- Proc. Nat. Acad. Sci. USA Vol. 72, No. 10, pp. 3952-3955, October 1975 Biochemistry
Chitin synthetase zymogen is attached to the yeast plasma membrane (yeast septum/gradient centrifugation/concanavalin A/glutaraldehyde)
ANGEL DURAN*, BLAIR BOWERSt, AND ENRICO CABIB*f *
National Institute of Arthritis, Metabolism and Digestive Diseases, and t National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland
20014
Communicated by T. M. Sonneborn, August 4,1975
specific activity 53.4 ,tCi/,umol) were products of New England Nuclear. Saccharomyces cerevisiAe, strain X2180 (American Type Culture Collection no. 26109) was grown in a minimal medium (8), and harvested during the logarithmic phase at an approximate cell density of 3.5 X 107 cells per ml (buds not counted as cells). Analytical. Active chitin synthetase and its zymogen were determined as described (9). Tris chloride at pH 7.5 was substituted for imidazole, except in the experiment of Table 1. Protein was assayed according to Lowry et al. (10). Protoplast Lysis and Membrane Purification. Centrifugations were carried out at about 20. Yeast protoplasts were obtained as previously reported (11) and suspended in 0.8 M sorbitol, in a final volume of I ml/g (wet weight) of yeast utilized. To 0.5 ml of this suspension, were added 3 ml of 0.8 M sorbitol, containing 0.05 M Tris chloride at pH 7.5 and 10 mM MgSO4, followed by 3.5 ml of the same solution, supplemented with 0.5 mg/ml of Con A. After 10 min at room temperature, the flocculated protoplasts were centrifuged at 2° for 1 min in a swinging bucket rotor at 250 X g. The pellet was carefully resuspended in 6.5 ml of the sorbitol-TrisMg++ mixture and recentrifuged at the same speed for 3 min. The final pellet was suspended in 8.5 ml of 10 mM Tris chloride at pH 7.5, containing 5 mM MgSO4, 1 mg/ml of deoxyribonuclease and 0.6 mM phenylmethylsulfonylfluoride. The mixture was stirred at 00 for 3 min in a Lourdes
ABSTRACT Pretreatment of yeast protoplasts with concanavalin A, according to the method used by- C. A. Scarborough for Neurospora (J. Biol. Chem. 250, 1106-1111, 1975), reinforced the plasma membranes, and helped to maintain their integrity during subsequent lysis of the protoplasts. After purification by centrifuging on a Renografin density gradient, practically intact membranes were obtained. Previous labeling of the protoplasts with 125I or with [3Hjconcanavalin A resulted in recovery of the radioactivity in the membrane fraction. The bulk of the chitin synthetase (chitin synthase; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4.-f-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) recovered in the gradient was also found in this fraction, in the zymogen form. About 20% of the activity sedimented in a plasmamembrane-free fraction at lower density. Glutaraldehyde inactivated chitin synthetase when it was added to a Iysate, but not when applied to intact protoplasts. It is concluded that chitin synthetase is so oriented in the membrane that it is only accessible from the inside of the cell. These results confirm our previous hypothesis that the chitin synthetase zymogen is associated with the plasma membrane, a basic assumption for the explanation of localized activation of the enzyme and initiation of septum formation. In a previous communication (1), we suggested that the inactive form of chitin synthetase (chitin synthase; UDP-acetamido-2-deoxy-D-glucose:chitin 4-,B-acetamidodeoxyglucosyltransferase; EC 2.4.1.16), the enzyme responsible for the formation of the yeast primary septum, is attached to the plasma membrane. This supposition proved difficult to substantiate because a reliable preparation of purified yeast plasmalemma was not available. Several such preparations have been described in the literature (2-6), but in every case they consisted of small membrane fragments and therefore it is difficult to exclude the possibility that they might be mixtures of intracellular and external membranes. Recently we have succeeded in obtaining yeast plasma membranes, essentially in a single piece, by a modification of the method used by Scarborough (7) for Neurospora. The method consists in reinforcing the membranes by coating them with concanavalin A (Con A) prior to cell rupture. After gradient centrifugation of the lysate, the bulk of the chitin synthetase zymogen has been found in association with the membranes.
Table 1. Glutaraldehyde inactivation of chitin synthetase before and after protoplast lysis
Acetylglucosamine incorporated into chitin
MATERIALS AND METHODS Materials. Meglumine diatrizoate (Renografin) was purchased from Squibb & Sons; bovine pancreas deoxyribonuclease, lactoperoxidase, and unlabeled concanavalin A were purchased from Sigma and glutaraldehyde from Ladd Research Industries, Burlington, Vermont. Carrier-free Na12I was obtained from Amersham-Searle, whereas [acetyl3H]concanavalin A (specific activity 149 mCi/,umol) and UDP['4C]GlcNAc (labeled in C-1 of the hexosamine moiety,
No.
