Arch. Microbiol. 103, 51--55 (1975) 9 by Springer-Verlag 1975
Subcellular Distribution of Yeast Invertase Isoenzymes J. MEYER and PH. MATILE Department of General Botany, Swiss Federal Institute of Technology, Zfirich Received August 26, 1974
Abstract. Homogenates from yeast cells contain 1 ~ or less of sedimentable invertase activity. Sedimentability is equally low in homogenates from cells repressed or derepressed with regard to invertase secretion. Intracellularly, the mannanprotein form of invertase is largely localized in
vacuoles whereas the small isoenzyme is largely present in the soluble cell fraction. These findings indicate that vesicles are not involved in the secretion of invertase. A soluble mode of invertase secretion is discussed.
Key words: Yeast -- Invertase -- Isoenzymes -- Localization -- Vacuoles -- Spheroplasts -- Lipid Granules -- Cytosol.
The external invertase of yeast is a mannanprotein which contains 50 7o of carbohydrate; its secretion is repressed to various degrees by hexoses (see Lampen, 1971). In spheroplasts a small form of invertase which contains no mannan is present together with the large glycoprotein form (Gascbn and Ottolenghi, 1967). Kinetics, immunological and other properties of the small invertase isoenzyme suggest that it is closely related with the secreted glycoprotein form of invertase (Gascbn e t a l . , 1968). Recent results obtained by Lampen et al. (1972) seem to disprove, however, that the small isoenzyme represents the precursor protein of the secreted enzyme. Another unsolved problem concerns the possible involvement of structures in the secretion of invertase. Beteta and Gascbn (1971) have demonstrated the presence of small and large invertase in preparations of isolated yeast vacuoles. Since the presence of various hydrolytic enzymes in the vacuole suggests a catabolic function of this organelle (Matile and Wiemken, 1967) its involvement in invertase secretion, as suggested by Beteta and Gascbn (1971), is rather unlikely. Lampen et al. (1972) have advanced a model in which secretory vesicles are responsible for the oriented and localized release of invertase in the region of buds (Tkacz and Lampen, 1973). Recently, Holley and Kidby (1973) have also proposed the involvement of vesicles in invertase secretion. However, the experimental data on the subcellular localization of invertase, that are required for supporting any of the proposed models, are not yet available. The results on enzyme localization depend considerably on the cell fractionation techniques employed. Since vacuoles seem to represent an important site of
invertase location, earlier findings have now been reexamined using an advanced technique for preparing purified vacuoles. We report findings on the intracellular localization of yeast invertase isoenzymes that seem to be incompatible with proposed conceptions of invertase secretion.
Materials and Methods Organism and Its Culture. The yeast strain 303-671 (prepared from crosses of Saceharomyces carlsbergensis, S. chevalieri and S. italieus) was used throughout this study. The culture medium Contained 20 g glucose, 2 g glutamic acid, 2 g yeast extract (Oxoid), 20 mg citric acid. H20, 3 g NH4NOs, 2 g KH~PO4, vitamins, salts and trace elements per liter. It was adjusted to pH 5.1 before autoclaving. Batch cultures were grown at 27~ using an oscillating shaker. Homogenization of Cells, Differential Centrifugation. Cells were harvested by centrifugation and washed twice with dist. water. After the final washing with the citrate-sorbitol medium (0.5 M sorbitol, 0.01 M citrate-NaOH buffer pH 6.5) they were disrupted by vigorous shaking in the presence of Ballotini-beads (diameter 0.45--0.5 mm) and medium. After 7 sec of shaking, the beads were removed and the homogenate diluted with medium. An initial centrifugation at 3000xg (10 min) yielded a cell free extract. Three consecutive centrifugations at 20000 x g (20 min), 50000xg (30 rain) and 270000xg (30 min) yielded the sediments 20, 50 and 270 and a final supernatant (soluble fraction). All of the sediments were washed once with medium. Preparation of Spheroplasts and Vacuoles. The methods used were essentially those developed by Wiemken (1969; see also Wiemken and Nurse, 1973). Spheroplasts suspended in a small volume of buffered osmoticum (0.7 M sorbitol, 1 Kindly provided by P. Ottolenghi and J. Friis, formerly at Carlsberg Laboratory, Copenhagen, Denmark.
