions in the regulation of cell proliferative responses

A 45-kDa human T cell surface glycoprotein which is tightly bound in the membrane of the resting T cell is released into the cell medium in soluble form after cell growth activation by phytohemagglutinin or neuraminidase/galactose oxidase treatments. In limited proteolysis by Staphylococcus aureus V8 protease, two major 35-kDa and 27-kDa peptide fragments of the surface-iodinated 45-kDa protein are common to the membrane-bound and the released forms, but a third 18-kDa fragment is observed exclusively with the released protein. The apparent molecular masses of the deglycosylated peptide backbones of the membrane-bound and the released molecule are 30 +/- 1 kDa, although a small size difference cannot be excluded. A polyclonal rabbit anti-(T cell membrane protein) antiserum precipitates the 45-kDa protein. A monoclonal anti-(45-kDa protein) antibody precipitates the membrane-bound 45-kDa protein solubilized with octyl glucoside, but does not precipitate the released protein. In cell culture assays, the monoclonal anti-(45-kDa protein) antibody specifically enhances the cell proliferative responses in phytohemagglutinin-treated and mixed lymphocyte cultures. These observations suggest that the 45-kDa protein has a specific receptor function in the regulation of cell proliferative responses.     

Effect of glycosylation inhibitors on the structure and function of the murine transferrin receptor

The murine transferrin receptor is a disulphide-linked dimer with three N-glycosylation sites. We have investigated the structural and functional properties of the transferrin receptor from murine plasmacytoma cells (NS-1 cells) treated with the glycosylation inhibitor, tunicamycin and the glycosylation-processing inhibitors, swainsonine and castanospermine. 1. Tunicamycin (1 microgram/ml) inhibited mannose incorporation in NS-1 cells by greater than 90%, but also inhibited methionine incorporation by up to 50%. Both swainsonine (1 microgram/ml) and castanospermine (50 micrograms/ml) resulted in mannose incorporation greater than 100% of untreated cells and neither drug affected methionine incorporation. 2. Incubation of NS-1 cells with tunicamycin resulted in a shift in the apparent molecular mass of the transferrin receptor from 96 kDa and 94 kDa to approximately 82 kDa. 3. Peptide N-glycosidase F digestion of the receptor from untreated cells resulted in the fully deglycosylated 82 kDa component as well as an 87 kDa component which represents partially deglycosylated receptor resistant to peptide N-glycosidase F digestion. 4. The receptor from swainsonine-treated cells was equally sensitive to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H (endo H; resulting in both 87-kDa and 82-kDa components), whereas the receptor from castanospermine-treated cells was only partially sensitive to endo H. 5. Analysis of mannose- and fucose-labelled cellular glycopeptides by concanavalin-A--Sepharose chromatography showed that swainsonine (1 microgram/ml) treatment resulted in approximately 90% inhibition of the synthesis of complex N-glycans and an accumulation of fucosylated hybrid structures. In contrast, castanospermine (100 micrograms/ml) treatment resulted in only partial inhibition (60%) of the synthesis of complex N-glycans. 6. Analysis of the receptor from tunicamycin, swainsonine and castanospermine treated cells under nonreducing conditions showed a single component corresponding to the dimer, indicating that dimerisation of newly synthesised murine receptor is independent of carbohydrate. 7. The non-glycosylated receptor from tunicamycin-treated cells appears to bind transferrin as demonstrated by interaction with transferrin-Sepharose. 8. Surface expression of the receptor was not significantly altered in the presence of either swainsonine or castanospermine as judged by flow cytometry.     

Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer.

An N-terminus sequence of human interleukin 1beta (hIL-1beta) was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte-colony stimulating factor (hG-CSF) and human growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF-alpha1 promoter. With the expression vector above, both recombinant proteins were well secreted into culture medium with high secretion efficiencies, and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fused hIL-1beta as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N-glycosylation resident in the hIL-1beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cerevisiae. When the N-glycosylation was completely blocked with the addition of tunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hIL-1beta fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N-terminus fusion of the hIL-1beta peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.     

The role of glucose and glycosylation in the regulation of lipoprotein lipase synthesis and secretion in rat adipocytes

Several studies have suggested that insulin and glucose increase adipose tissue lipoprotein lipase (LPL). To study the mechanism of the glucose-induced stimulation of LPL, the effects of glucose and glycosylation were examined in primary rat adipocyte cultures. In cells cultured in the presence of 1 mg/ml glucose, a 55-kDa LPL protein was synthesized and secreted into the medium, whereas cells cultured in glucose-free medium synthesized a 49-kDa form of LPL which was not secreted. The treatment of the mature 55-kDa form of LPL with peptide:N-glycosidase-F resulted in the formation of a 49-kDa form of LPL. When cells were cultured in the presence of tunicamycin, a 49-kDa form of LPL was synthesized by the cells but was not secreted. In addition, LPL activity was reduced by 90% when glycosylation was blocked by either tunicamycin or glucose deprivation. LPL synthetic rate was examined in cells cultured in a spectrum of glucose concentrations. LPL synthetic rate increased directly with medium glucose concentration and was decreased 80% in the absence of glucose compared to the synthetic rate in the presence of 1 mg/ml glucose. In addition, LPL synthetic rate in the presence of insulin was approximately 200% of the synthetic rate in untreated control cells at all glucose concentrations and even in the absence of glucose. In spite of the effect of glucose on LPL synthetic rate, glucose had no effect on the level of LPL mRNA. In contrast, the mRNA for the 78-kDa glucose-regulated protein (GRP78) was increased in adipocytes cultured in glucose-free medium. In summary, glucose was essential for glycosylation of LPL, and glycosylation was essential for LPL catalytic activity and secretion. In addition, glucose stimulated LPL synthetic rate and potentiated the stimulatory effects of insulin, but had no specific effect on LPL mRNA. Whereas insulin stimulates LPL by increasing the level of LPL mRNA, glucose stimulates LPL translation and post-translational processing.     