Treatment (in sequence)
(cpm)
1. 2. 3. 4. 5. 6. 7.
Con A, lysis Glutaraldehyde, Con A, lysis Con A, glutaraldehyde, lysis Lysis Glutaraldehyde, lysis Con A, lysis, glutaraldehyde Lysis, glutaraldehyde
921 919 904
711 750 85
70
Where indicated, glutaraldehyde was added to a final concentration of 1% to a suspension of protoplasts. After 30 sec at 0°, 20 volumes of 0.8 M sorbitol were added. Protoplasts were centrifuged for 5 min at 650 x g and washed again with 0.8 M sorbitol. Lysates were treated with glutaraldehyde in the same way, but dilution and washing were performed with 0.05 M imidazole, pH 6.5, containing 2 mM MgSQ4, and centrifugation was for 10 min at 20,000 X g. When treatment with Con A was omitted, lysis was carried out as previously reported (9, 12), because Con-A-free protoplasts did not break well under the conditions outlined under Materials and Methods. In every case, zymogen was activated with trypsin before assay for chitin synthetase activity.
Abbreviation: Con A, concanavalin A. t To whom reprint requests should be addressed.
3952
Biochemistry:
Dura'n et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
C H T N
FRACTION
U
Percent activity
mu/mg protein
before activation
0.81
3.5
100
Crude particles
-Band 1
2.8
0.21
n. d.
-Band 2
5.2
0.63
2
Band 3
75.6
3.1
0.5
- Band 4
0.6
0.39
n d.
FIG. 1. Distribution of chitin synthetase activity after centrifugation on a Renografin gradient. Crude particles refers to the sediment obtained by centrifuging the lysate 30 min at 105,000 X g. Zymogen activation was carried out with trypsin (9). mU, milliunits. n.d., not determined.
Multi-mix homogenizer at one-third of the maximum speed and incubated for 15 min at 30°. Portions of 3 ml were layered on 10-ml linear gradients of Renografin (5.8-50%), containing 20 mM Tris chloride, pH 7.5. The gradients were centrifuged for 1 hr in a SW 40 rotor with an L-2-65B Beckman centrifuge at 27,000 rpm (93,000 X g average). Fractions were removed from the top down with a J-shaped pipette connected to a peristaltic pump. Each fraction was diluted 10-fold with 5 mM Tris chloride, pH 7.5, containing 2 -40'
JR
3_
el
lk
p
..
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mM MgSO4 and centrifuged 30 min at 105,000 X g; the pellet was resuspended in the same buffer. Iodination. To 0.1 ml of a protoplast suspension obtained as described in the previous section, 2 ml of 0.8 M sorbitol, containing 0.1 M potassium phosphate at pH 7.5, were added and the suspension was centrifuged for 5 min at 650 X g. The pellet was suspended to a total volume of 1 ml in a mixture containing 0.8 M sorbitol, 0.1 M phosphate at pH 7.5, 15 jig/ml of lactoperoxidase, 47 ,LM hydrogen peroxide, and 1 ,uCi/ml of carrier-free NaI25I. After 10 min incubation at 250, the mixture was centrifuged for 5 min at 650 X g and the pellet was washed twice with 2 ml of 0.8 M sorbitol containing 0.1 M phosphate buffer at pH 7.5. The pellet was then suspended in a minimal volume of the same mixture and diluted with 0.4 ml of unlabeled protoplast suspension. Con A treatment, lysis, and gradient centrifugation were performed as described in the previous section. Electron Microscopy. Observations of membrane sections by electron microscopy were carried out as previously described (8).
SYNTHETASE
Spec if ic act iv ity
Percent recovery
3953
RESULTS Preparation of membranes and distribution of chitin synthetase activity Centrifugation of a lysate from Con-A-coated protoplasts on a linear Renografin gradient resulted in the separation of at least four well-defined bands (Fig. 1). Upon observation by phase contrast microscopy, band 1 was found to consist of very small particles, band 2 of somewhat larger vesicles, and band 4 of cell wall fragments and of damaged cells which had not been converted into protoplasts during the previous
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FIG. 2. Phase contrast (A) and electron microscope (B) images of purified membranes. membranes.
In B,
arrows
point
to vesicles associated with
Biochemistry: Duran et al.
