Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOSYNTHESIS OF CARBOXYPEPTIDASE Y IN YEAST. EVIDENCE FOR A PRECURSOR FORM OF THE GLYCOPROTEIN.
Andrej Hasilik and Widmar Tanner Fachbereich Biologie der Universit~t Regensburg 8#00 Regensburg, Universit~tsstr. 5d Received August 24,1976 A small but definite activity hydrolyzing N~acetyl-L-tyrosine ethyl ester was found in a membrane fraction from yeast. About half of it was due to carboxypeptidase Y. An antibody against this vacuolar enzyme precipitated a radioactive compound from the membrane fraction of cells labeled with a pulse of [5~ phenylalanine, which migrated more slowly on sodium dodecyl sulfate polyacrylamide gels than the authentic carboxypeptidase. Results obtained in pulse-chase experiments indicated that the slowly migrating material represents a precursor of the enzyme.
Carboxypeptidase
Y is a mannoprotein
(I) located in yeast
vacuoles (2,3). It has been chosen as an example for investigation of the biosynthesis of internal glycoproteins in yeast. In a previous study (1), the existence of a regulatory link between the synthesis of the protein moiety of carboxypeptidase Y and the reactions involved in its glycosylation has been postulated. To explore further the biosynthesis of this mannoprotein~ has been checked, whether carboxypeptidase
it
is present in the
membranous fraction used in this laboratory to study glycosylation reactions
(4). The data presented show that the membrane
fraction contains a precursor of carboxypeptidase X, which is larger than the final enzyme. METHODS Saccharom~ces 9erevisiae, strain X 2180 (a~) was used throughout this study. The maintenance of the organism and the
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
1430
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
preparation of the primary cultures were described previously (1). The main cultures, however, were grown in a synthetic medium containing per liter: 20 g glucose, 0.6 g ammonium sulfate, 27.2 g potassium dihydrogen phosphate, 0.1 g sodium chloride, 94 mg calcium chloride, 56 mg magnesium sulfate, 2.5 mg ferric chloride, 2 mg manganese dichloride, 25 mg myo-inositol, 10 mg calcium pantothenate, 5 mg thiamine hydrochloride, 5 mg pyridoxol hydrochloride, 5 mg nicotinic acid, 0.01 mg D(+) biotin. The growth was followed as optical density at 578 nm using photometer 1101M (Eppendorf, Hamburg). At an optical density of 3.2 the cells were rapidly spun down and resuspended in the synthetic medium at 1/3 of the original volume. 2.5 h later, at an optical density of about 16 L-phenyl[2,3-3H]alanine, I mCi/ml, 15.8 Ci/mmol (Amersham-Buchler, Braunschweig) was added. One half of the culture was analyzed for the pulse labeling, the other one was further incubated with non-radioactive L-phenylalanine, 0.12 mM. Once washed, the cells were taken up into 50 mM Tris chloride, pH 7.4, containing 10% mannitol and 1 mM magnesium chloride, 2 ml per g wet weight, and homogenized as described previously (1). After adjusting its pH to about 7.4, the homogenate was centrifuged for 10 min at 9,000 x g and the supernate obtained was further centrifuged for 1 h at 48,000 x The sediment obtained between 9,000 and 48,000 x g was washed twice with 10 ml of the homogenization buffer. To the membrane fraction thus obtained 50 mM Tris chloride, pH 7.4, 20% Triton XlO0 and 4 M KC1 were added to a final volume of 0.6 ml and final concentrations of 1% Triton XIO0 and 0.4 M KC1. 5 pg of purified carboxypeptidase Y (1), were added and the mixture was homogenized with aid of a dispensable pipette, incubated for 20 min at 57 ° and its pH was readjusted to 7.5. The cooled homogenate was centrifuged for 1 h at 48,000 x g. The immunoprecipitation was performed with both the 48,000 x g supernatant fraction of the cell homogenate and the membrane extract. These fractions were frozen and thawed, kept overnight at 4 ° and recentrifuged. 