52
Arch. Microbiot., Vol. t03, No. 1 (1975)
0.01 M citrate;NaOH buffer pH 6.5) were subjected to osmotic lysis by adding ca. 9 volumes of a hypotonic solution (0.15 M sorbitol, 8 ~ (w/v) Ficol! and 0.01 M citrateNAOH buffer pH 6.5). Stirring in a Sorvall Omnimixer removed efficiently the cytoplasmic material which initially adhered to the vacuoles released from bursting spheroplasts. The product of lysis was now overlayered with the above medium containing only 7.5 ~ (w/v) Ficoll. Upon centrifugation for 20 min at 10000• the vacuoles formed a white film at the surface of the 7.5 ~ Ficoll solution and could be removed with a Pasteur pipette. The few contaminating lipid granules present in this preparation could be removed by a second flotation of the vacuoles through a layer of 7.5 ~ Ficoll. Estimation of lnvertase Activity. 0.1 ml of enzyme plus 0.5 ml of substrate (1 ~ w/v sucrose in 0.05 M acetate buffer pH 5.0) were incubated at 30 ~C. The reaction was stopped by adding 0.5 ml 0.05 M phosphate buffer pH 7.0 and incubating the test tubes for 3 min in boiling water. The estimation of glucose formed by the action of invertase was performed using the glucose oxidase test system of Boehringer Mannheim GmbH, BRD. One unit of invertase activity equals I ~zmoleof sucrose hydrolized in 30 min at 30~C. Polyacrylamide-Gelelectrophoresis of lnvertase. Isoenzymes of invertase were separated using gels prepared after Maurer (1968) as follows. Stock solutions A, B, C and D contained in 100 ml of dist. water: 30 g acrylamide, 0.8 g bis-acrylamide (A); 0.14 g (NH02SzOs(B); 6.85 g Tris, 48 ml 1.0 N HC1, 0.46 ml Temed, pH 7.5 (C); 5 g Na-dodecylsulfate (D). These solutions were mixed to yield a 5.63 ~ (w/v) polyacrylamide gel: 1.5 vol A, 4 vol B, 1 vol C, 0.4 vol D and 1.1 vol dist. water. After polymerization 50 ~1 of enzyme +5 ~zl5 (w/v) Na-dodecylsulfate were layered onto the gel and stabilized by adding Sephadex G-25. The electrode buffer contained 5.52 g diethylbarbituric acid and 1.0 g Tris per liter (pH 7.0). Electrophoresis was carried out at constant current (2 mA per gel) for ca. 1.5 hrs. Thereafter the gels were sliced into 2 mm segments in which invertase activities were estimated. Sliced gels could be stored in the freezer for several days without loss of activity.
Results Sedimentability of Invertase The yeast strain 303-67 is highly sensitive to repression of invertase formation and secretion by glucose (Gasc6n and Ottolenghi, 1967). In the presence of glucose concentrations above ca. 0.1 ~ (w/v) secretion is repressed; in cultures growing on a 2 ~ (w/v) glucose medium secretion is derepressed as soon as the glucose level has fallen below this concentration (Meyer and Matile, 1974). If secretion of invertase would be mediated by a secretory vesicle one would expect to find a higher sedimentability of this enzyme in secreting cells as compared with repressed cells. It appears from the results summarized in Table 1 that sedimentability is equally low in extracts from repressed and derepressed cells, respectively. Vesicles with properties similar to glucanase-vesicles (Cortat et aL, 1972) would be present mainly in the 20000 and 50000• g sedi-
Table 1. Sedimentability of invertase in extracts of repressed and derepressed cells. Repressed cells were harvested from a batch culture on 2 ~o-glucose medium at a concentration of 3.1 • 107 cells/ml (glucose conc. > 1 ~), derepressed cells at 1.4• 10~ cells/ml (no detectable glucose in the medium) Fractions
Repressed cells
Derepressed cells
Cell free extract Sediment 20 Sediment 50 Sediment 270 Supernatant 270
1O0 0.24 0.58 0.31 98.87
1O0 0.07 0.24 0.54 99.I5
ments. Moreover, the total sedimentability of glucanase activity in budding cells is ca. 12 ~ as compared with ca. 1 ~ of sedimentable invertase. Invertase Activity in Subcellular Fractions of Spheroplasts A considerable proportion of the invertase activity present in the soluble cell fraction is external invertase but enzyme present in the cytosol and in the vacuoles (which are destroyed upon the rupture of cells) may also contribute to this fraction. The localization of invertase in vacuoles and cytosol has therefore been investigated using spheroplasts for liberating organelles by hypotonic treatment. Since the release of vacuoles and the isolation causes the loss of an unknown percentage of these fragile organelles the results from the fractionation of spheroplasts are given as specific activities of invertase (Table 2). It appears that the highest specific activity is present in the isolated vacuoles. The specific activity present in the soluble fraction is much lower than in isolated vacuoles; this is due to a low protein content of the vacuoles. In fact, the total vacuolar activity is only ca. 10--30 ~o of the soluble activity. Only traces of o~-glucosidase activity, which is known to be localized in the cytosoi, are present in the vacuolar preparation. It should be mentioned that t h e spheroplasts used for isolating vacuoles were derepressed with regard to invertase secretion (Meyer and Matile, 1974). Only scarce activities are, however, associated with sedimentable material; this finding strengthens the view that invertase containing small vesicles do not exist in yeast. In the case of glucanase activity, which is also secreted in spheroplasts, the sedimentability in preparations of lysed spheroplasts is as high as 77~o (Cortat et al., 1972). The invertase associated with isolated vacuoles is most probably a constituent of the vacuolar fluid. In preparations of intact vacuoles only ca. 50 ~ of the total activity (estimated in ultrasonicated samples) is
J. Meyer and Ph. Matile: Yeast Invertases
53
Table 2. Invertase activities present in subcellular fractions from invertase-secreting spheroplasts. The activities per mg protein in the'lysed spheroptasts are taken to be 1.0. The sediments 50 and 270 were obtained from the product of lysis of spheroplasts after flotation of vacuoles at 10000 • g; sediment 50:30 min 50000xg; sediment 270:30 rain 270000 • g. For comparison the distribution of fl-glucosidase, another secreted hydrolase, is also given Fractions
Localization of Invertase Isoenzymes
Specific activities
Spheroptasts Vacuoles Sediment 50 Sediment 270 Supernatant 270
Invertase
/~-Glucosidase
1.0 20.0 O.13 0.21 4.53
1.0 28.0 3.38 1.05 0.33
2.0 /ultrasonlcated ~LS~ vocuoles
~
I.0
~
-
vacuoles
eL5
0
]0
2 '0
" ~
30
vacuolar invertase activity is sedimentable with the vacuolar membranes; the corresponding sedimentability of an enzyme activity known to be associated with the tonoplast, 0r is ca. 84~ (van der VVilden et al., 1973).