3954
Proc. Nat. Acad. Sci. USA 72 (1975)
was found in the membrane fraction, as expected (see Fig. 3). Nevertheless, about 18%6P of the recovered 125I was in the fraction containing damaged cells, a disproportionate amount because less than one of those cells was present per every 100 protoplasts. This result appears to be due to permeability of the damaged cells to iodide and peroxidase, with subsequent labeling of internal protein. Even greater incorporation was observed when the damaged cells were first isolated in the gradient and then iodinated or when an equivalent number of protoplasts was lysed and the total intracellular protein was submitted to iodination. By the use of radioactive Con A it could be established that about 40% of the added lectin was taken up by the protoplasts. From the radioactivity associated with the membranes (Fig. 3B) it may be calculated that about 20% of their protein is Con A.
0. cL u
25
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20
-4
15
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Oz~ ~ ~ ~ ~ ~ W OX
10
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~~~~~~~Z
2
4
6
8
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12
FRACTION NUMBER
FIG. 3. Distribution of radioactivity and chitin synthetase after labeling of protoplasts with 125I (A)-or [3H]Con A (B). In both gradients 0.7 ml fractions were collected. With the 125I-labeled protoplasts, 30,000 cpm were layered onto the gradient and 22,400 were recovered in the sum of the fractions. For [3H]Con A the figures were 23,000 and 22,000, respectively. The drawing inserted between the graphs shows the approximate positions of the visible bands in the centrifuge tube.
incubation with snail enzyme. Band 3 consisted of very large membranes, about the size of the original protoplasts (Fig. 2A), some of them arranged in small clumps. An increase in the amount of Con A led to more clumping, with many membranes attached to each other back to back. Omission of the incubation of the lysate at 300 before centrifugation resulted in contamination of the membrane band with dark particles (phase contrast), possibly remnants of incompletely lysed nuclei. Electron micrographs of sections of the material from band 3 (Fig. 2B) showed long profiles and some vesicular material which appears to be attached to the membrane, since such vesicles are not seen free in the lumen of the convoluted profiles. Most of the chitin synthetase was recovered, with good yield, in the membrane fraction (Fig. 1). In 14 experiments the average specific activity of the enzyme in the membrane fractions was 3.0 times that of the crude particles. As observed in the last column of Fig. 1, practically all the chitin synthetase was in the inactive or zymogen state. However, if the activating factor present in the extract was not inhibited by addition of phenylmethylsulfonylfluoride, a large portion of the chitin synthetase was found in the activated state. Radioactive labeling of the protoplast surface When the protoplast surface was labeled with either 1251 or with [3H]Con A, most of the radioactivity in the gradient
A low-density particulate fraction containing chitin synthetase Whereas in the experiment of Fig. 1 only the main visible bands were isolated, in that of Fig. 3B the complete gradient was fractionated and analyzed. Surprisingly, a small peak of chitin synthetase activity was found in the higher portion of the gradient in a region that did not coincide with any of the major bands and was devoid of tritium or 1251 (Fig. 3). In this case also, less than 5% of the chitin synthetase was detected before treatment with trypsin. The peak was found reproducibly in several experiments and accounted for about 20% of the total recovered activity. When larger centrifuge tubes were used, a faint band of particulate material was found in the same area, but it coincided only in part with the peak of activity. Since the method used for the enzymatic assay is not specific for chitin (9), it was conceivable that the "'light" fraction might catalyze a different reaction. However, this was not the case because the reaction product of both the "light" and "heavy" enzymes was characterized as chitin by enzymatic degradation with chitinase and paper chromatography of the liberated diacetylchitobiose (12). The lack of radioactivity in the light fraction suggested that it might contain fragments from membranes which failed to adsorb Con A. Therefore, lectin treatment was omitted in some experiments and the protoplasts were lysed in 1 mM EDTA containing 0.02% Triton X-100, a treatment that results in the disruption of all membranes into very small fragments. After gradient centrifugation, the bulk of the chitin synthetase activity was found at the same position as for the Con-A-treated membranes. Some activity was again found trailing in the upper portion of the gradient, although this material had a somewhat different distribution.
The orientation of chitin synthetase in the membrane Some insight into the orientation of chitin synthetase in the membrane could be gained by studying the effect of glutaraldehyde on the activity under conditions in which either the external side or both sides of the membranes were exposed (Table 1). When protoplasts were briefly treated with glutaraldehyde prior to lysis, the subsequent yield of enzyme activity was not affected. When the same treatment was performed after the internal face of the membrane had been exposed by lysis, 90% of the activity was irreversibly lost. Essentially the same results were obtained whether the Con A treatment was included or not, and whether it was preceded or followed by exposure to glutaraldehyde (Table
Biochemistry:
Proc. Nat. Acad. Sci. USA 72 (1975)
Dura'n et al.
