Q 1987 by The Amencan Socrety of B~olotpeal Chemists, lnc. THE JOURNAL OF B!OLOGIC?L CHEMIST~Y Vol. 262, No. 9, l w e of March 25, pp. 43954401,1987

Printed in U.S.A.

Effect of Glycosylation on Yeast Invertase Oligomer Stability* (Received for publication, October 20,1986)

Markku TammiS, Lun Ballou, Alice Taylor, and Clinton E. BallouQ From the Department of Biochemistry, University of California, Berkeley, California 94720

Yeast external invertase is a glycoprotein that exists as a dimer that can associate to form tetramers, hex- amers, and octamers (Chu, F., Watorek, W., and Maley, F. (1983) Arch. Biochem. Biophys. 223, 543-555; Esmon, P. C., Esmon, B. E., Schauer, I. E., Taylor, A., and Schekman, R. (1987) J. Bioi. Chem., 262,4395- 4401), a process that is facilitated by the attached oligosaccharide chains. We have studied this associa- tion by high performance liquid chromatography on a gel filtration matrix, by which procedure wild-type bakers’ yeast invertase gives two peaks, and invertase from a core mutant (mnnl mnn9) of Saccharomyces cereuisiae X2180 gives three peaks. Concentration of an invertase solution by freezing drives the dimers into higher aggregates that, at 30 “C, re-equilibrate to a mixture of smaller forms, the composition of which depends on pH, concentration, and time. The invertase from a mutant, mnnl mnn9 dpgf, which underglyco- sylates its glycoproteins and produces invertase with 4-7 oligosaccharide chains, forms oligomers of much lower stability than the mnnl mnn9 invertase, which has 8-11 carbohydrate chains. Both of these mutants release external invertase from the periplasm into the medium during growth, but we conclude that defects in the cell wall structure may be more important in this release than an altered tendency of the invertases to aggregate. Investigation of aggregate formation by electron microscopy revealed that all invertases, in- cluding the internal nonglycosylated enzyme, form oc- tamers under appropriate conditions.

Internal, nonglycosylated bakers’ yeast invertase was shown by Gascon and Lampen (1) to have a molecular weight of 135,000, whereas Neumann and Lampen (2) reported that the external enzyme contained 50% carbohydrate and had a molecular weight of about 270,000, thus giving both enzymes about the same polypeptide molecular weight (3). Subse- quently, Trimble and Maley (4) demonstrated that the car- bohydrate-depleted external invertase produced by endoglu- cosaminidase H digestion was composed of two identical protein subunits of 60,000 daltons, whereas the amino acid sequence inferred from the DNA sequence of the cloned gene gives a polypeptide Mr of about 59,000 (5).

In sedimentation velocity studies, Neumann and Lampen (2) observed that the external invertase contained a major component with an s&+ of 10.4 S , and that “heavier compo-

* This work was supported by National Science Foundation Grant PCM~-00251 and United States Public Health Service Grant AI- 12522. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 Visiting professor from the University of Kuopio, Finland, and recipient of Fogarty International Fellowship 1 F05 TW03668.

0 To whom correspondence should be addressed.

nents which are also present in much smaller amounts are enzymatically active and indicate the presence of an associa- tion-~ssociation equilibrium.” This association was stated by Chu and Maley (6) to lead to octamer formation, a conclusion that was documented by Chu et al. (7). The tendency of glycosylated invertase to form multimers had also been noted by Esmon et al. (8) in studies on the invertase trapped in the endoplasmic reticulum as a result of a genetic defect in the secretory pathway, and a more detailed analysis by Esmon et al. (9) has provided clear documentation by gel electrophoresis and electron microscopy for the interaction of dimeric glyco- sylated invertase to form tetramers, hexamers, and octamers.

