Molecular Forms of Yeast Invertase

Eur. J. Biochem. 50, 571-579 (1975)

Molecular Forms of Yeast Invertase Fernando MORENO, Amparo G. OCHOA, Santiago GASCON, and Julio R. VILLANUEVA Departamento de Microbiologh, Consejo Superior de Investigaciones Cientificas, Facultad de Ciencias, Universidad de Salamanca

(Received April 1 /September 9, 1974)

The molecular forms of yeast invertase have been studied. It is shown that by gel filtration on Sephadex G-200 it is possible to demonstrate the presence not only of a light, carbohydrate-free, invertase, and a heavy invertase containing 50 % carbohydrate, but also of a continuous spectrum of molecular forms that probably represent the sequential addition of mannose to the light form during the secretion process, which culminates in the formation on the heavy enzyme that is found outside the cytoplasmic membrane. The elution volume-void volume ratio in Sephadex G-200 varies from 1.75 of the light to 1.05 of the heavy invertase. The separation of invertase has also been achieved by ion-exchange chromatography and by isoelectric focusing and is facilitated by removal of the heavy form by ammonium sulphate precipitation.

During the protoplasting process the removal of the cell wall is accompanied by the loss of most of the heavy form. The intermediate forms are exclusively detected inside the protoplast, together with the light invertase and a small amount of heavy invertase.

The effect of 2-deoxy-~-glucose and cycloheximide on the biosynthesis and distribution of molecular forms of yeast invertase has also been studied. In the presence of 10mM glucose Saccharomyes 303-67 repressed cells readily synthesize invertase during the two-hour incubation period. Upon the addition of 2-deoxy-~-glucose, at a concentration of 75 pg/ml, the observed inhibition in the cells is 60%, but if the activity is measured after breaking the cells, only a 31 % inhibition is found, revealing an accumulation of invertase inside the protoplast. 2-Deoxy-D-glucose originates a pile-up of the light and intermediate forms at the expense of the formation of the heavy enzyme, showing that the inhibition of the glycosilation and, therefore, the secretion process, has taken place.

In the absence of de n o w invertase synthesis originated by cycloheximide, the glycosilation process still takes place as indicated by the accumulation of the heavy form at the expense of the light, carbohydrate-free, enzyme.

Yeast invertase is an attractive system to study giycoprotein synthesis and secretion : The enzyme is present in two forms in yeast cells [l], a heavy invertase containing 50% mannan and 3 % glucosamine [2] and a light form which contains no carbohydrate [3,4].

Invertase content in yeast is dependent on growth conditions and specially on glucose concentration [5 - 81. In derepressed cells, that is in cells grown in low glucose media, most invertase is located outside the cytoplasmic membrane [9,10] and it is in the heavy form [l]. Inside the protoplasts there are small

En=-vmes. Invertase or /?-D-fructofuranoside fructohydro- lase (EC 3.2.1.26); glucose oxidase (EC 1.1.3.4); peroxidase (EC 1.11.1.7).

amounts of both heavy and light isoenzymes and they are localized in small vesicles or vacuoles [ll, 121.

The presence of only two forms of invertase would imply that the 50% content of mannan of the heavy enzyme is added simultaneously to the small invertase during the secretion process. However, this sounds improbable and we have been looking for the presence of invertase forms of intermediate molecular weight that might reveal the existence of a sequential addition of the carbohydrate moiety [13] which can probably be used as a model for the study of the biosynthesis of cell wall glycoproteins in yeast, a problem that has received considerable attention in studies performed with intact cells, protoplasts and particulated fractions [14- 181.

Eur. J . Biochem. 50 (1975)

572 Molecular Forms of Yeast Invertase

In 1971 [19] we reported the effect of cycloheximide and 2-deoxy-~-glucose on the biosynthesis and secretion of yeast invertase and concluded that although, as was already known [16,20- 231, they exert their action at two different points in the biosynthesis of glycoproteins, either of them can stop the process of synthesis and secretion of yeast invertase because, in the experimental conditions used, the relative amount of the heavy and light invertases remained constant. Most of the experiments were conducted with protoplasts from Saccharomyces strains 303-67 and FH4C.