0.1 to 0.5 ml of f-globulin fraction of a rabbit anti-carboxypeptidase Y antiserum specific for the protein moiety of the enzyme (1) was added to 0.1 to 0.45 ml aliquots of the samples which were mixed with 50 mM Tris chloride, pH 7.4, and concentrated solutions of Triton xqo0 and KC1 to get final concentrations of 1% Triton XIO0 and 0.4 M KC1 and a final volume such that the ~-globulin fraction was diluted not more than 5 times. The immunoprecipitation of the membrane extract was performed for only 50 min at room temperature and 90 min at # ° to reduce non-specific coprecipitation. The immunoprecipitates were washed as previously (1) with an additional washing with 0.2 ml acetone. The sediment was homogenized with 20 pl water and 50 pl of a solubilizer solution and the mixture was incubated at 95 ° for 4 min. The solubilizer solution contained per ml: 1.5 mg dithiothreitol, 25 mg sodium dodecyl sulfate (SDS), 50 pl 0.5 M sodium phosphate, pH 7.0 and 0.1 ml glycerol. The solubilized immunoprecipitates were subjected to SDS polyacrylamide gel electrophoresis (7.5% acrylamide) in the anionic system, pH 9.5, according to the Buchler manual. No stacking gel was used, however, and the gel and the cathode buffer contained 0.5 and 0.05% SDS, respectively. The radioactivity was determined in 1 mm gel slices (5).
1431
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Table I. Esterase activity in the membrane fraction. The cells were grown in YPD/2 medium (1) to a concentration of 16 g wet weight per 1. The twice washed membrane fraction (Methods) was taken up into 50 mM Tris chloride, pH 7.4, 1 mM magnesium chloride (0.5 ml per g cells used). The data in parentheses represent the activities as determined with an aliquot of the membrane preparation treated with 6.7% Triton XIO0; in the assay mixture the concentration was 0.9%. One of two parallel determinations was performed in the presence of 1.8 pg inhibitor of carboxypeptidase Y; the difference due to the inhibitor was used to calculate the sensitive activity. Membrane fraction
Esterase activity (mU/mg) total
inhibitor sensitive
total
8.3
(15.6)
supernate +
0.67 (14.0)
2.9
(7.4)
0.27 (5.3)
+ The supernate was prepared by centrifuging for 1 h at 48,000 x g either the untreated or the Triton XIO0 treated (the data in parentheses) membrane preparation. The values presented are based on the protein content of the preparation prior to the centrifugation.
The esterase activity was determined at 25 ° with N-acetylL-tyrosine ethyl ester in an spectrophotometric test coupled with alcohol dehydrogenase (6). Protein was determined according to Lowry et al. (7)RESULTS AND DISCUSSION The washed membrane fraction of S accharom~ces cerevisiae was found to contain an activityhydrolyzing
N-acetyltyrosine
ethyl ester. In Table 1 it is shown that by treating the membrane fraction with Triton XIOO the activity was enhanced and~ to a great part, rendered non-sedimentable.
In each case about
40% of the esterase activity was sensitive to the specific inhibitor of carboxypeptidase Y~ which also occurs in S.cerevisiae (6). By comparing this activity with the total one~ it was calculated that only about 0.2% of the carboxypeptidase Y of the
1432
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
400~
' I
i
I
, LIJ
-g
200
d
0
>, 1200 5~
o
800
121 0
-o 0
400
n,"
15
30
45 Stlce No
Fig. q Radioactivity immunoprecipitable from the supernatant fraction of the cell homogenate and from the membrane extra~t~ The culture, 100 ml, was labeled with 0.5 mCi L-phenyl|2,3- HJ alanine for 4.5 min (A), and chased for 30 min (B). Th~ immunoprecipitate of the supernatant fraction corresponded to 6.2 ml culture (o), that of the membrane extract corresponded to 40 ml culture (m). The broken line marks the migration distance of purified carboxypeptidase Y in a gel run in parallel.