Although internal nonglycosylated invertase forms a stable dimer, it yields unstable higher oligomers (lo), and the en- hanced ability of the glycosylated external invertase to form such oligomers depends on the extent of glycosylation (7, 9), as though the carbohydrate chains help to stabilize the mul- timeric state. In this report, we present additiona~ support for this view and demonstrate that the oligomerization is a freely reversible process that can be followed conveniently by high performance liquid chromatography. We have also studied the effect of mannoprotein glycosylation defects on the release of invertase into the medium during growth of yeast mannopro- tein mutants (11) and assessed the possible role of oligomer formation in retaining invertase in the periplasm.

EXPERIMENTAL PROCEDURES

General Methods-Chromatographic effluents were monitored for protein at 206 or 280 nm with an LKB Uvicord S 2138, and salt concentration was measured in gradients with a Bio-Rad Conductiv- ity Monitor equipped with a Standard Flow Cell. Protein was quan- titated by the Lowry procedure (12) with bovine serum albumin as the standard. For electron microscopy, desalted samples were applied to carbon-coated Pelco 8HGC grids (400 mesh), stained with uranyl acetate according to Kirschner et al. (13), and viewed in a Philips 201 electron microscope.

Cell mass was estimated from the wet packed volume or by dry weight after lyophilization, and cell number was determined by count- ing in a hemocytometer. For the mnn9 strains, which are clumpy, it was not possible to count cells in the usual manner, so cell number was estimated by assuming there are 10” cells/g of lyophilized cells. Separation of invertase oligomers was done by gel electrophoresis on native (8) and SDS’ gels (14) or by gel filtration on Bio-Gel A-5m, and high performance liquid chromatography was carried out on a Du Pont GF-450 gel filtration column (0.94 X 25 cm). To follow the rates of equilibration between oligomers, samples were frozen to drive the purified invertase into the higher oligomeric forms owing to concentration of the solution and, after thawing, were analyzed by HPLC.

For invertase assays, samples (5-50 pl) were incubated with 200 pl of 0.1 M sodium acetate, pH 5.1, for 2-20 min at 37 “C after the addition of 20 a1 of 40% sucrose. Glucose standards (5-50 fig) were used for calibration. The reaction was stopped by boiling the tubes after addition of 200 pl of 200 mM potassium phosphate, pH 7.0.

’ The abbreviations used are: SDS, sodium dodecyl sulfate; HPLC, high performance liquid chro~atography; man, mutation that affects mannoprotein glycosylation; dpg, mutation that affects dolichol phos- phoglucose synthesis.

.-

4395

Glucose oxidase reagent (1.0 ml) was added, and the tubes were incubated at 37 'C for 30 min. The absorbance was measured at 540 nm on a Spectronic 100 colorimeter after addition of 1.5 ml of 6 N

37 "C. HCl. One unit of enzyme activity released 1 pmol of glucose/min at

To compare invertase release by whole yeast cells, 5-ml YEPD liquid cultures were inoculated and shaken at 23 "C for 48 h. Assay for glucose in the medium showed that it was all consumed by 24 h, a t which time invertase synthesis and secretion had begun. The 48-h culture was centrifuged to separate cells and medium, and a portion of each was taken for invertase assay. The cells were washed with distilled water, lyophilized, and weighed. Invertase release is ex- pressed as the percent of the total invertase in the culture that was found in the medium.

Yeast Cultures-The yeast strains used were ~ ~ c ~ r o m y c e s cere- visiae X2180 (wild-type), mnnl mnn2 ( l l ) , mnnl mnn9 (15), and 4AL (mnnl mnn9 glsl dpgl) (16), and they were grown on a YEPD medium (1% yeast extract, 2% Bactopeptone, and 2% glucose). For large-scale growth of yeast, each 1-liter culture was inoculated with a 10-ml culture grown as above, and the 200-liter fermenter was inoc- ulated with 6 1-liter cultures. For small cultures, cells were isolated by centrifugation at 4000 X g for 10 min, whereas a Sharples contin- uous flow centrifuge was used for the 200-liter cultures. The yield of wet cell paste was 17-25 g/liter from the mnnl mnn9 and 4AL strains.