In this paper we present the existence of intermediate forms of invertase, demonstrated by chromatography and isoelectric focusing techniques. This prompted us to reexamine the results obtained before and we show that although cycloheximide and 2- deoxy-D-glucose can stop the synthesis of the heavy and light invertases, they act preferentially on the synthesis of the protein and the mannan respectively. In the presence of cycloheximide the amount of heavy enzyme in the cells of Saccharomyces 303-67 increases at the expense of the light form. With 2-deoxy- D-glucose there is an increase of the light and intermediate forms and a relatively lower level of the heavy invertase in the cells.

EXPERIMENTAL PROCEDURES

Materials

Glucose oxidase type V, peroxidase type 111, 0-dianisine, cycloheximide, 2-deoxy-~-glucose ; and 2-mercaptoethanol were from Sigma Chemical CO., Sephadex G-200, Sepharose 6B, DEAE-Sephadex A-50, sulphopropyl-Sephadex C-50 and blue dextran were from Pharmacia; Aquacide 2 from Calbiochem; the snail enzyme (sue digestifd'Helixpomatia stabilisd) was from L'industrie Biologique FranGaise (Genne- villiers, France); the yeast extract was from Difco Laboratories; and the Ampholines from LKB.

Yeast Strains

Two yeast strains were used, Saccharomyces 303-67 [24], kindly supplied by Dr P. Ottolenghi, and a mutant of this, strain FH4C [25] generously supplied by Dr J. 0. Lampen. Extensive work on yeast invertase has been conducted in both strains [l - 4,8,9,11,19, 241.

The parent Saccharomyces strain 303-67 is diploid and homozygous for the Suz gene (R2 of Winge and Roberts [24]). It synthesizes invertase in appreciable amounts only after the glucose has disappeared from the medium [8,9] and it does not hydrolyze maltose.

The mutant strain FH4C was obtained by Montene- court et al. [25] by ultraviolet irradiation of strain 303-67. It produces high levels of invertase even when growing in the presence of glucose.

Culture Condition, Preparation of Protoplasts and Cell-Free Extracts

The yeast were inoculated into flasks with 100 ml medium containing 1 % yeast extract and glucose and incubated in a rotatory shaker at 28 "C for 16 h. The medium for preparation of derepressed cells, or low glucose cells, contained 1 % glucose and this disappeared before 14 h of cultivation. For repressed cells, or high glucose cells, 3% glucose was used and had more than 1% left in the culture supernatant at the time of harvesting. The cells were collected and washed twice with distilled water by centrifugation at 4000 x g for 10 min.

The protoplasts were obtained as described previously [3], centrifuged at 10000 x g for 10 min and then resuspended and washed twice in 0.05 M Tris- HCl buffer, pH 7.5, containing 0.6 M KCl and 0.01 M magnesium sulphate. Protoplasts were lysed by resuspension in 0.05 M Tris-HC1 buffer, pH 7.5 and the supernatant was obtained by centrifugation at 20 000 x g for 20 min.

The cells were broken in a Braun MSK homogenizer with glass beads and the cell-free extract was obtained by centrifugation at 20000 x g for 20 min.

ANALYTTCAL PROCEDURES

Invertase Assay

The invertase was assayed in a two-step method as described previously [3]. For the second step, 1.4 U glucose oxidase per ml were used. One unit of invertase is defined as the amount of enzyme which hydro- lizes 1 pmol sucrose in 1 min at 30 "C in 0.05 M sodium acetate buffer, pH 5.0, containing 0.125 M sucrose.

Analytical Gel Filtration

The quantitative analysis of invertase forms was performed in a Sephadex (3-200 column (2.5 x 90 cm) equilibrated and eluted at 4 "C with 0.05 M Tris-HC1 buffer pH 7.5, using reversed flow. The void volume (V,) was determined from the elution volume of blue dextran, detected by its absorbance at 600 nm.

Fractions containing invertase were concentrated, when needed, by dialysis at 4 "C against sodium salts of carboxymethyl-cellulose (Aquacide 2).