......... rain
Fig. 1. Latency of vacuolar invertase. Total activity: isolated vacuoles were ultrasonicated for 10 sec prior to incubation. Substrate accessible activity: untreated, intact vacuoles. The incubation mixtures contained 0.2 M sorbitol as osmoticum
substrate accessible (Fig. 1). Under the conditions of the invertase assay the isolated vacuoles are stable. Hence, a latency of 50 ~ could either indicate that half of the enzyme is associated with the vacuolar membrane in a substrate accessible fashion or, else, that the tonoplast is not perfectly impermeable for sucrose, Indeed, vacuoles released from spheroplasts upon osmotic shock are permeable for micromolecules (Schaffner and Wiemken, unpublished results). If isolated vacuoles are ruptered, only 7 ~ of the total
In vacuoles isolated from the yeast strain 303-67 Beteta and Gasc6n (t971) have detected roughly equal activities of large and small invertase. Using an improved method for isolation and purification of vacuoles (see Wiemken and Nurse, t973) and polyacrylamide-geMectrophoresis for separating invertase isoenzymes we found that a major fraction of the vacuolar invertase is large isoenzyme. Fig.2 shows a typical electropherogramme of vacuolar invertase: the activity of the mannanprotein present in the peak with the lower mobility is 4 times higher than the activity of the small invertase. This unexpected result has been checked with other yeast strains. In two strains of Saccharomyces cerevisiae (4H4F 2 and LBG H 10223) practically all of the invertase activity was found to be large isoenzyme. These findings are consistant with the localization of the small invertase isoenzyme. In the soluble fraction of lysed spheroplasts (270000• supernatant) the small invertase is predominant (Fig. 3). Taking into consideration that a fraction of vacuoles (containing mainly large invertase) is disrupted upon the osmotic lysis of spheroplasts it may be concluded that the small amount of glycoprotein invertase present in the soluble fraction is in fact vacuolar invertase. Hence, the small isoenzyme may be the only form of invertase present in the cytosol. Holley and Kidby (1973) have suggested that the invertase activity associated with isolated vacuoles may, in fact, be present in (secretory) vesicles which Obtained from P. Ottolenghi and J. Friis, formerly at Carlsberg Laboratory, Copenhagen, Denmark. Obtained from Department of Microbiology, Swiss Federal Institute of Technology, Zfirich, Switzerland.
klS = 0.35
a-,
L
0
02
OA
OfiRf
02
a~
a6
2 3 Fig. 2. Distribution of invertase isoenzymes in polyacrylamide get after electrophoresis of a preparation of vacuoies. The peak at Rf = 0.2 represents the large mannanprotein form, the small peak at R~ -= 0.55-0.6 is the small isoenzyme Fig. 3. Invertase isoenzymes present in the soluble fraction of lysed spheroplasts (270000 • g supernatant)
54
Fig. 4. Electron micrograph of lipid granules (spherosomes) isolated from lysed spheroplasts. Note the single electron dense contour of the membrane. • 41800
contaminate the vacuolar fractions. The only class of organelles that may contaminate the preparations of vacuoles obtained upon flotation are lipid granules. Indeed, these spherosomes isolated from spheroplasts contain some invertase activity, the specific activity being comparatively high due to the extremely low protein content. In order to investigate this localization further lipid granules have been isolated from whole cells by flotation through layers of buffered 0.2 M sorbitol. Electron micrographs demonstrate the homogeneity of the preparations (Fig. 4) and also the existence of a typical spherosomal membrane having only one electron opaque leaflet instead of two leaflets of the triplelayered "unit membranes". It appeared that the invertase present in the fraction lipid granules is superficially and loosely attached to these organelles. Upon repeated flotation it can be progressively removed. Furthermore the isoenzyme composition in lipid granules resembles the composition in the cells used for the isolation. Hence, it seems that a small fraction of the free invertase molecules present in homogenates is unspecifically attached to the lipid granules. Discussion In models of invertase secretion proposed to date a granulocrine mode of enzyme release has been considered. Either the vacuoles (Beteta and Gascbn, 1971) or some sort of vesicles (Lampen et al., 1972; Holley and Kidby, 1973) have been charged with the required transport and eventual glycosylation of invertase. An
Arch, Microbiol., Vol. 103, No. 1 (1975) oriented and localized release of invertase from secretory vesicles would, indeed, allow to explain the fact that the newly secreted enzyme is present only in the growing bud (Tkacz and Lampen, 1973). Moreover, the synthesis of mannan is associated with membranes (Cortat et al., 1973); a Golgi-like vesicle appears, therefore, as the appropriate structure for the glycosylation of a precursor invertase and subsequent release into the extracytoplasmic space (Lampen et al., 1972). Data presented in this report suggest that secretory vesicles containing invertase do not exist in yeast ceils. Not only is sedimentability of invertase very low, it is equally low whether the cells are repressed or derepressed with regard to invertase secretion. In addition, the lysis of spheroplasts which must be considered as a gentle method of