1). It may be concluded that chitin synthetase is accessible only from the inside of the cell. DISCUSSION The size and morphology of the isolated membranes together with the results of 125I and [3H]Con A labeling clearly indicate that these are indeed the plasma membranes, with very little contamination from other organelles. The bulk of the chitin synthetase is in the zymogen form and is associated with this fraction. There is a 3-fold increase in specific activity with respect to the crude particles from the lysate. The magnitude of the increase in specific activity seems reasonable, since the membranes are the major particulate component of the yeast lysate, as observed by phase contrast microscopy. Besides its importance for the mechanism of chitin synthesis, this result furnishes a well-proven enzymatic marker for the yeast plasma membrane. The overall process of chitin synthesis and translocation into the periplasmic space may involve several components of the membrane. At least some of these components must be facing the interior of the cell, since UDPGlcNAc, the substrate of chitin synthetase, is intracellular. This supposition was strikingly confirmed by the fact that glutaraldehyde can only inactivate the enzyme when acting on the internal face of the membrane. The recovery of some chitin synthetase activity in the upper portion of the gradients poses a puzzle. If the light fraction were composed of plasma membranes which failed to take up Con A, lysis in the absence of the lectin would have shifted the position of the bulk of the activity to the upper zone of the gradient, but this was not the case. Two other possibilities suggest themselves: (a) That chitin synthetase is not integrated into the membrane, but is linked to some vesicles or particles of lighter density. These particles would be normally attached to the membrane, but some of them might be liberated upon lysis and homogenization. (b) That the light fraction consists of precursor structures, which would normally end up in the membrane in due time. The fact that lysis under very drastic conditions, i.e., in the presence of EDTA and Triton X-100, did not increase the proportion of enzyme in the light fraction is not consistent with the first hypothesis, although it does not eliminate it. The solution of this problem must await further purification of the
3955
light fraction and study of its behavior in different physiological states. Our working hypothesis about septum initiation proposed that vesicles carrying the activating factor of chitin synthetase would fuse with the plasma membrane and activate the zymogen attached to the latter (1). Two major assumptions of this hypothesis, i.e., the localization of the activating factor in vesicles (13) and that of the zymogen association with the plasma membrane, have now been confirmed. It remains to be seen whether proteolytic action (14) is indeed the mechanism by which chitin synthetase is activated in vio.
We are indebted to J. Lunney for help with the iodination procedure and to Drs. G. G. Ashwell, J. Braatz, W. B. Jakoby, and R. Ulane for discussions and criticism. A.D. was a fellow of the Comision de Intercambio Cultural entre Espafia y los Estados Unidos de America. 1. Cabib, E. & Farkas, V. (1971) Proc. Nat. Acad. Sci. USA 68, 2052-2056. 2. Christensen, M. S. & Cirillo, V. P. (1972) J. Bacteriol. 110, 1190-1205. 3. Fuhrmann, G. F., Wehrli, E. & Boehm, C. (1974) Biochim. Biophys. Acta 363,295-310. 4. Kosinova', A., Farkas, V., Machala, S. & Bauer, S. (1974) Arch. Microbiol. 99,255-263. 5. Matile, P., Moor, H. & Muhlethaler, K. (1967) Arch. Mikrobiol. 58, 201-211. 6. Schibeci, A., Rattray, J. B. M. & Kidby, D. K. (1973) Biochim. Biophys. Acta 311, 15-25. 7. Scarborough, G. A. (1975) J. Biol. Chem. 250, 1106-1111. 8. Bowers, B., Levin, G. & Cabib, E. (1974) J. Bacteriol. 119, 564-575. 9. Cabib, E. (1972) in Methods in Enzymology, ed. Ginsburg, V. (Academic Press, New York and London), Vol. 28, pp. 572580. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 11. Cabib, E. (1971) in Methods in Enzymology, ed. Jakoby, W. B. (Academic Press, New York and London), Vol. 22, pp.
120-122. 12. Keller, F. A. & Cabib, E. (1971) J. Biol. Chem. 246,160-166. 13. Cabib, E., Ulane, R. & Bowers, B. (1973) J. Biol. Chem. 248, 1451-1458. 14. Cabib, E. & Ulane, R. (1973) Biochem. Biophys. Res. Commun. 50, 186-191.