Eur. J. Biochem. 50 (1975)

F. Moreno, A. G. Ochoa, S . Gas&, and J. R. Villanueva

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Fig. 1. Gel filtration in Sephadex G-200 of cell-free extracts from Saccharomyces 303-67. In A and C the cells were derepressed; that is, they had grown in I % glucose and this disappeared before 14.h growth. In B, D and E, repressed cells were used and more than 1 % glucose was left in the culture

0.02

Isoelectric Focusing

This was performed at 6 “C in a 110-ml LKB column filled with a glycerol gradient, containing 1 ampholine. After 50 h at 400 V, 2-ml fractions were collected. Where necessary, the fractions were ex- haustively dialyzed against 0.05 M Tris-HC1, pH 7.5, pooled and concentrated.

E 1

- 1.75 - 0.02

RESULTS

Characterization of Molecular Forms of Invertase by Gel Filtration in Sephadex G-200

Yeast invertase can be separated into several molecular forms as may be appreciated in Fig. 1. The presence of a light and a heavy form is well documented and typical profiles are shown in Fig. 1 A and B, which represent the usual pattern of invertase distribution, as detected by Sephadex G-200 filtration, of cell-free

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supernatant at the time of harvesting (see Experimental Procedure). The fractions from E with a V J V , ratio from 1.3 to 2.0 (bracket) were pooled concentrated and applied to the column (F). The extracts used contained 20.0 invertase units in A; 9.0 in B; 25.0 in C ; 4.0 in D; 2.0 in E and 1.5 in F

extracts obtained from derepressed and repressed yeast cells respectively. These characteristic profiles were originally obtained using columns 2 cm in diam- eter and 40- 60 cm long [l].

Intermediate molecular forms are frequently observed with reversed flow and longer columns, as may be seen in Fig. 1 C and D.

Even when the intermediate molecular forms can- not be readily appreciated, they can be detected by rechromatography and one example of this is shown in Fig. 1. In Fig. 1 E, a cell-free extract from repressed cells was subjected to gel filtration on Sephadex G-200 and fractions which should apparently contain a mixture of heavy and light invertase were pooled, concentrated and applied again to the Sephadex G-200 (Fig. 1 F). Besides the heavy (VJV, = 1.05) and the light (VJ V, = 1.75) forms that one would expect, an intermediate molecular form with a VJV0 of 1.32, was found in this experiment. This result is obtained


514 Molecular Forms of Yeast lnvertase

consistently every time when a rechromatography is made from fractions having a Ve/Vo from 1.2 to 1.5, although is should be noted that the intermediate form found varies in the Ve/Vo ratio from experiment to experiment. This probably represents the existence of a continuous spectrum of invertase forms, going from the light to the heavy invertase, and that the observed change in the intermediate from only represents

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v v, Fig. 2. Gel filtration c.f an ammonium ,sulphute precipitate of' cell-jirw extrac.ts,from derepressed cells. Extracts from Sacchu- romyces strain 303-67 were made 70 saturation by the addition of solid ammonium sulphate, and the solution was stored overnight at 4 "C. The precipitate was collected at 20000 x g and dissolved in 0.05 M Tris-HC1 buffer, pH 7.5; 50.0 invertase units were applied to the Sephadex G-200 column

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Fig. 3. Gelfiltration ofthe supernatant of lysedprotoplasts and eniyme released during protoplast formation. (A) Supernatant of lysed protoplasts from Saccharomyces FH4C; (B) supernatant of lysed protoplast from Saccharomyces 303-67 ; (C) enzyme released to the medium during protoplasting in

the predominant one under these experimental conditions.

The investigation of intermediate forms in cell- free extracts from derepressed cells, where the heavy form is extremely abundant, is facilitated by first removing most of the heavy invertase which might mask the presence of the other forms. This removal can be accomplished by repeated chromatography as shown in Fig. 1 E and F; by ammonium sulphate precipitation, or by digestion of the cell wall during protoplast formation.

