Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y

The structural gene for yeast vacuolar carboxypeptidase Y (PRC1) has been cloned by complementation of the prc1-1 mutation. As much as an eightfold elevation in the level of carboxypeptidase Y (CPY) results when a multiple-copy plasmid containing the PRC1 gene is introduced into yeast. Unlike the situation with a single copy of PRC1 in which newly synthesized CPY is efficiently localized to the vacuole, plasmid-directed overproduction results in secretion of greater than 50% of the protein as the precursor form. Secretion is blocked in a mutant that is defective at a late stage in the transport of periplasmic proteins. Unlike normal cell surface glycoproteins, secreted CPY precursor acquires no additional oligosaccharide modifications beyond those that accompany normal transport to the vacuole. In the periplasm, the CPY precursor is proteolytically activated to an enzymatically active form by an enzyme that is unrelated to the vacuolar processing enzyme. These findings suggest that proper sorting and transport of CPY is saturable. This may reflect limiting amounts of a CPY-sorting receptor, or of CPY-modifying machinery that is essential for recognition by such a receptor.     

Proteinase C (carboxypeptidase Y) mutant of yeast.

A mutant of yeast lacking proteinase C (carboxypeptidase Y) activity has been found by using a histochemical stain to screen mutagenized colonies. This defect segregates 2:2 in meiotic tetrads. Cell extracts lacked the esterolytic, amidase, and proteolytic activities associated with proteinase C. The absence of proteinase C does not affect mitotic growth and has no obvious effect on the formation of viable ascospores or meiotic segregation. The mutant grows on peptides known to be cleaved by proteinase C in vitro. This finding is consistent with the idea that other enzymes exist in vivo with overlapping substrate specificities.     

Inactivation of yeast enzymes by proteinase A and B and carboxypeptidase Y from yeast.

Changes in the activities of 15 different enzymes during incubation of a crude yeast extract with the purified yeast proteinases A and B, and carboxypeptidase Y, respectively, have been measured. The spectrum of action of the three proteinases on the enzymes measured differs significantly, increasing or decreasing their activities or having no effect. Incubation of purified cytoplasmic malate dehydrogenase or purified mitochondrial malate dehydrogenase with proteinases A and B results in selective inactivation of the cytoplasmic enzyme, whereas the mitochondrial activity is not affected. Carboxypeptidase Y has no effect on the activity of either dehydrogenase. The results support the idea of selective proteolysis as the mechanism of the earlier observed inactivation of cytoplasmic malate dehydrogenase, initiated by the addition of glucose to intact yeast cells grown on acetate as carbon source ("glucose effect").     

Studies on a carboxypeptidase Y mutant of yeast and evidence for a second carboxypeptidase Activity.

Immunological studies on the carboxypeptidase Y mutant prcl-l of Saccharomyces cerevisiae revealed the origin of mutation in the structural gene of carboxypeptidase Y. The absence of carboxypeptidase Y has no effect on growth, even after drastic changes of growth conditions. A double mutant (prc 1- leu2-) lacking carboxypeptidase Y and auxotrophic for leucine is able to grow on the peptide benzyloxycarbonylglycylleucine (Cbz-Gly-Leu) as sole nitrogen source, indicating the existence of a second carboxypeptidase. Using a new peptidase test, the existence of this second enzyme, called carboxypeptidase S, was confirmed biochemically.     

Carbohydrate-free carboxypeptidase Y is transferred into the lysosome-like yeast vacuole

Carboxypeptidase Y, localized in the lysosome-like yeast vacuole, has been metabolically labeled with [2-³H]mannose. After immunoprecipitation the carbohydrate moieties were released by treatment with endo-β-N-acetyl-glucosaminidase H and separated by paper electrophoresis. Evidence for the presence of phospho-monoester and -diester groups in the molecule has been obtained. In the latter phosphate links C-1 of mannose or of mannosyl 1,3-mannose to C-6 of a mannose residue within a larger oligomannose moiety. In the presence of tunicamycin yeast cells synthesize a carbohydrate-free carboxypeptidase Y, which could be traced after metabolic labeling with [¹⁴C]-phenylalanine. The carbohydrate-free enzyme was segregated into the vacuoles to the same extent as the intact glycoprotein.     

Effects of yeast proteinase A, proteinase B and carboxypeptidase Y on yeast phosphofructokinase.