homogenization does not yield a subcellular fraction of structures that could represent the hypothetical secretory vesicles. It should be emphasized that in the case of glucanases, the granulocrine secretion of which has been demonstrated, ca. 77 ~ of the total activity is sedimentable upon osmotic lysis of spheroplasts (Cortat et al., 1972). A mode of secretion without the involvement of vesicles would be eccrine, that is, transport of the enzyme across the plasma membrane. Since the glycoprotein form of invertase seems to be largely absent from the cytosol it might be hypothesized that it is formed in the plasmalemma, glycosylation of the precursor protein being coupled with transport of the invertase into the external space. The localization of mannan synthetase in the yeast plasmalemma (Cortat et al., 1973) would support this hypothesis. However, the small invertase isoenzyme which initially has been considered to represent the precursor protein of the secreted invertase does not seem to be involved in secretion; upon the chase of radioactive label in large and small enzyme it appears that only a small fraction of the latter is eventually secreted (Lampen et al., 1972). Hence, the bulk of small invertase in the cytosol has an unknown function. Still another problem concerns the vacuolar invertases. On the one hand the presence in vacuoles of a low activity of small invertase could be explained by the degradation of large invertase (Lampen et al., 1972). The vacuolar localization of mannosidase (van der Wilden et al., 1973) would support this view. Moreover, the presence in vacuoles of various proteolytic enzymes (Matile and Wiemken, 1967; results to be published) could explain the complete disappearence in stationary cells of internal large invertase (Meyer and Matile, 1974) which is largely localized in the vacuoles. On the other hand the origin of the vacuolar large invertase can certainly not be explained if its formation in the plasmalemma is assumed. It would be necessary to
J. Meyer and Ph. Matile: Yeast Invertases introduce a second site of invertase glycosytation in the tonoplast. In this context it is interesting to note that substantial amounts of mannan are indeed localized in the yeast vacuole and preliminary experiments suggest that mannan synthetase may be localized in the vacuolar membrane (MatiIe, unpublished results). Thus, the vacuole may function as a digestive compartment in which intracellular invertases without a metabolic function are eventually broken down.
Acknowledgements. The present investigation was supported by the Swiss National Science Foundation.
References Beteta, P., Gasc6n,S.: Localization of invertase in yeast vacuoles. FEBS Letters 13, 297-300 (1971) Cortat, M., Matile, Ph., Kopp,Y. : Intracellular localization of mannan synthetase activity in budding baker's yeast. Biochem. biophys. Res. Commun. 53, 482-489 (1973) Cortat, M., Matile, Ph., Wiemken, A. : Isolation of glucanasecontaining vesicles from budding yeast. Arch. Mikrobiol. 82, 189-205 (1972) Gasc6n,S., Neumann, P., Lampen,J.O.: Comparative study of the purified internal and external invertases from yeast. J. biol. Chem. 243, 1573-1577 (1968)
55 Gascon, S., Ottolenghi, P. : Invertase isoenzymes and their localization in yeast. C. R. Lab. Carlsberg 36, 8 5 - 9 3 (1967) Hotley, R. A., Kidby, D. K. : Role of vacuoles and vesicles in extra-cellular enzyme secretion from yeast. Canad. J. Microbiol. 19, t 13 - i 17 (1973) Lampen, J.O.: Yeast invertase. In: The enzymes, P. D. Boyer, ed, vol. V, pp. 291-316. New York: Academic Press t971 Lampen,J.O., Kuo, S.-C., Cano, F.R., Tkacz, J.S.: Structural features in synthesis of external enzymes by yeast. Proc. 4th Int. Fermentation Symposium, Kyoto, pp. 122-128 (1972) Matile, Ph., Wiemken, A. : The vacuole as the lysosome of the yeast cell. Arch. Mikrobiol. 56, 148-t55 (1967) Maurer, tS.R.: Disk-Elektrophorese. Berlin: de Gruyter 1968 Meyer, L, Matite, Ph. : Regulation of isoenzymes and secretion of invertase in baker's yeast. Biochem. Physiol. Pflanzen fin press) Tkacz, 3. S., Lampen, 3'.O. : Surface distribution of invertase on growing Saccharomyces cells. J. Bact. 113, 1073t075 (1973) Wiemken,A.: Eigenschaften der Hefevakuole. Thesis Nr. 4340, ETH Z~Jrich (1969) Wiemken,A., Nurse, P. : The vacuole as a compartment of amino acid pools in yeast. Proc. Third Int. Specialized Symp. on Yeasts, part II, pp. 331-347 (1973) van der Wilden, W., Matile, Ph., Schellenberg, M., Meyer, J., Wiemken,A.: Vacuolar membranes: Isolation from yeast cells. Z. Naturforsch. 28e, 416--421 (1973)
Prof. Dr. Ph. Matile Institut f/ar Allgemeine Botanik der Eidgen6ssischen Technischen Hochschule Sonneggstr. 5, CH-8006 Zfirich, Switzerland
Subcellular Distribution of Yeast Invertase Isoenzymes J. MEYER and PH. MATILE Department of General Botany, Swiss Federal Institute of Technology, Zfirich Received August 26, 1974
Abstract. Homogenates from yeast cells contain 1 ~ or less of sedimentable invertase activity. Sedimentability is equally low in homogenates from cells repressed or derepressed with regard to invertase secretion. Intracellularly, the mannanprotein form of invertase is largely localized in
vacuoles whereas the small isoenzyme is largely present in the soluble cell fraction. These findings indicate that vesicles are not involved in the secretion of invertase. A soluble mode of invertase secretion is discussed.