cell was associated with the membrane fraction isolated. Since the membranes prepared in a similar way have been found to contain several glycosyltransferase
activities
and since the synthesis of a number of glycoproteins to be associated with membranes membrane-bound
is known
(9) it was assumed, that the
enzyme might be a precursor of the final vacuo-
lar form. In an attempt to investigate
this possibility carboxy-
peptidase Y was isolated by immunoprecipitation brane and the supernatant acrylamide
(4,8),
fractions
gel electrophoresis.
from the mem-
and analyzed by SDS poly-
Surprisingly,
active component of the immunoprecipitates
the major radio-
obtained from the
subcellular fractions after a short pulse of[3~phenylalanine was more slowly moving than the authentic carboxypeptidase (Fig. IA). Whereas in the supernatant
fraction at least a shoul-
der was visible in the region of carboxypeptidase
1433
Y
Y, in a twice-
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
washed membrane fraction sedimenting between 9,000 and 4 8 , 0 0 0 x g exclusively the large form was present. After a 30 min chase with non-radioactive phenylalanine the immunoprecipitable radioactive material completely disappeared from the washed membrane fraction. In the supernate only one peak of radioactivity was observed~ which exactly coincided with the authentic carboxypeptidase Y. It is suggested that the large short-lived substance immunoprecipitable from extracts of pulse-labeled cells is a precursor of carboxypeptidase Y. The amount of radioactivity in the endproduct form as compared to that in the precursor (Fig. l) did not balance quantitatively,
since much of the precursor was
discarded in the 9,000 x g sediment. Indeed, a large amount of the precursor was found in this fraction in an independent experiment using different conditions for homogenization and extraction; more than half of the slowly migrating precursor was found in the 100,000 x g sediment fraction of the homogenate from pulse labeled cells and after a chase the increase in the radioactivity in the endproduct was well accounted for by the decrease in the radioactivity in the precursor. Conversion of the large into the small carboxypeptidase Y could also be demonstrated by incubating a supernatant fraction containing both forms of the enzyme (Fig. 2) in vitro at pH 5.0. 70% of the total radioactivity were recovered after the incubation; the radioactive precursor was no longer detectable, whereas there was a 2.3 fold increase in the radioactivity in the endproduct. The change was qualitatively the same as the one observed after in vivo conversion (Fig. 2). The low molecular weight material often observed at about the position of the tracking dye (Fig. 2) is assumed to be precipitated due to formation of a complex with carboxypeptidase
1434
Y.
Vol. 72, No. 4, 1 9 7 6
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS I
I
I
I
E 1000t 800 I >'• 600I o 400
200[_ o elsmmmm~
15
30
/.,5 Slice No
Fig. 2 In vitro conversion of the slowly migrating form of carboxypeptidase Y to the fast migrating one. The culture, 130 ml, was labeled with 1 mCi L-phenylL2,3-3H]alanine for 4.5 min and chased for 30 min. Pulse labeled supernatant fraction of the cell homogenate (a), pH 5 - treated supernatant fraction of the pulse labeled cells (e), supernatant fraction of the pulse-chase labeled cells (o). The pH 5 treatment was performed as described previously for the activation of proteinases in the crude extracts (q). Aliquots corresponding to 9 ml culture were analyzed in each case.
The difference in the molecular weight of the two forms has been estimated with protein standards on the same SDS gels to be about 5,000 D. This indicated that the precursor form of carboxypeptidase Y is not a complex of the enzyme with its specific inhibitor (6), since the molecular weight of the latter is nearly 25,000 D (10). Furthermore,
it was observed that the
enzyme inhibitor complex formed in vitro from the purified components dissociates under the experimental conditions used. Recently, Boer et al. have reported a membrane-associated precursor of secreted acid phosphatase in yeast spheroplasts (11). In this case the precursor seems to contain less sugar than the endproduct. A difference in size of the two acid phosphatase forms was not demonstrated. The bulk of carboxypeptidase Y is confined to the vacuolar compartment of the cell (2,3). There might exist an analogy,
1435
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
therefore, between the mode of synthesis of this glycoprotein in yeast and that of the secretory proteins in higher eucaryotes. The large carboxypeptidase Y may contain an extra extension of the polypeptide chain as known to occur at the N-termini of e.g. pancreatic secretory proteins (12).