Isolation of Inuertase from Cells-The method of Lehle et al. (17) was used with minor modifications. The cell paste from a 6-liter culture was suspended in 400 ml of 10 mM sodium phosphate, pH 6.5, containing 1 mM phenylmethylsulfonyl fluoride, and homogenized either by a Braun homogenizer (5 times for 1 min each of a 10% cell paste suspension in a 1:2 v/v ratio of liquid to glass beads) or by a Biospec Bead-Beater (5 times for 1 min each of a 50% cell paste suspension in a 1:1 v/v ratio of liquid to beads). The homogenate was centrifuged for 90 min at 14,000 X g, the precipitate was discarded, Streptomycin sulfate was added to the crude extract (1 g/80 g of cell paste), and the solution was stirred for 1 h at 4 "C. After centrifuga- tion at 14,000 X g for l h, the precipitate was discarded, the solution was kept at 50 "C for 30 min and again centrifuged at 14,000 X g for 1 h, and the precipitate was discarded. Addition of ammonium sulfate to 60% of saturation precipitated internal nonglycosylated invertase, whereas addition to 80% saturation was required to precipitate 4AL external invertase, at which concentration the mnnl mnn9 external invertase remained in solution.

Each ammonium sulfate fraction was dialyzed against 10 mM sodium phosphate, pH 6.5, and applied to a DEAE-Sephacel column (1.4 X 12 em) equilibrated in the same buffer. Elution of external invertases was accomplished with a 600-ml NaCl gradient (0-0.4 M), whereas a gradient from 0 to 0.6 M was used to elute internal invertase. A flow rate of 30 ml/h was used and 5-ml fractions were collected. The fractions with invertase activity were combined, dialyzed against 10 mM sodium citrate, pH 3.7, and applied to an SP-Trisacryl column (1.4 X 8 cm) equilibrated in the same buffer. External invertases were eluted with a 400-ml NaCl gradient (0-0.3 M ) at a flow rate of 30 ml/ h, whereas a gradient to 0.4 M salt was used to elute internal invertase.

Combined peak fractions from the SP-Trisacryl column were con- centrated on an Amicon PM-30 filter to about 2 ml and the solution was applied to a Bio-Gel A-5m column (2.5 X 92 em) and eluted at 13 ml/h with 10 mM sodium acetate, pH 5.0, containing 0.1 M NaCI. The highly associated invertase (tetramer to octamer) was obtained in a peak free from impurities. Concentration and rechromatography of the lower molecular weight invertase fraction increased the yield of the pure, highly associated form. The fractions that contained enzyme activity were combined and dialyzed against distilled water before l y o p h i ~ ~ t i o n or against 10 mM sodium phosphate, pH 6.5, before further purification on a hydroxylapatite column (1.4 X 8 cm). This column was pre-equilibrated with 10 mM phosphate, pH 6.5, and the elution was carried out with 200 ml of a 10-200 mM sodium phosphate gradient of the same pH. Fractions containing invertase were combined, dialyzed against distilled water and lyophilized. The recovery of mnnl mnn9 invertase from the crude cell extract was about 20%, whereas the yield of internal invertase amounted to 16% of the corresponding purified external invertase.

l ~ o ~ u t ~ o n of Inuertase from Culture Media-For isolation of inver- tase from the medium of a 200-liter fermentation, sodium dihydrogen phosphate was added to a concentration of 10 mM and adjusted to pH 6.5 with NaOH. The cells were removed with a Sharples centri- fuge, sodium azide was added to the effluent to a concentration of 0.02%, and the solution was stored at 4 "C until it could be processed. All subsequent steps were carried out at 4 "C in the cold room. The

invertase was concentrated by passing the medium through a DEAE- Sephadex A-50 column (8 X 32 cm) at a flow rate of 400 ml/h. The effluent was monitored daily for invertase activity and, when inver- tase began to appear, the column was eluted with a 3-liter NaCl gradient (0-0.7 M) in the same buffer. The medium from the mnnl mnn9 fermentation was processed in seven batches, whereas that from the 4AL strain required only four cycles owing to a higher affinity of the latter enzyme for the ion-exchanger. The major peak of enzyme activity from each batch, which was eluted at about 0.1 M salt, was collected separately from a later fraction that was eluted at a higher salt concentration and was saved for isolation of internal invertase.