Key words: Yeast -- Invertase -- Isoenzymes -- Localization -- Vacuoles -- Spheroplasts -- Lipid Granules -- Cytosol.
The external invertase of yeast is a mannanprotein which contains 50 7o of carbohydrate; its secretion is repressed to various degrees by hexoses (see Lampen, 1971). In spheroplasts a small form of invertase which contains no mannan is present together with the large glycoprotein form (Gascbn and Ottolenghi, 1967). Kinetics, immunological and other properties of the small invertase isoenzyme suggest that it is closely related with the secreted glycoprotein form of invertase (Gascbn e t a l . , 1968). Recent results obtained by Lampen et al. (1972) seem to disprove, however, that the small isoenzyme represents the precursor protein of the secreted enzyme. Another unsolved problem concerns the possible involvement of structures in the secretion of invertase. Beteta and Gascbn (1971) have demonstrated the presence of small and large invertase in preparations of isolated yeast vacuoles. Since the presence of various hydrolytic enzymes in the vacuole suggests a catabolic function of this organelle (Matile and Wiemken, 1967) its involvement in invertase secretion, as suggested by Beteta and Gascbn (1971), is rather unlikely. Lampen et al. (1972) have advanced a model in which secretory vesicles are responsible for the oriented and localized release of invertase in the region of buds (Tkacz and Lampen, 1973). Recently, Holley and Kidby (1973) have also proposed the involvement of vesicles in invertase secretion. However, the experimental data on the subcellular localization of invertase, that are required for supporting any of the proposed models, are not yet available. The results on enzyme localization depend considerably on the cell fractionation techniques employed. Since vacuoles seem to represent an important site of
invertase location, earlier findings have now been reexamined using an advanced technique for preparing purified vacuoles. We report findings on the intracellular localization of yeast invertase isoenzymes that seem to be incompatible with proposed conceptions of invertase secretion.
Materials and Methods Organism and Its Culture. The yeast strain 303-671 (prepared from crosses of Saceharomyces carlsbergensis, S. chevalieri and S. italieus) was used throughout this study. The culture medium Contained 20 g glucose, 2 g glutamic acid, 2 g yeast extract (Oxoid), 20 mg citric acid. H20, 3 g NH4NOs, 2 g KH~PO4, vitamins, salts and trace elements per liter. It was adjusted to pH 5.1 before autoclaving. Batch cultures were grown at 27~ using an oscillating shaker. Homogenization of Cells, Differential Centrifugation. Cells were harvested by centrifugation and washed twice with dist. water. After the final washing with the citrate-sorbitol medium (0.5 M sorbitol, 0.01 M citrate-NaOH buffer pH 6.5) they were disrupted by vigorous shaking in the presence of Ballotini-beads (diameter 0.45--0.5 mm) and medium. After 7 sec of shaking, the beads were removed and the homogenate diluted with medium. An initial centrifugation at 3000xg (10 min) yielded a cell free extract. Three consecutive centrifugations at 20000 x g (20 min), 50000xg (30 rain) and 270000xg (30 min) yielded the sediments 20, 50 and 270 and a final supernatant (soluble fraction). All of the sediments were washed once with medium. Preparation of Spheroplasts and Vacuoles. The methods used were essentially those developed by Wiemken (1969; see also Wiemken and Nurse, 1973). Spheroplasts suspended in a small volume of buffered osmoticum (0.7 M sorbitol, 1 Kindly provided by P. Ottolenghi and J. Friis, formerly at Carlsberg Laboratory, Copenhagen, Denmark.