ACKNOWLEDGEMENTS The authors thank Drs. Heidrun Natern and Renate Barth, Biochem. Inst., Univ. Freiburg i.Br. for samples of the purified inhibitor of carboxypeptidase Y, and Dr. R. N. Wohlhueter for reading the manuscript. The technical assistance of Miss R. Leonhart is acknowledged. The present work was supported by a grant from Deutsche Forschungsgemeinschaft.
REFERENCES
1. Hasilik, A., and Tanner, W. (1976) J. Bacteriol., in print. 2. Lenney, J. F., Matile, Ph., Wiemken, A., Schellenberg, N., and Meyer, J. (1974) Biochem. Biophys. Res. Commun. 60,
1378-1383. 3. Hasilik, A., MUller, H., and Holzer, H. (1974) Eur. J. Biochem. 48, 111-117. 4. Lehle, L., and Tanner, W. (1974) Biochim. Biophys. Acta 350 225-235. 5- Zaitlin, M., and Hariharasubramanian, V. (1970) Anal. Biochem. 35, 296-297. 6. Matern, H., Betz, H., and Holzer, H. (1974) Biochem. Biophys. Res. Commun. 60, 1051-1057. 7. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 8. Nakajima, T., and Ballou, C. E. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3912-3916. 9. Schachter, H. (1974) Biochem. Soc. Symp. 40, 57-71. 10. Matern, H., Hoffmann, M., and Holzer, H. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4874-4878. 11. Boer, P.,van Rijn, H. J. M., Reinking, A., and Steyn-Parv~, E. P. (1975) Biochim. Biophys. Acta 377, 331-342. 12. Devillers-Thiery, A., Kindt, T., Scheele, G., and Blobel, G. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 5016-5020.
1436
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOSYNTHESIS OF CARBOXYPEPTIDASE Y IN YEAST. EVIDENCE FOR A PRECURSOR FORM OF THE GLYCOPROTEIN.
Andrej Hasilik and Widmar Tanner Fachbereich Biologie der Universit~t Regensburg 8#00 Regensburg, Universit~tsstr. 5d Received August 24,1976 A small but definite activity hydrolyzing N~acetyl-L-tyrosine ethyl ester was found in a membrane fraction from yeast. About half of it was due to carboxypeptidase Y. An antibody against this vacuolar enzyme precipitated a radioactive compound from the membrane fraction of cells labeled with a pulse of [5~ phenylalanine, which migrated more slowly on sodium dodecyl sulfate polyacrylamide gels than the authentic carboxypeptidase. Results obtained in pulse-chase experiments indicated that the slowly migrating material represents a precursor of the enzyme.