External invertase from the mnnl mnn9 medium was recovered in the supernatant liquid after adding ammonium sulfate to the com- bined DEAE-Sephadex fractions to 80% of saturation at 4 "C, whereas the 4AL invertase was found in the precipitate obtained between 55 and 90% of saturation of the corresponding DEAE-Sephadex frac- tions. Internal invertase was precipitated at 55% of saturation by ammonium sulfate from the late fractions of enzyme activity obtained from the DEAE-Sephadex column.

The invertases obtained in the ammonium sulfate step were di- alyzed against 10 mM sodium phosphate, pH 6.5, and purified as for the cellular invertases. From the medium of a 200-liter mnnl mnn9 culture, 34% of the invertase activity was recovered, 19.1 mg of protein (12), whereas the 4AL medium yielded 31.6 mg of invertase protein, a 36% recovery.

Hydroxylapatite Chromatography of Purqieied Invertases-A hydroxylapatite (high resolution, Behring Diagnostics) column (0.7 X 17 cm) was operated at a flow rate of 13 ml/h with a peristaltic pump located before the column. The column was equilibrated in 10 mM sodium p h ~ p h a t e , pH 6.5, samples of less than 2 mg of protein were introduced, and the invertases were eluted with sodium phos- phate gradients, pH 6.5, up to 200 mM. Salt concentration and absorbance at 280 nm were recorded with flow cells installed between the column and fraction collector. The column was washed with 400 mM phosphate between samples, and fractions to be rechromato- graphed were equilibrated in the loading buffer on an Amicon PM- 30 membrane concentrator.

Electrophoresis-A Hoefer vertical high-separating slab gel system was used (0.75 mm thick, 14 ern wide, and 10 cm high), with a 2.5-cm 3% stacking gel. The nondenatured multimeric forms of invertase were analyzed by the Laemmli SDS-polyacrylamide gel electropho- resis system (14) in a 4-12.5% gradient gel a t 4 "C. For separation of invertase monomers, the samples were mixed in a 1:l (v/v) ratio with buffer, containing 2.5% SDS and 6.25% mercaptoethanol, and were boiled before application. A constant current of 20-30 mA was used. For quantitation, gels were scanned on a Kratos Sp~trodensitometer Model SD3000 at 540 nm for the activity stain and at 620 nm for the silver stain. All gels were cast on Gel-Bond PAG (FMC C o ~ r a t i o n ) plastic supports. The gel was cut into halves so that the nondenatured part could be stained for invertase activity and the part with the denatured samples and molecular weight standards could be visual- ized with the silver stain of Morissey (18) but omitting the glutaral- dehyde treatment. Protein molecular weight standards (Pharmacia P-L Biochemicals) were used for calibration.

RESULTS

Isohtion of Invertase from Cells and Growth Medium of mnnl mnn9 and 4AL Strains and Separation of Oligomers by Gel Filtration-Although wild-type strains of S. cereuisiae, such as X2180, retain most of the secreted invertase in the periplasm, strains containing the mnn9 mannoprotein muta- tion release about 20% of the external invertase into the medium (Table I). The invertase in the medium from a 200- liter culture was concentrated by passing the solution through a large DEAE-Sephadex A-50 column. The bound invertase was eluted with a NaCl gradient and subjected to ammonium sulfate fractionation followed by chromato~aphy on DEAE- Sephacel and SP-Trisacryl and gel filtration on Bio-Gel A- 5m to yield the pure enzyme. In some instances, an additional hydroxylapatite step was employed. Details and differences in the behavior of invertases from different strains are given under "Experimental Procedures." The recovery of invertase was about 35%. Similar steps were used to isolate invertase