Most of the heavy invertase is soluble in 70% saturation ammonium sulphate. The precipitate contains the light form and 20 to 30% of the total heavy form [3]. Fig.2 shows the results obtained by gel filtration on Sephadex G-200, of a 70% ammonium sulphate precipitate of a cell-free extract from Succha- romyces 303-67 derepressed cells. Besides the predominant light form ( Ve/ Vo = 1.75), two intermediate invertase forms ( Ve/ Vo of 1.20 and 1.34, respectively) are readily distinguishable. With the whole cell-free extract, these forms would only have been appreciated as a shoulder of the heavy invertase.

As was already known 131, the enzyme liberated into the protoplasting media is in the large form, and the removal of the heavy invertase by this means facilitates the demonstration of intermediate molecular forms in the protoplasts. Fig. 3 summarizes the results obtained by gel filtration on Sephadex C-200

r 1 I

experiment A ; (D) enzyme released during protoplast formation in experiment B. Protoplasts were obtained as indicated in Experimental Procedure. The extracts applied to the Sephadex G-200 column contained 2.0 invertase units in A; 2.0 in B; 150.0 in C and 18.0 in D


F. Moreno, A. G. Ochoa. S. Gascon, and J. R. Villanueva 575

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Fig.4. Fractionation of the molecular forms of invertase on DEAE-Sephades A-50 and characterization of the fractions by gel filtration in Sephudex G-200. In (A) a 70 % ammonium sulphate precipitate, obtained as indicated in Fig.2 from a cell-free extract of Saccharomyces strain 303-67 repressed cells containing 10.0 invertase units, was dialyzed against 0.05 M Tris-HCI buffer; pH 7.5 and applied to a DEAE- Sephadex A-50 column (25 x 25 cm) previously equilibrated with the same buffer. The column was washed with 200 ml of this buffer, and a linear gradient was established to 0.8 M NaCl in the same buffer. The total volume of the gradient was

of lysed protoplasts and the enzyme released into the supernatant during protoplast formation. The results for Saccharomyces FH4C and for Saccharomyces 303-67 are similar. The removal of most of the large form makes it possible to obtain results practically identical with those commented on above for the use of ammonium sulphate precipitation. In both strains, the enzyme outside the cytoplasmic membrane is in the large form, whereas inside the protoplasts there seems to exist a continuous spectrum of invertase molecules whose molecular weights go from the light to the large form, which is the one secreted.

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400 ml; ( e - o ) , invertase activity; (+ - - - ~ +), molarity of the eluent. Fractions from A marked 1(250 to 300 ml containing 0.5 invertase units); I1 (340 to 365 ml containing 1 .O invertase units) and I11 (370 to 500 ml containing 6.0 invertase units) were pooled, concentrated and their invertase forms investigated by gel filtration in Sephadex G-200 columns. The results for samples I, I1 and I11 are shown in B, C and D respectively. Column D was further analyzed and fractions with a VJV0 ratio from 1.2 to 1.5 (bracket) were pooled, concentrated and subjected to gel filtration in Sephadex G-200 (E)

Fractionation on DEAE-Sephadex A-50 It was already known that heavy invertase at pH 7.5

shows lower affinity for DEAE-Sephadex than the light form, and this is one crucial step in the purifica- tion of both forms [2,3]. After the finding of intermediate forms described above, we thought that it might be possible to differentiate between them by ion-exchange chromatography. The results obtained are illustrated for Saccharomyces 303-67 in Fig. 4, which shows that after the removal of most of the heavy invertase by ammonium sulphate precipitation, there appear several peaks of activity (Fig. 4A) which


576 Molecular Forms of Yeast Invertase

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Fig. 5. Zsoelectricfocusing ofinvertuse (A) Ammonium sulphate precipitate (70% saturation) of a cell-free extract from repressed Succhuromyces strain 303-67 ; (B) Ammonium sulphate precipitate (70 "/, saturation) of a cell-free extract from derepressed Saccharomyces strain FH4C. (D) Super- natant obtained from a culture medium where Sacchurornyces strain FH4C had grown for 16 h. It contained heavy invertase with a V J V , ratio of 1.05 as determined by gel filtration in

were later analyzed by gel filtration on Sephadex G-200. The first peak corresponds to heavy invertase (Fig. 4B); the second to an intermediate form, only slightly larger than the small invertase, with a VJ V, of 1.64 (Fig.4C) and the third is composed mainly of light invertase (Fig. 4D), although small amounts of intermediate forms are also present, as Fig.4E shows.