Carboxypeptidase
Y is a mannoprotein
(I) located in yeast
vacuoles (2,3). It has been chosen as an example for investigation of the biosynthesis of internal glycoproteins in yeast. In a previous study (1), the existence of a regulatory link between the synthesis of the protein moiety of carboxypeptidase Y and the reactions involved in its glycosylation has been postulated. To explore further the biosynthesis of this mannoprotein~ has been checked, whether carboxypeptidase
it
is present in the
membranous fraction used in this laboratory to study glycosylation reactions
(4). The data presented show that the membrane
fraction contains a precursor of carboxypeptidase X, which is larger than the final enzyme. METHODS Saccharom~ces 9erevisiae, strain X 2180 (a~) was used throughout this study. The maintenance of the organism and the
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
1430
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
preparation of the primary cultures were described previously (1). The main cultures, however, were grown in a synthetic medium containing per liter: 20 g glucose, 0.6 g ammonium sulfate, 27.2 g potassium dihydrogen phosphate, 0.1 g sodium chloride, 94 mg calcium chloride, 56 mg magnesium sulfate, 2.5 mg ferric chloride, 2 mg manganese dichloride, 25 mg myo-inositol, 10 mg calcium pantothenate, 5 mg thiamine hydrochloride, 5 mg pyridoxol hydrochloride, 5 mg nicotinic acid, 0.01 mg D(+) biotin. The growth was followed as optical density at 578 nm using photometer 1101M (Eppendorf, Hamburg). At an optical density of 3.2 the cells were rapidly spun down and resuspended in the synthetic medium at 1/3 of the original volume. 2.5 h later, at an optical density of about 16 L-phenyl[2,3-3H]alanine, I mCi/ml, 15.8 Ci/mmol (Amersham-Buchler, Braunschweig) was added. One half of the culture was analyzed for the pulse labeling, the other one was further incubated with non-radioactive L-phenylalanine, 0.12 mM. Once washed, the cells were taken up into 50 mM Tris chloride, pH 7.4, containing 10% mannitol and 1 mM magnesium chloride, 2 ml per g wet weight, and homogenized as described previously (1). After adjusting its pH to about 7.4, the homogenate was centrifuged for 10 min at 9,000 x g and the supernate obtained was further centrifuged for 1 h at 48,000 x The sediment obtained between 9,000 and 48,000 x g was washed twice with 10 ml of the homogenization buffer. To the membrane fraction thus obtained 50 mM Tris chloride, pH 7.4, 20% Triton XlO0 and 4 M KC1 were added to a final volume of 0.6 ml and final concentrations of 1% Triton XIO0 and 0.4 M KC1. 5 pg of purified carboxypeptidase Y (1), were added and the mixture was homogenized with aid of a dispensable pipette, incubated for 20 min at 57 ° and its pH was readjusted to 7.5. The cooled homogenate was centrifuged for 1 h at 48,000 x g. The immunoprecipitation was performed with both the 48,000 x g supernatant fraction of the cell homogenate and the membrane extract. These fractions were frozen and thawed, kept overnight at 4 ° and recentrifuged. 0.1 to 0.5 ml of f-globulin fraction of a rabbit anti-carboxypeptidase Y antiserum specific for the protein moiety of the enzyme (1) was added to 0.1 to 0.45 ml aliquots of the samples which were mixed with 50 mM Tris chloride, pH 7.4, and concentrated solutions of Triton xqo0 and KC1 to get final concentrations of 1% Triton XIO0 and 0.4 M KC1 and a final volume such that the ~-globulin fraction was diluted not more than 5 times. The immunoprecipitation of the membrane extract was performed for only 50 min at room temperature and 90 min at # ° to reduce non-specific coprecipitation. The immunoprecipitates were washed as previously (1) with an additional washing with 0.2 ml acetone. The sediment was homogenized with 20 pl water and 50 pl of a solubilizer solution and the mixture was incubated at 95 ° for 4 min. The solubilizer solution contained per ml: 1.5 mg dithiothreitol, 25 mg sodium dodecyl sulfate (SDS), 50 pl 0.5 M sodium phosphate, pH 7.0 and 0.1 ml glycerol. The solubilized immunoprecipitates were subjected to SDS polyacrylamide gel electrophoresis (7.5% acrylamide) in the anionic system, pH 9.5, according to the Buchler manual. No stacking gel was used, however, and the gel and the cathode buffer contained 0.5 and 0.05% SDS, respectively. The radioactivity was determined in 1 mm gel slices (5).
1431
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Table I. Esterase activity in the membrane fraction. The cells were grown in YPD/2 medium (1) to a concentration of 16 g wet weight per 1. The twice washed membrane fraction (Methods) was taken up into 50 mM Tris chloride, pH 7.4, 1 mM magnesium chloride (0.5 ml per g cells used). The data in parentheses represent the activities as determined with an aliquot of the membrane preparation treated with 6.7% Triton XIO0; in the assay mixture the concentration was 0.9%. One of two parallel determinations was performed in the presence of 1.8 pg inhibitor of carboxypeptidase Y; the difference due to the inhibitor was used to calculate the sensitive activity. Membrane fraction
Esterase activity (mU/mg) total
inhibitor sensitive
total
8.3
(15.6)
supernate +
0.67 (14.0)
2.9
(7.4)
0.27 (5.3)
+ The supernate was prepared by centrifuging for 1 h at 48,000 x g either the untreated or the Triton XIO0 treated (the data in parentheses) membrane preparation. The values presented are based on the protein content of the preparation prior to the centrifugation.