Invertase Oligomer Stability 4397 TABLE I

Release of external invertase into the medium "_____.I

Invertase activity Strain Dry cell -

Cells" Released

mg unitslg dry cells % X2180 90 290 12 3.8 mnnl mnn2 90 864 66 7.1 mnnl mnnZmnn.9 90 3010 790 20.7 4AL 80 2075 676 24.6

".

a Activity was assayed with whofe cells and is normalized for differences in cell mass. The higher activity with cells containing mnn mutations is not due to a greater ease of diffusion by substrate and products across the cell wall because cell homogenates gave similar values.

* Native gel electrophoresis confirmed that all of the activity in the medium was due to glycosylated invertase, which eliminated cell lysis as a mechanism for enzyme release.

"

from the homogenized cell paste, which led to a 20% recovery of the external invertase in the cells and some internal non- glycosylated invertase, which equaled 16% of the external form retained by the cells. Only minor amounts of nonglyco- sylated invertase were found in the medium, which suggests that lysis was not involved in release of invertase from the cells.

The Bio-Gel A-5m chromatography step was importantly affected by the pH and invertase concentration, owing to their influence on aggregation of the dimeric enzyme to larger oligomers. In some instances, the mnnl mnn9 invertase was eluted in a major part consisting of the larger oligomers, whereas in other experiments three peaks of activity were observed (Fig. 1). External invertase from mnnl mnn9 cells gave three peaks when chromatographed on Bio-Gel A-5m, the fastest of which migrated with thyroglobulin (MI 670,000) and the slowest with ferritin half-unit (MI 220,000). These probably represent an octamer-hexamer mixture and dimer, respectively, whereas an intermediate peak (M, 400,000) cor- responds to tetramer. The expected sizes of the oligomers are 180,000 (dimer), 360,000 (tetramer), 540,000 (hexamer) and 720,000 (octamer), and why four peaks were not observed is unclear. Rechromatography of the fastest and slowest eluting material gave a similar pattern of three peaks, demonstrating the ease with which re-equilibration of the separated oligo- mers can occur. Gel filtration of the 4AL external invertase gave a less well-defined pattern, with a major peak corre- sponding to dimer (calculated M, 150,000) and other material eluting in the region for higher oligomeric forms (data not shown).

Observance of Invertase Oligomers by Gel Electrophoresis- Wild-type external invertase is not resolved into oligomeric bands by gel electrophoresis, apparently owing to the large and heterogeneous carbohydrate component (4). The first demonstration of such resolution came from studies on the invertase accumulated in the see18 mutant in which the carbohydrate chains have a uniform Man8GlcNAc2-structure (8). Subsequently, the invertase of the mnn9 mutant, which possesses Manlo_13GlcNAcz-oligosaccharide chains, was found to give a similar gel electrophoretic pattern of four enzymat- ically active bands (9). In the present study, we have con- firmed this observation using the mnnl mnn9 invertase, which has uniform ManloGlcNAc2-chains, and compared its gel pattern with that of the 4AL strain. Freshly thawed invertase samples were electrophoresed at 4 "C on a gradient gel, in the presence of 0.1% SDS but without boiling, and the enzyme was visualized either with an activity stain (8) or a silver stain (18). Four bands were observed for the mnnl