Incubation of a crude yeast extract containing phosphofructokinase with proteinase A, proteinase B or carboxypeptidase Y gave the following results: Proteinase B and carboxypeptidase Y did not change the activity of phosphofructokinase during incubation. On the other hand, incubation with proteinase A resulted in a 40-100% activation; continued incubation, however, led to an inactivation of the enzyme. Addition of allosteric effectors did not change the activation or inactivation process. The activated phosphofructokinase was not changed with respect to pH optimum and ATP inhibition. Molecular weight determination of phosphofructokinase in crude extracts in the presence of inhibitors of proteinase A indicated a molecular weight of 700000. Without inhibitors of proteinase A, the molecular weight was determined to be 600 000, while after 40-100% activation by proteinase A, a molecular weight of 500 000 was obtained. The activity profile of proteinase A in density gradients indicated that this enzyme is bound to variety of cellular proteins.     

Compartmentation of inhibitors of proteinases A and B and carboxypeptidase Y in yeast

After centrifugal fractionation at 40,000 × g of a metabolic lysate from yeast spheroplasts proteinases A and B, and carboxypeptidase Y were found exclusively in the sediment, whereas inhibitors of these proteinases were present only in the supernatant. Immunoprecipitation with an antiserum prepared against the pure heat-stable proteinase B-inhibitor occured in the supernatant but not in the extract of the particulate fraction.     

Synthesis and maturation of the yeast vacuolar enzymes carboxypeptidase Y and aminopeptidase I

We have studied the two vacuolar enzymes carboxypeptidase Y and aminopeptidase I from Saccharomyces cerevisiae with respect to biosynthesis, maturation and transfer from their site of synthesis into the organelle. The levels of translatable mRNA for these two proteins increase more than 10-fold at the end of the exponential growth period on glucose as carbon source and decrease again in the stationary phase. Two precursors of carboxypeptidase Y have been identified by in vivo pulse-labelling with [35S]methionine. These differ in their amount of carbohydrate as shown by inhibition of N-linked glycosylation with tunicamycin. The first is a protein with an apparent molecular weight of 67 kDa, which can be converted into the mature 60-kDa protein via an intermediate of 69 kDa. In the pep4-3 mutant, which is disturbed in the maturation of several vacuolar enzymes (Hemmings, B.A., Zubenko, G.S., Hasilik, A. and Jones, E.W. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 435-439), the 69-kDa precursor accumulates in the vacuole. This suggests that the final proteolytic cleavage of carboxypeptidase Y can occur in the vacuole.     

Refolding and purification of yeast carboxypeptidase Y expressed as inclusion bodies in Escherichia coli

The genes encoding carboxypeptidase Y (CPY) and CPY propeptide (CPYPR) from Saccharomyces cerevisiae were cloned and expressed in Escherichia coli. Six consecutive histidine residues were fused to the C-terminus of the CPYPR for facilitated purification. High-level expression of CPY and CPYPR-His(6) was achieved but most of the expressed proteins were present in the form of inclusion bodies in the bacterial cytoplasm. The CPY and CPYPR-His(6) produced as inclusion bodies were separated from the cells and solubilized in 6 and 3 M guanidinium chloride, respectively. The denatured CPYPR-His(6) was refolded by dilution 1:30 into the renaturation buffer (50 mM Tris-HCl containing 0.5 M NaCl and 3 mM EDTA, pH 8.0), and the refolded CPYPR-His(6) was further purified to 90% purity by single-step immobilized metal ion affinity chromatography. The denatured CPY was refolded by dilution 1:60 into the renaturation buffer containing CPYPR-His(6) at various concentrations. Increasing the molar ratio of CPYPR-His(6) to CPY resulted in an increase in the CPY refolding yield, indicating that the CPYPR-His(6) plays a chaperone-like role in in vitro folding of CPY. The refolded CPY was purified to 92% purity by single-step p-aminobenzylsuccinic acid affinity chromatography. When refolding was carried out in the presence of 10 molar eq CPYPR-His(6), the specific activity, N-(2-furanacryloyl)-l-phenylalanyl-l-phenylalanine hydrolysis activity per milligram of protein, of purified recombinant CPY was found to be about 63% of that of native S. cerevisiae CPY.     

Multiple endoplasmic reticulum-associated pathways degrade mutant yeast carboxypeptidase Y in mammalian cells

The degradation of misfolded and unassembled proteins by the endoplasmic reticulum (ER)-associated degradation (ERAD) has been shown to occur mainly through the ubiquitin-proteasome pathway after transport of the protein to the cytosol. Recent work has revealed a role for N-linked glycans in targeting aberrant glycoproteins to ERAD. To further characterize the molecular basis of substrate recognition and sorting during ERAD in mammalian cells, we expressed a mutant yeast carboxypeptidase Y (CPY*) in CHO cells. CPY* was retained in the ER in un-aggregated form, and degraded after a 45-min lag period. Degradation was predominantly by a proteasome-independent, non-lysosomal pathway. The inhibitor of ER mannosidase I, kifunensine, blocked the degradation by the alternate pathway but did not affect the proteasomal fraction of degradation. Upon inhibition of glucose trimming, the initial lag period was eliminated and degradation thus accelerated. Our results indicated that, although the proteasome is a major player in ERAD, alternative routes are present in mammalian cells and can play an important role in the disposal of both glycoproteins and non-glycoproteins.     

Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide

We have isolated cis-acting mutations in the gene encoding the yeast vacuolar protein carboxypeptidase Y (CPY) that result in missorting and aberrant secretion of up to 95% of newly synthesized CPY. The CPY polypeptides synthesized by these mutants use the late secretory pathway to exit the cell, since the late-acting sec1 mutation prevents their secretion. The mutant versions of CPY are secreted as the proCPY zymogen and are enzymatically activatable in vivo and in vitro. All the mutations, including small deletions and an amino acid substitution, map to the amino-terminal propeptide region and define a discrete yeast vacuolar localization domain whose integrity is required for efficient sorting of the CPY zymogen. Thus, the N-terminal propeptide of CPY carries out at least three functions: it mediates translocation across the endoplasmic reticulum, renders the enzyme inactive during transit, and targets the molecule to the vacuole.     

Regulated secretion

A report from the minisymposium on regulated secretion at the 39th Annual Meeting of the American Society for Cell Bilogy, Washington DC, December 11-15, 1999     

Stabilization of the structure and activity of yeast carboxypeptidase Y by its high-mannose oligosaccharide chains

Sodium dodecyl sulfate was shown to promote both the inactivation and proteolytic degradation of the yeast glycoprotein, carboxypeptidase Y, with the former effect occurring six times faster than the latter. Although the proteolysis, as judged by polyacrylamide gel electrophoresis, was inhibited by pepstatin, which implicates the presence of proteinase A, the possibility of autodigestion could not be ruled out. A contributing role of the enzyme's carbohydrate moiety to these two processes was revealed by treating carboxypeptidase Y with endo-β-N-acetylglucosaminidase H. This treatment removes all four of the enzyme's Oligosaccharide chains in sodium dodecyl sulfate and as a consequence increases the rate of inactivation of the resulting carboxypeptidase Y by twofold and its proteolytic degradation by threefold relative to that of untreated enzyme. It thus appears that carboxypeptidase Y is a glycoprotein whose structural integrity and functional activity are influenced by its associated carbohydrate component.     

Catalytic activity of carboxypeptidase B and of carboxypeptidase Y with anisylazoformyl substrates

Anisylazoformyllysine (CH3OC6H4-N = N-CO-Lys-OH) is rapidly hydrolyzed at the acyl-lysine linkage by the zinc-enzyme porcine carboxypeptidase B. The catalytic reaction is readily monitored spectrophotometrically by disappearance of the intense absorption (348.5 nm, epsilon 18400) of the azo chromophore, which chemically fragments after substrate cleavage. Carboxypeptidase Y has no activity toward this type of substrate.     

Peptide synthesis with immobilized carboxypeptidase Y

Enzymatic peptide synthesis was investigated using carboxypeptidase Y immobilized with glutaraldehyde on 10 mum microparticulate amino-silica. Carboxypeptidase Y was immobilized with 98.5% recovery of active enzyme to yield the immobilized enzyme having 0.55 units esterase activity/mg amino-silica support. The stability of the immobilized enzyme was examined as a function of pH, temperature, and reactant concentrations. Immobilized Carboxypeptidase Y was used in stirred batch and recirculating packed-bed reactors for peptide synthesis. Packed-bed reactors (40 x 4.6 mm, 60 x 4.6 mm) were used to catalyze the synthesis of 170 mg N-benzoyl-L-arginyl-L-methioninamide, 380 mg N-benzoyl-L-arginyl-L-methionyl-L-leucinamide, and 200 mg N-benzoyl-L-arginyl-L-methionyl-L-leucyl-L-phenylalaninamide in 8, 3, and 1 hour, respectively, as intermediates in the synthesis of L-methionyl-L-leucyl-L-phenylalanine. No inactivation of the immobilized enzyme was observed during the course of the reactions. The N-benzoyl-L-arginyl group served to increase the water solubility of the peptides and was removed by immobilized trypsin at the end of synthesis to obtain the final product. While the first two syntheses were conducted with aqueous reaction mixtures, the synthesis of N-benzoyl-L-arginyl-L-methionyl-L-leucyl-L-phenylalaninamide was carried out in a reaction mixture containing dimethylformamide to avoid precipitation of the product. HPLC and amino acid analysis confirmed the high purity and amino acid composition of the final product.