The esterase activity was determined at 25 ° with N-acetylL-tyrosine ethyl ester in an spectrophotometric test coupled with alcohol dehydrogenase (6). Protein was determined according to Lowry et al. (7)RESULTS AND DISCUSSION The washed membrane fraction of S accharom~ces cerevisiae was found to contain an activityhydrolyzing
N-acetyltyrosine
ethyl ester. In Table 1 it is shown that by treating the membrane fraction with Triton XIOO the activity was enhanced and~ to a great part, rendered non-sedimentable.
In each case about
40% of the esterase activity was sensitive to the specific inhibitor of carboxypeptidase Y~ which also occurs in S.cerevisiae (6). By comparing this activity with the total one~ it was calculated that only about 0.2% of the carboxypeptidase Y of the
1432
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
400~
' I
i
I
, LIJ
-g
200
d
0
>, 1200 5~
o
800
121 0
-o 0
400
n,"
15
30
45 Stlce No
Fig. q Radioactivity immunoprecipitable from the supernatant fraction of the cell homogenate and from the membrane extra~t~ The culture, 100 ml, was labeled with 0.5 mCi L-phenyl|2,3- HJ alanine for 4.5 min (A), and chased for 30 min (B). Th~ immunoprecipitate of the supernatant fraction corresponded to 6.2 ml culture (o), that of the membrane extract corresponded to 40 ml culture (m). The broken line marks the migration distance of purified carboxypeptidase Y in a gel run in parallel.
cell was associated with the membrane fraction isolated. Since the membranes prepared in a similar way have been found to contain several glycosyltransferase
activities
and since the synthesis of a number of glycoproteins to be associated with membranes membrane-bound
is known
(9) it was assumed, that the
enzyme might be a precursor of the final vacuo-
lar form. In an attempt to investigate
this possibility carboxy-
peptidase Y was isolated by immunoprecipitation brane and the supernatant acrylamide
(4,8),
fractions
gel electrophoresis.
from the mem-
and analyzed by SDS poly-
Surprisingly,
active component of the immunoprecipitates
the major radio-
obtained from the
subcellular fractions after a short pulse of[3~phenylalanine was more slowly moving than the authentic carboxypeptidase (Fig. IA). Whereas in the supernatant
fraction at least a shoul-
der was visible in the region of carboxypeptidase
1433
Y
Y, in a twice-
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
washed membrane fraction sedimenting between 9,000 and 4 8 , 0 0 0 x g exclusively the large form was present. After a 30 min chase with non-radioactive phenylalanine the immunoprecipitable radioactive material completely disappeared from the washed membrane fraction. In the supernate only one peak of radioactivity was observed~ which exactly coincided with the authentic carboxypeptidase Y. It is suggested that the large short-lived substance immunoprecipitable from extracts of pulse-labeled cells is a precursor of carboxypeptidase Y. The amount of radioactivity in the endproduct form as compared to that in the precursor (Fig. l) did not balance quantitatively,
since much of the precursor was
discarded in the 9,000 x g sediment. Indeed, a large amount of the precursor was found in this fraction in an independent experiment using different conditions for homogenization and extraction; more than half of the slowly migrating precursor was found in the 100,000 x g sediment fraction of the homogenate from pulse labeled cells and after a chase the increase in the radioactivity in the endproduct was well accounted for by the decrease in the radioactivity in the precursor. Conversion of the large into the small carboxypeptidase Y could also be demonstrated by incubating a supernatant fraction containing both forms of the enzyme (Fig. 2) in vitro at pH 5.0. 70% of the total radioactivity were recovered after the incubation; the radioactive precursor was no longer detectable, whereas there was a 2.3 fold increase in the radioactivity in the endproduct. The change was qualitatively the same as the one observed after in vivo conversion (Fig. 2). The low molecular weight material often observed at about the position of the tracking dye (Fig. 2) is assumed to be precipitated due to formation of a complex with carboxypeptidase
1434
Y.