0.8

c-)l

0 .t IO 0.6

Y d

>. t l- 1. 2 0.4

W cn 4 l- a: W > 0.2 z

7 0 80 90 100 1 1 0

FRACTION FIG. 1. Purification and fractionation of external invertase

by gel filtration on a Bio-Gel A-5m column. Invertase-contain- ing fractions, from chromatography of purified mnnl mnn9 external invertase on a Bio-Gel A-5m column (2.5 X 93 cm) eluted with 100 mM NaCl in 10 mM sodium acetate buffer, pH 5, were combined as shown in the inset, and the forms of highest and lowest molecular weight (peaks I and I I I ) were concentrated on an Amicon PM-30 filter and rechromatographed separately on a Bio-Gel A-5m column (1 X 195 cm) at 4 "C. Open circles, peak I; closed circles, peak 111; A, elution position of thyroglobulin (M, 670,000); B, elution position of ferritin half-unit (Mr 220,000); C, elution position of aldolase (M, 158,000).

mnn9 invertase with both reagents, the fastest moving having an apparent M, 200,000 and corresponding to the dimer, with the other bands at Mr 360,000, 560,000, and 700,000 (Fig. 2). In contrast, the 4AL invertase showed one major band that moved slightly faster than the mnnl mnn9 invertase dimer (apparent Mr 160,01)0), a slower migrating minor band (Mr 320,000), and two barely visible bands that correspond to higher oligomers. Under these same conditions, internal non- glycosylated invertase showed an enzymically inactive protein band of the monomer at MI 60,000 and a second diffuse band that ran slightly slower than the monomer and showed inver- tase activity. Thus, absence of carbohydrate chains destabil- izes the dimeric invertase so that it dissociates under these conditions. The enzymatic activity may reflect reassociation of monomer in the gel to yield active dimer, since published evidence suggests that the monomer is inactive (7).

As observed in the gel filtration experiments, the pretreat- ment of an invertase sample had a major effect on the pattern of oligomers observed following gel electrophoresis. Freezing the sample at low pH increased the amount of higher oligo- mers, whereas warming the sample at higher pH led to dis- sociation to the dimer.

Interconversion of Invertase Oligomers Observed by HFLC- Purified invertase shows multiple peaks when analyzed by HPLC on a Du Pont GF-450 column that includes mouse IgM (Mr 900,000) and resolves proteins down to M, 17,500 (myoglobin) when eluted with 0.2 M sodium phosphate, pH 7.5. Most invertase preparations show three peaks that cor- respond in molecular weights to dimer, tetramer and hexamer + octamer, the latter two apparently failing to separate from each other. Because the oligomeric composition of the inver- tase does not change perceptively during chromatography,

4398 Invertase Oligomer Stability

DISTANCE ALONG GEL FIG. 2. Gel electrophoresis of native external invertase

multimers. Samples were applied in a 10-fold dilution of the gel buffer with 10% glycerol and electrophoresed on a 4-12.5% gradient gel at 4 "C in a Tris-glycine buffer, pH 8.3, containing 0.1% SDS (14). The invertase multimers were detected with the activity stain and were compared quantitatively by densitometer scan at 540 nm. Top panel, men1 men9 invertase in which the four peaks correspond, left to right, to octamer (28%), hexamer (27%), tetramer (29%), and dimer (16%). Bottom panel, 4AL invertase, in which the two distinct peaks correspond to tetramer (25%) and dimer (56%), with smaller amounts of octamer (8%) and hexamer (11%). The faster migration and predominance of the dimeric form in the 4AL invertase reflect its lower carbohydrate content. Molecular mass markers (m) are indicated in the middle panel

which takes only a few minutes, the interconversion of mul- timers can be followed conveniently. Freezing an invertase solution leads to formation of the higher oligomers, probably owing to concentration of the glycoprotein. Upon thawing and chromatography, the sample shows one or more peaks that re-equilibrate to a new mixture of oligomers at a rate dependent on concentration, pH, temperature, and carbohy- drate structure of the invertase.