Cell type dependent inhibition of transport of cathepsin D in HepG2 cells and fibroblasts exposed to deoxy-manno-nojirimycin and deoxynojirimycin

The synthesis, transport and processing of lysosomal enzymes was examined in human hepatoma HepG2 cells and in human fibroblasts exposed to the Golgi alpha-mannosidase I inhibitor 1-deoxy-manno-nojirimycin. In HepG2 cells cathepsin D, beta-hexosaminidase and arylsulfatase B synthesized in the presence of 5 mM 1-deoxy-manno-nojirimycin contained exclusively endo-beta-N-acetylglucosaminidase H-cleavable oligosaccharides, indicating that alpha-mannosidase I had been inhibited efficiently. The proteolytic processing of intracellularly retained cathepsin D was retarded and the fraction of secreted cathepsin D was increased two-fold. In fibroblasts neither segregation nor maturation of cathepsin D were affected by 1-deoxy-manno-nojirimycin in spite of the inhibition of oligosaccharide processing. In the presence of the glucosidase I inhibitor 1-deoxynojirimycin, the precursor of cathepsin D (larger by about 1 kDa than the secreted form) accumulated transiently in light membranes in HepG2 cells. Release from the site of accumulation was accompanied by a decrease in size by about 1 kDa. This change was attributed to the removal of glucose residues. In fibroblasts the transient accumulation of larger precursors in the presence of 1-deoxynojirimycin was more pronounced than in HepG2 cells. The differential effects of alpha-mannosidase I and glucosidase I inhibitors on the transport of cathepsin D in HepG2 cells and fibroblasts may indicate that different intermediates in the biosynthetic pathway of asparagine-linked oligosaccharides participate in the transport of lysosomal enzymes in the two cell types.     

Effects of replacing serine and threonine residues within the signal peptide on the secretion of the major outer membrane lipoprotein of Escherichia coli

We have investigated the importance of serine and threonine residues within the signal peptide in the secretion and processing of the major outer membrane lipoprotein precursor prolipoprotein in Escherichia coli. This was accomplished by systematically replacing these residues with alanine utilizing oligodeoxyribonucleotide-directed mutagenesis. The results demonstrated that the replacement of serine 15 but not threonine 16 alone caused an initial accumulation of membrane-bound unmodified prolipoprotein. In addition, replacement of both serine 15 and threonine 16 resulted in a greater accumulation of this membrane-bound precursor. The accumulated prolipoprotein could be matured to lipoprotein in a quantitative manner, and this process was inhibited by globomycin and carbonyl cyanide m-chlorophenylhydrazone. These results will be discussed in terms of the contribution that serine and threonine have in determining the overall secondary structure of the signal peptide and its importance in secretion and/or processing.     

Accumulation and secretion of exoglucanase activity in yeast secretory mutants

Representative conditional yeast secretory mutants, blocked in transport of secretory and plasma membrane proteins from the endoplasmic reticulum (sec 18), from the Golgi body (sec 7) and in transport of secretory vesicles (sec 1), accumulated exoglucanase, a constitutive yeast activity, when incubated at the restrictive temperature (37°C). Different proportions of the accumulated activity were released by mutant cells under permissive conditions. The presence or absence of cycloheximide during the secretion period made no differences in the results. More than 90% of the internal activity was bound to membrane in wild type cells. However, only the soluble pool underwent changes during the accumulation or secretion periods. The bulk of secretory invertase accumulated by sec 1 was also soluble. By contrast sec 7 and sec 18 accumulated membrane-bound as well as soluble invertase forms and both were secreted in similar proportions in each mutant. More than 90% of the accumulated invertase was secreted at the permissive temperature in sec 18 cells. That percentage was significantly lower for exoglucanase (<65%). Concomitantly, invertase accumulated by this mutant exited from the cells with a lower half time (t 1/2=150 min). These results may be interpreted assuming that exoglucanase is exported by a passive flow of the soluble pool.Non-standard abbreviations p-NPG p-nitrophenyl--d-glucopyranoside - Con A concanavalin A - Tris tris(hydroxymethyl)-amino-methane     

Biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes.

The biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloroquine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1+2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultaneously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region.(ABSTRACT TRUNCATED AT 250 WORDS)