Isoelectric Focusing of Invertase Forms

It has been reported that it is possible to separate several invertase forms by isoelectric focusing from homogenates of yeast [26,27]. In order to charac- terize them, we looked for the isoelectric points of the

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Sephadex G-200; (E) Partially purified light invertase obtained by repeated ammonium sulphate precipitation and later isolation in Sephadex G-200 (Ve/Vo ratio 1.75). (C) Fractions 53 to 60 (bracket) from B were subjected to gel filtration in Sephadex G-200. The pH range (+ - - ~ ~ +) of the carrier ampholines was from 3 to 10 in A and B, and from 3 to 6 in D and E. The sample contained 20.0 invertase units in A; 50.0 in B; 7.0 in C ; 50.0 in D and 2.0 in E

different forms of invertase, using cell-free extracts and partially purified heavy and light forms, previously dialyzed against 0.05 M Tris-HCl buffer pH 7.5. The results are shown in Fig. 5. The isoelectric focusing of an ammonium sulphate precipitate (70 % saturation) of cell-free extracts from repressed Saccharomyces strain 303-67 (Fig. 5 A), and derepressed Saccharomyces FH4C (Fig. 5 B), shows the existence of at least three forms that can be separated by the differences in their isoelectric point. Fig.1D and E show that the isoelectric points of the heavy and light forms are 3.4 and 4.2 respectively. The invertase forms with an isoelectric point close to 5.0 that appear in Fig. 5A and B correspond to intermediate forms as demonstrated by


F. Moreno, A. G. Ochod, S. Gascon, and J. R. Villanueva 577

Table 1. Eflect of 2-deoxy-D-glucose und cycloheximide on the synthesis of invertase by Saccharomyces 303-67 repressed cells 3 ml of a suspension containing 5 x lo9 cells per ml were added to 100 ml of a medium with 1 % yeast extract and 10 mM glucose. The additions when listed were made at zero time and except for the zero-time control the mixture was incubated with shaking at 28 "C for 2 h. Glucose had disappeared from the supernatant before the 120-min incubation. No growth of the cells could be detected by absorbance at 600 nm. The cells were collected and washed twice and resuspended in 11 ml distilled water. 10 ml of this suspension were broken in a Braun MSK homogenizer with glass beads. The cell-free extract was obtained by centrifugation at 20000 x g for 20 min and the homogenate was filtered through a Millipore filter (pore size 0.45 pm). All values for activity are expressed as invertase units per 1.4 x lo9 cells or their equivalent in the cell-free extracts. Inhibition is given as a percentage of the total increase of invertase activity in the control. Cycloheximide was 0.5 pg/ml and 2-deoxy-~-glucose 75 pg/ml where added

Source of enzyme Invertase activity -~ ~~ -

at time zero, no additions no additions D-glucose hexlmide

after 120 min, after 120 min with 2-deoxy- after 120 min with cyclo-

~

U % U %

Cells 0.11 3.4 1.4 60 0.18 98 Cell-free extract 0.32 4.5 3.2 31 0.4 98

gel filtration in Sephadex G-200 (Fig. 5C). The invertase having an isoelectric points of 4.8 is probably equivalent to that in Fig. 1 C which has a Ve/Vo of 1.2.

Gel Filtration on Sepharose-6B and Chromatography on Sulphopropyl-Sephadex C-50

The heavy invertase has a Ve/Vo of 1.05 in Sephadex G-200, which is too close to the void volume of the column and it is therefore difficult to analyze the possible existence of invertase forms with molecular weight close to the heavy enzyme.