The invertase from the mnnl mnn9 strain has 8-11 car- bohydrate chains, each with 10 mannose units, and the HPLC pattern (Fig. 3A) of a freshly thawed sample, 3 mg/ml, shows one main peak (apparent M, 600,000) that slowly changes over time at pH 5 and 30 "C to a mixture of three peaks (Fig. 3, B-F). At equilibrium, the predominant peak (41% of the t~otal) is the dimer along with tetramer (33%) and hexamer- octamer (26%) (Fig. 3F). All peaks show the same specific enzyme activity. When the same invertase was analyzed in a similar manner but at a concentration of 0.03 mg/ml, the dimer eventually reached 85% of the total invertase. At 3 mg/ ml and pH 8.2, the dimeric form makes up 75% of the total invertase at equilibrium (Fig. 3, G-L). The rates at which the relative amounts of the three peaks change are plotted in Fig. 4, where the decrease in the octamer-hexamer peak (Fig. 4A) can be compared with the changes in the tetramer peak (Fig. 4B) and steady increases in the dimer peak (Fig. 4C) a t different concentrations and pH values. The rates of re- equilibration are clearly lower at pH 8.2 (ctosed circles) than at pH 5.0 (open circles) and at a concentration of 0.03 mg/ml (open triangles) than at 3 mg/ml (open circles), the latter result reflecting the reversibility of the reaction.

The 4AL invertase has 4-7 carbohydrate chains, each with

H

~ K

T-rT" 9 10 1% 12

I

"T-r 9 to 11 12

TIME AFTER INJECTION (rnin) FIG. 3. Time course for re-equilibration of mnnl mnni) in-

vertase multimers by HPLC. The invertase solution, 3 mg/ml in 50 mM sodium phosphate, pH 5, was stored frozen. After thawing it was kept at 30 "C and 1.4-fll samples were taken at different times for analysis by HPLC on the Du Pont GF-450 column by elution with 0.3 M sodium phosphate, pH 5.5, at 1 mi/min. In the top panel of six figures, incubation at 30 "C was for 0 min (A) , 15 min (B) , and 1 (C) , 2 (D), 4 (E) , and 7 h (F). The same experiment done at pH 8.2 gave the results in the lower panel of six figures, at 0 min (GI, 30 min (H), and 1 (I), 2 (J),4 ( K ) , and 22 h (L). The small peak of protein eluting at 10.2 min in L did not have invertase activity and is assumed to be an impurity or denatured enzyme. At pH 5, the equilibrium mixture had the composition shown in F, whereas at pH 8.2 the invertase was converted totally to the dimer shown in L.

10 mannose units, and the HPLC pattern obtained for the freshly thawed sample shows three poorly resolved peaks that, a t p H 5 and 30 "C, equilibrate to a mixture in which the dimer predominates (51%) but with a significant amount of tetramer (33%) still present (data not shown). The rates at which the changes occur for the three different peaks (Fig. 5), compared with those for the mnnl mnn9 invertase under the same conditions, show that re-equilibration has a strong inverse relationship to the carbohydrate content of the protein.

Wild-type invertase (Sigma) shows two peaks when freshly thawed, which equilibrate so that the lower molecular weight

Invertase Oligomer Stability 4399

I I I I 1 2 3 4 5 6

HOURS AT 3OoC

FIG. 4. Rates of re-equilibration of mnnl mnn9 invertase multimers by HPLC. The invertase solution was frozen to enhance formation of higher oligomers, and then thawed and assayed by HPLC at various times after incubation at 30 'C to follow relative changes in the three protein peaks. A is peak I, B is peak 11, and C is peak 111. The invertase solutions were at 3 mg/ml and pH 5 (open circles), 3 mJm1 and pH 8.2 (closed circles), or 0.03 mg/ml and pH 5 (open triangles).

4 0 L A

2 4 0 k z 0

HOURS AT 30°C

FIG. 5. Rates of re-equilibration of 4AL invertase multi- mers by HPLC. The conditions and symbols are as in Fig. 4.

species predominates. The rates of these changes are shown in Fig. 6. Nonglycosylated invertase showed only one peak, corresponding to dimer in size, probably because equilibration was too rapid to allow detection of different intermediates even with HPLC.