52
Arch. Microbiot., Vol. t03, No. 1 (1975)
0.01 M citrate;NaOH buffer pH 6.5) were subjected to osmotic lysis by adding ca. 9 volumes of a hypotonic solution (0.15 M sorbitol, 8 ~ (w/v) Ficol! and 0.01 M citrateNAOH buffer pH 6.5). Stirring in a Sorvall Omnimixer removed efficiently the cytoplasmic material which initially adhered to the vacuoles released from bursting spheroplasts. The product of lysis was now overlayered with the above medium containing only 7.5 ~ (w/v) Ficoll. Upon centrifugation for 20 min at 10000• the vacuoles formed a white film at the surface of the 7.5 ~ Ficoll solution and could be removed with a Pasteur pipette. The few contaminating lipid granules present in this preparation could be removed by a second flotation of the vacuoles through a layer of 7.5 ~ Ficoll. Estimation of lnvertase Activity. 0.1 ml of enzyme plus 0.5 ml of substrate (1 ~ w/v sucrose in 0.05 M acetate buffer pH 5.0) were incubated at 30 ~C. The reaction was stopped by adding 0.5 ml 0.05 M phosphate buffer pH 7.0 and incubating the test tubes for 3 min in boiling water. The estimation of glucose formed by the action of invertase was performed using the glucose oxidase test system of Boehringer Mannheim GmbH, BRD. One unit of invertase activity equals I ~zmoleof sucrose hydrolized in 30 min at 30~C. Polyacrylamide-Gelelectrophoresis of lnvertase. Isoenzymes of invertase were separated using gels prepared after Maurer (1968) as follows. Stock solutions A, B, C and D contained in 100 ml of dist. water: 30 g acrylamide, 0.8 g bis-acrylamide (A); 0.14 g (NH02SzOs(B); 6.85 g Tris, 48 ml 1.0 N HC1, 0.46 ml Temed, pH 7.5 (C); 5 g Na-dodecylsulfate (D). These solutions were mixed to yield a 5.63 ~ (w/v) polyacrylamide gel: 1.5 vol A, 4 vol B, 1 vol C, 0.4 vol D and 1.1 vol dist. water. After polymerization 50 ~1 of enzyme +5 ~zl5 (w/v) Na-dodecylsulfate were layered onto the gel and stabilized by adding Sephadex G-25. The electrode buffer contained 5.52 g diethylbarbituric acid and 1.0 g Tris per liter (pH 7.0). Electrophoresis was carried out at constant current (2 mA per gel) for ca. 1.5 hrs. Thereafter the gels were sliced into 2 mm segments in which invertase activities were estimated. Sliced gels could be stored in the freezer for several days without loss of activity.
Results Sedimentability of Invertase The yeast strain 303-67 is highly sensitive to repression of invertase formation and secretion by glucose (Gasc6n and Ottolenghi, 1967). In the presence of glucose concentrations above ca. 0.1 ~ (w/v) secretion is repressed; in cultures growing on a 2 ~ (w/v) glucose medium secretion is derepressed as soon as the glucose level has fallen below this concentration (Meyer and Matile, 1974). If secretion of invertase would be mediated by a secretory vesicle one would expect to find a higher sedimentability of this enzyme in secreting cells as compared with repressed cells. It appears from the results summarized in Table 1 that sedimentability is equally low in extracts from repressed and derepressed cells, respectively. Vesicles with properties similar to glucanase-vesicles (Cortat et aL, 1972) would be present mainly in the 20000 and 50000• g sedi-
Table 1. Sedimentability of invertase in extracts of repressed and derepressed cells. Repressed cells were harvested from a batch culture on 2 ~o-glucose medium at a concentration of 3.1 • 107 cells/ml (glucose conc. > 1 ~), derepressed cells at 1.4• 10~ cells/ml (no detectable glucose in the medium) Fractions
Repressed cells
Derepressed cells
Cell free extract Sediment 20 Sediment 50 Sediment 270 Supernatant 270
1O0 0.24 0.58 0.31 98.87
1O0 0.07 0.24 0.54 99.I5
ments. Moreover, the total sedimentability of glucanase activity in budding cells is ca. 12 ~ as compared with ca. 1 ~ of sedimentable invertase. Invertase Activity in Subcellular Fractions of Spheroplasts A considerable proportion of the invertase activity present in the soluble cell fraction is external invertase but enzyme present in the cytosol and in the vacuoles (which are destroyed upon the rupture of cells) may also contribute to this fraction. The localization of invertase in vacuoles and cytosol has therefore been investigated using spheroplasts for liberating organelles by hypotonic treatment. Since the release of vacuoles and the isolation causes the loss of an unknown percentage of these fragile organelles the results from the fractionation of spheroplasts are given as specific activities of invertase (Table 2). It appears that the highest specific activity is present in the isolated vacuoles. The specific activity present in the soluble fraction is much lower than in isolated vacuoles; this is due to a low protein content of the vacuoles. In fact, the total vacuolar activity is only ca. 10--30 ~o of the soluble activity. Only traces of o~-glucosidase activity, which is known to be localized in the cytosoi, are present in the vacuolar preparation. It should be mentioned that t h e spheroplasts used for isolating vacuoles were derepressed with regard to invertase secretion (Meyer and Matile, 1974). Only scarce activities are, however, associated with sedimentable material; this finding strengthens the view that invertase containing small vesicles do not exist in yeast. In the case of glucanase activity, which is also secreted in spheroplasts, the sedimentability in preparations of lysed spheroplasts is as high as 77~o (Cortat et al., 1972). The invertase associated with isolated vacuoles is most probably a constituent of the vacuolar fluid. In preparations of intact vacuoles only ca. 50 ~ of the total activity (estimated in ultrasonicated samples) is
J. Meyer and Ph. Matile: Yeast Invertases
53
Table 2. Invertase activities present in subcellular fractions from invertase-secreting spheroplasts. The activities per mg protein in the'lysed spheroptasts are taken to be 1.0. The sediments 50 and 270 were obtained from the product of lysis of spheroplasts after flotation of vacuoles at 10000 • g; sediment 50:30 min 50000xg; sediment 270:30 rain 270000 • g. For comparison the distribution of fl-glucosidase, another secreted hydrolase, is also given Fractions
Localization of Invertase Isoenzymes
Specific activities
Spheroptasts Vacuoles Sediment 50 Sediment 270 Supernatant 270
Invertase
/~-Glucosidase
1.0 20.0 O.13 0.21 4.53
1.0 28.0 3.38 1.05 0.33
2.0 /ultrasonlcated ~LS~ vocuoles
~
I.0
~
-
vacuoles
eL5
0
]0
2 '0
" ~
30
vacuolar invertase activity is sedimentable with the vacuolar membranes; the corresponding sedimentability of an enzyme activity known to be associated with the tonoplast, 0r is ca. 84~ (van der VVilden et al., 1973).