Vol. 72, No. 4, 1 9 7 6
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS I
I
I
I
E 1000t 800 I >'• 600I o 400
200[_ o elsmmmm~
15
30
/.,5 Slice No
Fig. 2 In vitro conversion of the slowly migrating form of carboxypeptidase Y to the fast migrating one. The culture, 130 ml, was labeled with 1 mCi L-phenylL2,3-3H]alanine for 4.5 min and chased for 30 min. Pulse labeled supernatant fraction of the cell homogenate (a), pH 5 - treated supernatant fraction of the pulse labeled cells (e), supernatant fraction of the pulse-chase labeled cells (o). The pH 5 treatment was performed as described previously for the activation of proteinases in the crude extracts (q). Aliquots corresponding to 9 ml culture were analyzed in each case.
The difference in the molecular weight of the two forms has been estimated with protein standards on the same SDS gels to be about 5,000 D. This indicated that the precursor form of carboxypeptidase Y is not a complex of the enzyme with its specific inhibitor (6), since the molecular weight of the latter is nearly 25,000 D (10). Furthermore,
it was observed that the
enzyme inhibitor complex formed in vitro from the purified components dissociates under the experimental conditions used. Recently, Boer et al. have reported a membrane-associated precursor of secreted acid phosphatase in yeast spheroplasts (11). In this case the precursor seems to contain less sugar than the endproduct. A difference in size of the two acid phosphatase forms was not demonstrated. The bulk of carboxypeptidase Y is confined to the vacuolar compartment of the cell (2,3). There might exist an analogy,
1435
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
therefore, between the mode of synthesis of this glycoprotein in yeast and that of the secretory proteins in higher eucaryotes. The large carboxypeptidase Y may contain an extra extension of the polypeptide chain as known to occur at the N-termini of e.g. pancreatic secretory proteins (12).
ACKNOWLEDGEMENTS The authors thank Drs. Heidrun Natern and Renate Barth, Biochem. Inst., Univ. Freiburg i.Br. for samples of the purified inhibitor of carboxypeptidase Y, and Dr. R. N. Wohlhueter for reading the manuscript. The technical assistance of Miss R. Leonhart is acknowledged. The present work was supported by a grant from Deutsche Forschungsgemeinschaft.
REFERENCES
1. Hasilik, A., and Tanner, W. (1976) J. Bacteriol., in print. 2. Lenney, J. F., Matile, Ph., Wiemken, A., Schellenberg, N., and Meyer, J. (1974) Biochem. Biophys. Res. Commun. 60,
1378-1383. 3. Hasilik, A., MUller, H., and Holzer, H. (1974) Eur. J. Biochem. 48, 111-117. 4. Lehle, L., and Tanner, W. (1974) Biochim. Biophys. Acta 350 225-235. 5- Zaitlin, M., and Hariharasubramanian, V. (1970) Anal. Biochem. 35, 296-297. 6. Matern, H., Betz, H., and Holzer, H. (1974) Biochem. Biophys. Res. Commun. 60, 1051-1057. 7. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 8. Nakajima, T., and Ballou, C. E. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3912-3916. 9. Schachter, H. (1974) Biochem. Soc. Symp. 40, 57-71. 10. Matern, H., Hoffmann, M., and Holzer, H. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4874-4878. 11. Boer, P.,van Rijn, H. J. M., Reinking, A., and Steyn-Parv~, E. P. (1975) Biochim. Biophys. Acta 377, 331-342. 12. Devillers-Thiery, A., Kindt, T., Scheele, G., and Blobel, G. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 5016-5020.
1436