Sepharose-6B is known to have a separation range useful for larger molecules than those that can be analyzed on Sephadex G-200 and we therefore did some preliminary experiments on gel filtration in a 2 x 60-cm Sepharose-6B column, equilibrated and eluted at 4 "C with 0.05 M Tris-HC1 buffer, pH 7.5, previously calibrated with blue dextran. Partially purified heavy and light invertase were subjected to gel filtration on Sepharose-6B and they showed symmetrical peaks with a Ve/Vo ratio of 1.4 and 1.6 respectively. A mixture of both gave a symmetrical peak with a Ve/Vo of 1.5 and we therefore concluded that this is not an analytical tool useful for the purpose of this study.

The heavy and the light invertase can be separated not only by DEAE-Sephadex A-50 but also in sulphopropyl-Sephadex C-50 [3]. In some preliminary experiments we were able to demonstrate that it is possible to obtain at least four peaks with invertase activity by chromatography of a cell-free extract from Saccharo- myces 303-67 on a sulphopropyl-Sephadex C-50 column run in 0.05 M acetate buffer, pH 4.0, with a continuous gradient up to 1 M NaCI. We did not explore these results further, because the light inver-

tase is not stable at low pH and satisfactory separation can be obtained by other methods.

Effect of 2-Deoxy-~-glucose and Cycloheximide on the Biosynthesis and Distribution of Molecular Forms of Invertase in Yeast Cells

We have used essentially identical conditions as those described in our previous study on the effect of cycloheximide and 2-deoxy-~-glucose [ 191 except for the fact that now we have used repressed cells instead of yeast protoplast obtained from derepressed cells. We have chosen intact cells and not protoplasts because we were interested in studying quantitatively the full spectrum of invertase forms, and not only those found in protoplasts. Repressed cells have been selected because in this system, the zero-time controls have reduced amounts of heavy invertase.

Utilization of sucrose by Saccharomyces is dependent on the hydrolytic activities of the invertase bound to the cells, but external to the cytoplasmic membrane [28]. The enzyme inside the protoplast is not detected in the invertase assay with intact cells, because sucrose does not penetrate the cytoplasmic membrane and this explains the increase on invertase activity found on cell-free extracts as compared with the cells (Table 1). The inhibition originated by cycloheximide is similar to that previously described [19] and the same happens with 2-deoxy-~-glucose. Here however an interesting fact is that, whereas the inhibition in cells is 60%, in cell-free extracts it is only 31 %, indicating that 2- deoxy-D-glucose originates an accumulation of invertase inside the protoplasts, and therefore, a concom- itant partial inhibition of the secretion process.

The distribution of invertase forms is shown in Fig. 6. In repressed cells (zero-time control) the small


578 Molecular Forms of Yeast lnvertase

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141 4 Fig. 6. Eflect of 2-dt.oxy-~-glucose and cycloheximide on the distribulion of invertase molecular forms in yeast cells. Samples identical to those described in Table 1 were applied to a Se- phadex (3-200 column; 8 ml samples were used containing 2.5 invertase units in the zero-time control (&-- O), 36.0 in the 120-min control (M), 25.6 units from the 2-deoxy- D-glucose sample (A-A), and 3.2 units in that of cycloheximide (A 4)

amount of invertase is distributed evenly between the heavy and the light forms. The incubation during 120min in optimal conditions for the synthesis of invertase, originates a 30-fold increase in the specific activity of the cells (Table 1) but, as there is no growth, the small forms of the enzyme remains constant and the increase appears as heavy invertase. The peak of heavy invertase is not symmetrical, and there probably exist several forms of enzyme of molecular weight close to that of the heavy enzyme (Fig.6). Although this has not been represented, cells incubated under identical conditions but in the presence of 1 % glucose, which does not disappear before the 120-min incubation, duplicate in number as well as in invertase in cells and cell-free extracts; upon filtration in Sephadex G-200 the amount of heavy and light enzymes have also been doubled.

The effect of 2-deoxy-~-ghcose and cycloheximide on the relative proportion of invertase forms is shown in Fig.6. The distribution indicates that 2- deoxy-D-glucose originates an accumulation of the light invertase and an intermediate molecular form also appears, at the expense of the formation of the heavy enzyme, showing that the inhibition of the glyco-

silation process has taken place. The action of cycloheximide is the opposite to that observed with 2- deoxy-D-glucose. The inhibition obtained with 0.5 pgi ml cycloheximide is practically total (Table 1) and in the absence of invertase synthesis de novo, the glycosilation process still takes place as indicated by the accumulation of the heavy form at the expense of the light, carbohydrate-free, enzyme.