......... rain
Fig. 1. Latency of vacuolar invertase. Total activity: isolated vacuoles were ultrasonicated for 10 sec prior to incubation. Substrate accessible activity: untreated, intact vacuoles. The incubation mixtures contained 0.2 M sorbitol as osmoticum
substrate accessible (Fig. 1). Under the conditions of the invertase assay the isolated vacuoles are stable. Hence, a latency of 50 ~ could either indicate that half of the enzyme is associated with the vacuolar membrane in a substrate accessible fashion or, else, that the tonoplast is not perfectly impermeable for sucrose, Indeed, vacuoles released from spheroplasts upon osmotic shock are permeable for micromolecules (Schaffner and Wiemken, unpublished results). If isolated vacuoles are ruptered, only 7 ~ of the total
In vacuoles isolated from the yeast strain 303-67 Beteta and Gasc6n (t971) have detected roughly equal activities of large and small invertase. Using an improved method for isolation and purification of vacuoles (see Wiemken and Nurse, t973) and polyacrylamide-geMectrophoresis for separating invertase isoenzymes we found that a major fraction of the vacuolar invertase is large isoenzyme. Fig.2 shows a typical electropherogramme of vacuolar invertase: the activity of the mannanprotein present in the peak with the lower mobility is 4 times higher than the activity of the small invertase. This unexpected result has been checked with other yeast strains. In two strains of Saccharomyces cerevisiae (4H4F 2 and LBG H 10223) practically all of the invertase activity was found to be large isoenzyme. These findings are consistant with the localization of the small invertase isoenzyme. In the soluble fraction of lysed spheroplasts (270000• supernatant) the small invertase is predominant (Fig. 3). Taking into consideration that a fraction of vacuoles (containing mainly large invertase) is disrupted upon the osmotic lysis of spheroplasts it may be concluded that the small amount of glycoprotein invertase present in the soluble fraction is in fact vacuolar invertase. Hence, the small isoenzyme may be the only form of invertase present in the cytosol. Holley and Kidby (1973) have suggested that the invertase activity associated with isolated vacuoles may, in fact, be present in (secretory) vesicles which Obtained from P. Ottolenghi and J. Friis, formerly at Carlsberg Laboratory, Copenhagen, Denmark. Obtained from Department of Microbiology, Swiss Federal Institute of Technology, Zfirich, Switzerland.
klS = 0.35
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0
02
OA
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02
a~
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2 3 Fig. 2. Distribution of invertase isoenzymes in polyacrylamide get after electrophoresis of a preparation of vacuoies. The peak at Rf = 0.2 represents the large mannanprotein form, the small peak at R~ -= 0.55-0.6 is the small isoenzyme Fig. 3. Invertase isoenzymes present in the soluble fraction of lysed spheroplasts (270000 • g supernatant)
54
Fig. 4. Electron micrograph of lipid granules (spherosomes) isolated from lysed spheroplasts. Note the single electron dense contour of the membrane. • 41800
contaminate the vacuolar fractions. The only class of organelles that may contaminate the preparations of vacuoles obtained upon flotation are lipid granules. Indeed, these spherosomes isolated from spheroplasts contain some invertase activity, the specific activity being comparatively high due to the extremely low protein content. In order to investigate this localization further lipid granules have been isolated from whole cells by flotation through layers of buffered 0.2 M sorbitol. Electron micrographs demonstrate the homogeneity of the preparations (Fig. 4) and also the existence of a typical spherosomal membrane having only one electron opaque leaflet instead of two leaflets of the triplelayered "unit membranes". It appeared that the invertase present in the fraction lipid granules is superficially and loosely attached to these organelles. Upon repeated flotation it can be progressively removed. Furthermore the isoenzyme composition in lipid granules resembles the composition in the cells used for the isolation. Hence, it seems that a small fraction of the free invertase molecules present in homogenates is unspecifically attached to the lipid granules. Discussion In models of invertase secretion proposed to date a granulocrine mode of enzyme release has been considered. Either the vacuoles (Beteta and Gascbn, 1971) or some sort of vesicles (Lampen et al., 1972; Holley and Kidby, 1973) have been charged with the required transport and eventual glycosylation of invertase. An