Chitin synthetase zymogen is attached to the yeast plasma membrane (yeast septum/gradient centrifugation/concanavalin A/glutaraldehyde)
ANGEL DURAN*, BLAIR BOWERSt, AND ENRICO CABIB*f *
National Institute of Arthritis, Metabolism and Digestive Diseases, and t National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland
20014
Communicated by T. M. Sonneborn, August 4,1975
specific activity 53.4 ,tCi/,umol) were products of New England Nuclear. Saccharomyces cerevisiAe, strain X2180 (American Type Culture Collection no. 26109) was grown in a minimal medium (8), and harvested during the logarithmic phase at an approximate cell density of 3.5 X 107 cells per ml (buds not counted as cells). Analytical. Active chitin synthetase and its zymogen were determined as described (9). Tris chloride at pH 7.5 was substituted for imidazole, except in the experiment of Table 1. Protein was assayed according to Lowry et al. (10). Protoplast Lysis and Membrane Purification. Centrifugations were carried out at about 20. Yeast protoplasts were obtained as previously reported (11) and suspended in 0.8 M sorbitol, in a final volume of I ml/g (wet weight) of yeast utilized. To 0.5 ml of this suspension, were added 3 ml of 0.8 M sorbitol, containing 0.05 M Tris chloride at pH 7.5 and 10 mM MgSO4, followed by 3.5 ml of the same solution, supplemented with 0.5 mg/ml of Con A. After 10 min at room temperature, the flocculated protoplasts were centrifuged at 2° for 1 min in a swinging bucket rotor at 250 X g. The pellet was carefully resuspended in 6.5 ml of the sorbitol-TrisMg++ mixture and recentrifuged at the same speed for 3 min. The final pellet was suspended in 8.5 ml of 10 mM Tris chloride at pH 7.5, containing 5 mM MgSO4, 1 mg/ml of deoxyribonuclease and 0.6 mM phenylmethylsulfonylfluoride. The mixture was stirred at 00 for 3 min in a Lourdes
ABSTRACT Pretreatment of yeast protoplasts with concanavalin A, according to the method used by- C. A. Scarborough for Neurospora (J. Biol. Chem. 250, 1106-1111, 1975), reinforced the plasma membranes, and helped to maintain their integrity during subsequent lysis of the protoplasts. After purification by centrifuging on a Renografin density gradient, practically intact membranes were obtained. Previous labeling of the protoplasts with 125I or with [3Hjconcanavalin A resulted in recovery of the radioactivity in the membrane fraction. The bulk of the chitin synthetase (chitin synthase; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4.-f-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) recovered in the gradient was also found in this fraction, in the zymogen form. About 20% of the activity sedimented in a plasmamembrane-free fraction at lower density. Glutaraldehyde inactivated chitin synthetase when it was added to a Iysate, but not when applied to intact protoplasts. It is concluded that chitin synthetase is so oriented in the membrane that it is only accessible from the inside of the cell. These results confirm our previous hypothesis that the chitin synthetase zymogen is associated with the plasma membrane, a basic assumption for the explanation of localized activation of the enzyme and initiation of septum formation. In a previous communication (1), we suggested that the inactive form of chitin synthetase (chitin synthase; UDP-acetamido-2-deoxy-D-glucose:chitin 4-,B-acetamidodeoxyglucosyltransferase; EC 2.4.1.16), the enzyme responsible for the formation of the yeast primary septum, is attached to the plasma membrane. This supposition proved difficult to substantiate because a reliable preparation of purified yeast plasmalemma was not available. Several such preparations have been described in the literature (2-6), but in every case they consisted of small membrane fragments and therefore it is difficult to exclude the possibility that they might be mixtures of intracellular and external membranes. Recently we have succeeded in obtaining yeast plasma membranes, essentially in a single piece, by a modification of the method used by Scarborough (7) for Neurospora. The method consists in reinforcing the membranes by coating them with concanavalin A (Con A) prior to cell rupture. After gradient centrifugation of the lysate, the bulk of the chitin synthetase zymogen has been found in association with the membranes.
Table 1. Glutaraldehyde inactivation of chitin synthetase before and after protoplast lysis
Acetylglucosamine incorporated into chitin
MATERIALS AND METHODS Materials. Meglumine diatrizoate (Renografin) was purchased from Squibb & Sons; bovine pancreas deoxyribonuclease, lactoperoxidase, and unlabeled concanavalin A were purchased from Sigma and glutaraldehyde from Ladd Research Industries, Burlington, Vermont. Carrier-free Na12I was obtained from Amersham-Searle, whereas [acetyl3H]concanavalin A (specific activity 149 mCi/,umol) and UDP['4C]GlcNAc (labeled in C-1 of the hexosamine moiety,
No.