DISCUSSION

Fig.1 summarizes the main findings of this study. The predominant forms in every instance are those described previously [l]. In Saccharomyces 303-67 the heavy and light invertase are in similar concentrations in repressed cells ; in derepressed cells the heavy invertase increases and accounts for more than 80 of the total enzyme. We have not made any attempt to determine the relative proportion of intermediate forms, but our data indicate that they probably represent no more than 20-30% of the total enzyme of the cell.

The intermediate molecular forms are present in derepressed Saccharomyces 303-67 as well as in SQC- charomyces FH4C (Fig. 2) and this can be demonstrated by removal of the predominant heavy invertase, which is soluble in 70% saturation ammonium sulphate. The same result is obtained if the large amounts of the heavy form, present in the cell wall of intact yeast, is liberated during protoplasting (Fig. 3). This experimental approach allows not only the presence of intermediate molecular forms to be demonstrated but, what is probably more important, an interesting conclusion on the cellular localization of the different forms. The enzyme found in the protoplasting media is in the large form in both strains (Fig. 3 C and D), whereas the light and the intermediary forms are found exclusively in the protoplasts.

In the presence of 2-deoxy-~-glucose, the light and intermediate molecular forms accumulate, and it is tempting to speculate on the relation between this result and that shown in Table 1, which indicates a greater inhibition in intact cells than when invertase is assayed in cell-free extracts. We believe that this could be explained if 2-deoxy-~-glucose inhibited the glycosilation and therefore the secretion process. This would result in an accumulation of intermediate and light forms of the enzyme inside the protoplasts and, as we have indicated earlier, this activity is not accessible to the substrate in intact cells. This hypothesis can be tested experimentally and we are working that direction.

There has been some controversy on the events that ultimately lead to the secretion of exocellular


F. Moreno, A. G. Ochoa, S. Gascon, and J. R. Villanueva 579

enzymes in yeast [13,29]. Lampen et al. believe that the small invertase is not a precursor of the large enzyme but a metabolic dead-end [13]. On the other hand we still think that the working hypothesis pro- posed by us [ll], modified to fit on it the finding of intermediate forms, is still valid. This would imply in the first place the synthesis of the enzymatic protein (small form), and later a sequential addition of carbohydrate to it. This sequential addition of mannose would account for the continuous spectrum of invertase forms with molecular weights increasing accord- ing to its mannan content. The heavy enzyme, containing 50”/, mannan, would the be secreted into the periplasmic space. The transformation of the light enzyme in the heavy form, in the absence of protein synthesis, seems to be in favour of our hypothesis, and the same is probably true in view of the fact that the presence of 2-deoxy-~-g~ucose, under conditions of partial inhibition of invertase synthesis, originates an accumulation of the light and intermediate molecular forms (Fig. 6 ) as a consequence of the interference with the glycosilation process, which would take place in the rough endoplasmic reticulum and in vesicles derived from it.

The existence of different forms within the heavy enzyme has been suggested [l] but we believe that the form present outside the cytoplasmic membrane is homogeneous. It should be pointed out, however, that our analytical methods do not allow us to discard the possibility of some microheterogeneity.

It would be interesting to test whether the differences found in the elution volume in Sephadex G-200 are correlated to changes in the mannan content of the intermediate forms. The obvious way is to purify one of these forms, and this seems possible in view of the differences found in their isoelectric points (Fig. 5 ) and in chromatography on DEAE-Sephadex (Fig. 4).

This work was supported in part by a grant from the Funducibn Juan March.

REFERENCES 1. Gascon, S. & Ottolenghi, P. (1967) C . R . Trav. Lab.

2. Neumann, N. P. & Lampen, J. 0. (1967) Biochemistry, 6, Carlsherg, 36, 85- 93.

468 - 475.

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S. Gasc6n.s present address: Departamento lnterfacultativo de Bioquhica, Facultad de Medicina, Universidad de Oviedo. Oviedo, Spain

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