Arch, Microbiol., Vol. 103, No. 1 (1975) oriented and localized release of invertase from secretory vesicles would, indeed, allow to explain the fact that the newly secreted enzyme is present only in the growing bud (Tkacz and Lampen, 1973). Moreover, the synthesis of mannan is associated with membranes (Cortat et al., 1973); a Golgi-like vesicle appears, therefore, as the appropriate structure for the glycosylation of a precursor invertase and subsequent release into the extracytoplasmic space (Lampen et al., 1972). Data presented in this report suggest that secretory vesicles containing invertase do not exist in yeast ceils. Not only is sedimentability of invertase very low, it is equally low whether the cells are repressed or derepressed with regard to invertase secretion. In addition, the lysis of spheroplasts which must be considered as a gentle method of homogenization does not yield a subcellular fraction of structures that could represent the hypothetical secretory vesicles. It should be emphasized that in the case of glucanases, the granulocrine secretion of which has been demonstrated, ca. 77 ~ of the total activity is sedimentable upon osmotic lysis of spheroplasts (Cortat et al., 1972). A mode of secretion without the involvement of vesicles would be eccrine, that is, transport of the enzyme across the plasma membrane. Since the glycoprotein form of invertase seems to be largely absent from the cytosol it might be hypothesized that it is formed in the plasmalemma, glycosylation of the precursor protein being coupled with transport of the invertase into the external space. The localization of mannan synthetase in the yeast plasmalemma (Cortat et al., 1973) would support this hypothesis. However, the small invertase isoenzyme which initially has been considered to represent the precursor protein of the secreted invertase does not seem to be involved in secretion; upon the chase of radioactive label in large and small enzyme it appears that only a small fraction of the latter is eventually secreted (Lampen et al., 1972). Hence, the bulk of small invertase in the cytosol has an unknown function. Still another problem concerns the vacuolar invertases. On the one hand the presence in vacuoles of a low activity of small invertase could be explained by the degradation of large invertase (Lampen et al., 1972). The vacuolar localization of mannosidase (van der Wilden et al., 1973) would support this view. Moreover, the presence in vacuoles of various proteolytic enzymes (Matile and Wiemken, 1967; results to be published) could explain the complete disappearence in stationary cells of internal large invertase (Meyer and Matile, 1974) which is largely localized in the vacuoles. On the other hand the origin of the vacuolar large invertase can certainly not be explained if its formation in the plasmalemma is assumed. It would be necessary to
J. Meyer and Ph. Matile: Yeast Invertases introduce a second site of invertase glycosytation in the tonoplast. In this context it is interesting to note that substantial amounts of mannan are indeed localized in the yeast vacuole and preliminary experiments suggest that mannan synthetase may be localized in the vacuolar membrane (MatiIe, unpublished results). Thus, the vacuole may function as a digestive compartment in which intracellular invertases without a metabolic function are eventually broken down.
Acknowledgements. The present investigation was supported by the Swiss National Science Foundation.
References Beteta, P., Gasc6n,S.: Localization of invertase in yeast vacuoles. FEBS Letters 13, 297-300 (1971) Cortat, M., Matile, Ph., Kopp,Y. : Intracellular localization of mannan synthetase activity in budding baker's yeast. Biochem. biophys. Res. Commun. 53, 482-489 (1973) Cortat, M., Matile, Ph., Wiemken, A. : Isolation of glucanasecontaining vesicles from budding yeast. Arch. Mikrobiol. 82, 189-205 (1972) Gasc6n,S., Neumann, P., Lampen,J.O.: Comparative study of the purified internal and external invertases from yeast. J. biol. Chem. 243, 1573-1577 (1968)
55 Gascon, S., Ottolenghi, P. : Invertase isoenzymes and their localization in yeast. C. R. Lab. Carlsberg 36, 8 5 - 9 3 (1967) Hotley, R. A., Kidby, D. K. : Role of vacuoles and vesicles in extra-cellular enzyme secretion from yeast. Canad. J. Microbiol. 19, t 13 - i 17 (1973) Lampen, J.O.: Yeast invertase. In: The enzymes, P. D. Boyer, ed, vol. V, pp. 291-316. New York: Academic Press t971 Lampen,J.O., Kuo, S.-C., Cano, F.R., Tkacz, J.S.: Structural features in synthesis of external enzymes by yeast. Proc. 4th Int. Fermentation Symposium, Kyoto, pp. 122-128 (1972) Matile, Ph., Wiemken, A. : The vacuole as the lysosome of the yeast cell. Arch. Mikrobiol. 56, 148-t55 (1967) Maurer, tS.R.: Disk-Elektrophorese. Berlin: de Gruyter 1968 Meyer, L, Matite, Ph. : Regulation of isoenzymes and secretion of invertase in baker's yeast. Biochem. Physiol. Pflanzen fin press) Tkacz, 3. S., Lampen, 3'.O. : Surface distribution of invertase on growing Saccharomyces cells. J. Bact. 113, 1073t075 (1973) Wiemken,A.: Eigenschaften der Hefevakuole. Thesis Nr. 4340, ETH Z~Jrich (1969) Wiemken,A., Nurse, P. : The vacuole as a compartment of amino acid pools in yeast. Proc. Third Int. Specialized Symp. on Yeasts, part II, pp. 331-347 (1973) van der Wilden, W., Matile, Ph., Schellenberg, M., Meyer, J., Wiemken,A.: Vacuolar membranes: Isolation from yeast cells. Z. Naturforsch. 28e, 416--421 (1973)
Prof. Dr. Ph. Matile Institut f/ar Allgemeine Botanik der Eidgen6ssischen Technischen Hochschule Sonneggstr. 5, CH-8006 Zfirich, Switzerland