Treatment (in sequence)
(cpm)
1. 2. 3. 4. 5. 6. 7.
Con A, lysis Glutaraldehyde, Con A, lysis Con A, glutaraldehyde, lysis Lysis Glutaraldehyde, lysis Con A, lysis, glutaraldehyde Lysis, glutaraldehyde
921 919 904
711 750 85
70
Where indicated, glutaraldehyde was added to a final concentration of 1% to a suspension of protoplasts. After 30 sec at 0°, 20 volumes of 0.8 M sorbitol were added. Protoplasts were centrifuged for 5 min at 650 x g and washed again with 0.8 M sorbitol. Lysates were treated with glutaraldehyde in the same way, but dilution and washing were performed with 0.05 M imidazole, pH 6.5, containing 2 mM MgSQ4, and centrifugation was for 10 min at 20,000 X g. When treatment with Con A was omitted, lysis was carried out as previously reported (9, 12), because Con-A-free protoplasts did not break well under the conditions outlined under Materials and Methods. In every case, zymogen was activated with trypsin before assay for chitin synthetase activity.
Abbreviation: Con A, concanavalin A. t To whom reprint requests should be addressed.
3952
Biochemistry:
Dura'n et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
C H T N
FRACTION
U
Percent activity
mu/mg protein
before activation
0.81
3.5
100
Crude particles
-Band 1
2.8
0.21
n. d.
-Band 2
5.2
0.63
2
Band 3
75.6
3.1
0.5
- Band 4
0.6
0.39
n d.
FIG. 1. Distribution of chitin synthetase activity after centrifugation on a Renografin gradient. Crude particles refers to the sediment obtained by centrifuging the lysate 30 min at 105,000 X g. Zymogen activation was carried out with trypsin (9). mU, milliunits. n.d., not determined.
Multi-mix homogenizer at one-third of the maximum speed and incubated for 15 min at 30°. Portions of 3 ml were layered on 10-ml linear gradients of Renografin (5.8-50%), containing 20 mM Tris chloride, pH 7.5. The gradients were centrifuged for 1 hr in a SW 40 rotor with an L-2-65B Beckman centrifuge at 27,000 rpm (93,000 X g average). Fractions were removed from the top down with a J-shaped pipette connected to a peristaltic pump. Each fraction was diluted 10-fold with 5 mM Tris chloride, pH 7.5, containing 2 -40'
JR
3_
el
lk
p
..
A,4
co:
mM MgSO4 and centrifuged 30 min at 105,000 X g; the pellet was resuspended in the same buffer. Iodination. To 0.1 ml of a protoplast suspension obtained as described in the previous section, 2 ml of 0.8 M sorbitol, containing 0.1 M potassium phosphate at pH 7.5, were added and the suspension was centrifuged for 5 min at 650 X g. The pellet was suspended to a total volume of 1 ml in a mixture containing 0.8 M sorbitol, 0.1 M phosphate at pH 7.5, 15 jig/ml of lactoperoxidase, 47 ,LM hydrogen peroxide, and 1 ,uCi/ml of carrier-free NaI25I. After 10 min incubation at 250, the mixture was centrifuged for 5 min at 650 X g and the pellet was washed twice with 2 ml of 0.8 M sorbitol containing 0.1 M phosphate buffer at pH 7.5. The pellet was then suspended in a minimal volume of the same mixture and diluted with 0.4 ml of unlabeled protoplast suspension. Con A treatment, lysis, and gradient centrifugation were performed as described in the previous section. Electron Microscopy. Observations of membrane sections by electron microscopy were carried out as previously described (8).
SYNTHETASE
Spec if ic act iv ity
Percent recovery
3953
RESULTS Preparation of membranes and distribution of chitin synthetase activity Centrifugation of a lysate from Con-A-coated protoplasts on a linear Renografin gradient resulted in the separation of at least four well-defined bands (Fig. 1). Upon observation by phase contrast microscopy, band 1 was found to consist of very small particles, band 2 of somewhat larger vesicles, and band 4 of cell wall fragments and of damaged cells which had not been converted into protoplasts during the previous
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/4
,
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10 pm
I
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.......
-d
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B
FIG. 2. Phase contrast (A) and electron microscope (B) images of purified membranes. membranes.
In B,
arrows
point
to vesicles associated with
Biochemistry: Duran et al.
3954
Proc. Nat. Acad. Sci. USA 72 (1975)
was found in the membrane fraction, as expected (see Fig. 3). Nevertheless, about 18%6P of the recovered 125I was in the fraction containing damaged cells, a disproportionate amount because less than one of those cells was present per every 100 protoplasts. This result appears to be due to permeability of the damaged cells to iodide and peroxidase, with subsequent labeling of internal protein. Even greater incorporation was observed when the damaged cells were first isolated in the gradient and then iodinated or when an equivalent number of protoplasts was lysed and the total intracellular protein was submitted to iodination. By the use of radioactive Con A it could be established that about 40% of the added lectin was taken up by the protoplasts. From the radioactivity associated with the membranes (Fig. 3B) it may be calculated that about 20% of their protein is Con A.
0. cL u
25
5 B
z 0
20
-4
15
1
Oz~ ~ ~ ~ ~ ~ W OX
10
2
z
'0
00
~~~~~~~Z
2
4
6
8
10O
12
FRACTION NUMBER
FIG. 3. Distribution of radioactivity and chitin synthetase after labeling of protoplasts with 125I (A)-or [3H]Con A (B). In both gradients 0.7 ml fractions were collected. With the 125I-labeled protoplasts, 30,000 cpm were layered onto the gradient and 22,400 were recovered in the sum of the fractions. For [3H]Con A the figures were 23,000 and 22,000, respectively. The drawing inserted between the graphs shows the approximate positions of the visible bands in the centrifuge tube.
incubation with snail enzyme. Band 3 consisted of very large membranes, about the size of the original protoplasts (Fig. 2A), some of them arranged in small clumps. An increase in the amount of Con A led to more clumping, with many membranes attached to each other back to back. Omission of the incubation of the lysate at 300 before centrifugation resulted in contamination of the membrane band with dark particles (phase contrast), possibly remnants of incompletely lysed nuclei. Electron micrographs of sections of the material from band 3 (Fig. 2B) showed long profiles and some vesicular material which appears to be attached to the membrane, since such vesicles are not seen free in the lumen of the convoluted profiles. Most of the chitin synthetase was recovered, with good yield, in the membrane fraction (Fig. 1). In 14 experiments the average specific activity of the enzyme in the membrane fractions was 3.0 times that of the crude particles. As observed in the last column of Fig. 1, practically all the chitin synthetase was in the inactive or zymogen state. However, if the activating factor present in the extract was not inhibited by addition of phenylmethylsulfonylfluoride, a large portion of the chitin synthetase was found in the activated state. Radioactive labeling of the protoplast surface When the protoplast surface was labeled with either 1251 or with [3H]Con A, most of the radioactivity in the gradient
A low-density particulate fraction containing chitin synthetase Whereas in the experiment of Fig. 1 only the main visible bands were isolated, in that of Fig. 3B the complete gradient was fractionated and analyzed. Surprisingly, a small peak of chitin synthetase activity was found in the higher portion of the gradient in a region that did not coincide with any of the major bands and was devoid of tritium or 1251 (Fig. 3). In this case also, less than 5% of the chitin synthetase was detected before treatment with trypsin. The peak was found reproducibly in several experiments and accounted for about 20% of the total recovered activity. When larger centrifuge tubes were used, a faint band of particulate material was found in the same area, but it coincided only in part with the peak of activity. Since the method used for the enzymatic assay is not specific for chitin (9), it was conceivable that the "'light" fraction might catalyze a different reaction. However, this was not the case because the reaction product of both the "light" and "heavy" enzymes was characterized as chitin by enzymatic degradation with chitinase and paper chromatography of the liberated diacetylchitobiose (12). The lack of radioactivity in the light fraction suggested that it might contain fragments from membranes which failed to adsorb Con A. Therefore, lectin treatment was omitted in some experiments and the protoplasts were lysed in 1 mM EDTA containing 0.02% Triton X-100, a treatment that results in the disruption of all membranes into very small fragments. After gradient centrifugation, the bulk of the chitin synthetase activity was found at the same position as for the Con-A-treated membranes. Some activity was again found trailing in the upper portion of the gradient, although this material had a somewhat different distribution.
The orientation of chitin synthetase in the membrane Some insight into the orientation of chitin synthetase in the membrane could be gained by studying the effect of glutaraldehyde on the activity under conditions in which either the external side or both sides of the membranes were exposed (Table 1). When protoplasts were briefly treated with glutaraldehyde prior to lysis, the subsequent yield of enzyme activity was not affected. When the same treatment was performed after the internal face of the membrane had been exposed by lysis, 90% of the activity was irreversibly lost. Essentially the same results were obtained whether the Con A treatment was included or not, and whether it was preceded or followed by exposure to glutaraldehyde (Table
Biochemistry:
Proc. Nat. Acad. Sci. USA 72 (1975)
Dura'n et al.
1). It may be concluded that chitin synthetase is accessible only from the inside of the cell. DISCUSSION The size and morphology of the isolated membranes together with the results of 125I and [3H]Con A labeling clearly indicate that these are indeed the plasma membranes, with very little contamination from other organelles. The bulk of the chitin synthetase is in the zymogen form and is associated with this fraction. There is a 3-fold increase in specific activity with respect to the crude particles from the lysate. The magnitude of the increase in specific activity seems reasonable, since the membranes are the major particulate component of the yeast lysate, as observed by phase contrast microscopy. Besides its importance for the mechanism of chitin synthesis, this result furnishes a well-proven enzymatic marker for the yeast plasma membrane. The overall process of chitin synthesis and translocation into the periplasmic space may involve several components of the membrane. At least some of these components must be facing the interior of the cell, since UDPGlcNAc, the substrate of chitin synthetase, is intracellular. This supposition was strikingly confirmed by the fact that glutaraldehyde can only inactivate the enzyme when acting on the internal face of the membrane. The recovery of some chitin synthetase activity in the upper portion of the gradients poses a puzzle. If the light fraction were composed of plasma membranes which failed to take up Con A, lysis in the absence of the lectin would have shifted the position of the bulk of the activity to the upper zone of the gradient, but this was not the case. Two other possibilities suggest themselves: (a) That chitin synthetase is not integrated into the membrane, but is linked to some vesicles or particles of lighter density. These particles would be normally attached to the membrane, but some of them might be liberated upon lysis and homogenization. (b) That the light fraction consists of precursor structures, which would normally end up in the membrane in due time. The fact that lysis under very drastic conditions, i.e., in the presence of EDTA and Triton X-100, did not increase the proportion of enzyme in the light fraction is not consistent with the first hypothesis, although it does not eliminate it. The solution of this problem must await further purification of the
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light fraction and study of its behavior in different physiological states. Our working hypothesis about septum initiation proposed that vesicles carrying the activating factor of chitin synthetase would fuse with the plasma membrane and activate the zymogen attached to the latter (1). Two major assumptions of this hypothesis, i.e., the localization of the activating factor in vesicles (13) and that of the zymogen association with the plasma membrane, have now been confirmed. It remains to be seen whether proteolytic action (14) is indeed the mechanism by which chitin synthetase is activated in vio.
We are indebted to J. Lunney for help with the iodination procedure and to Drs. G. G. Ashwell, J. Braatz, W. B. Jakoby, and R. Ulane for discussions and criticism. A.D. was a fellow of the Comision de Intercambio Cultural entre Espafia y los Estados Unidos de America. 1. Cabib, E. & Farkas, V. (1971) Proc. Nat. Acad. Sci. USA 68, 2052-2056. 2. Christensen, M. S. & Cirillo, V. P. (1972) J. Bacteriol. 110, 1190-1205. 3. Fuhrmann, G. F., Wehrli, E. & Boehm, C. (1974) Biochim. Biophys. Acta 363,295-310. 4. Kosinova', A., Farkas, V., Machala, S. & Bauer, S. (1974) Arch. Microbiol. 99,255-263. 5. Matile, P., Moor, H. & Muhlethaler, K. (1967) Arch. Mikrobiol. 58, 201-211. 6. Schibeci, A., Rattray, J. B. M. & Kidby, D. K. (1973) Biochim. Biophys. Acta 311, 15-25. 7. Scarborough, G. A. (1975) J. Biol. Chem. 250, 1106-1111. 8. Bowers, B., Levin, G. & Cabib, E. (1974) J. Bacteriol. 119, 564-575. 9. Cabib, E. (1972) in Methods in Enzymology, ed. Ginsburg, V. (Academic Press, New York and London), Vol. 28, pp. 572580. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 11. Cabib, E. (1971) in Methods in Enzymology, ed. Jakoby, W. B. (Academic Press, New York and London), Vol. 22, pp.
120-122. 12. Keller, F. A. & Cabib, E. (1971) J. Biol. Chem. 246,160-166. 13. Cabib, E., Ulane, R. & Bowers, B. (1973) J. Biol. Chem. 248, 1451-1458. 14. Cabib, E. & Ulane, R. (1973) Biochem. Biophys. Res. Commun. 50, 186-191.