The concentration dependence of the oligomeric equilib-

HOURS AT 30° C

FIG. 6. Rates of re-equilibration of wild-type invertase multimers by HPLC. The conditions are as in Fig. 4, except that only two peaks were observed by HPLC and the invertase solutions were 1.5 mg/ml and pH 5 (open circles) or 0.015 mg/ml and pH 5 (open triangles).

D

J ~

TIME AFTER INJECTION ( m i d FIG. 7. Dependence of oligomeric composition on external

invertase concentration. The invertase was dissolved in 50 mM sodium phosphate, pH 5.0, and kept at least 12 h a t room temperature (20-22 "C) before analysis by HPLC. A-D, mnnl mnn9 invertase at 0.3 (A}, 3 (B) , 10 (C) , and 20 mg/ml (D). E-H, 4AL invertase at 0.3 ( E ) , 10 (F), 20 (GI, and 40 mg/ml (H).

rium composition of mnnl mnn9 and 4AL external invertases are compared in Fig. 7. At pH 5 and a concentration of 20 mg/ml or higher, the mnnl mnn9 invertase in a solution equilibrated overnight at 20 "C exists exclusively as the octa- mer (Fig. 7D) , whereas the 4AL invertase is still mainly dissociated to dimer and tetramer at a concentration of 40 mg/ml {Fig. 7H).

Confirmation of Invertase Oligomer Formation by Electron Microscopy-The procedure of Esmon et ai. (9) was used to observe oligomer formation by electron microscopy (Fig. 8).

4400 Invertase Oligomer Stability

A

C

B i

FIG. 8. Negatively stained electron micrographs of invertase multimers. Samples in distilled water were frozen and then thawed just before spreading on the grids. A, commercial bakers' yeast invertase; B, mnnl mnn9 external invertase; C, 4AL external invertase; and D, 5'. ceTeuisiue internal invertase. B shows a preponderance of octamers, whereas C shows dimers, tetramers, hexamers, and octamers. The distribution of multimers observed on the micrographs paralleled that seen in HPLC patterns of the same sample. Note that the octamers in A, B, and C remain open on one side, whereas many of those in D have a closed conformation. Magnification is X 172,000.

The photographs for wild-type (Fig. 8A) and mnnl mnn9 (Fig. 8B) external invertases are similar to those already published (9), whereas the results for the 4AL invertase (Fig. 8C) and for nonglycosylated internal invertase (Fig. 8D) are novel, although evidence that the latter does associate a t a suffi- ciently high concentration has been reported (10).

In our investigation, all invertase preparations formed oc- tamers to various degrees depending on conditions. Unfortu- nately, we were unable to assess oligomer composition in fractions obtained directly from the HPLC column because elution required the presence of salt, which had to be removed before electron microscopy could be performed. Preparations of the mnnl mnn9 and 4AL external invertases, which had been allowed to equilibrate under similar conditions, were photographed and then counted to determine the oligomeric

composition. The results were consistent with those obtained by HPLC.

DISCUSSION

The association of yeast external invertase dimers into higher oligomers, postulated by Neumann and Lampen (1) and documented by Chu et al. (7), Esmon et al. (9), and the present work, can be observed by gel electrophoresis, gel filtration, sedimentation velocity, HPLC, and electron mi- croscopy. Oligomer formation is enhanced by low pH, high ionic strength, and a high concentration as well as by glyco- sylation of the polypeptide chains. Here we have shown that freezing of samples is a convenient way to drive the invertase into the octameric form and that HPLC is an excellent procedure for assessing the oligomeric composition of a prep-

Invertase Oligomer Stability 4401

aration. S. cerevisiae strains containing the mnnl mnn9 mu- tations make invertase with uniformly sized carbohydrate chains, which facilitates its study, although such invertase is still heterogeneous in that individual polypeptide chains carry 8-11 oligosaccharide units (16). A mutation in dolichol phos- phoglucose synthesis superimposed on the mnnl mnn9 back- ground (mnnl mnn9 dpgl ) leads to the strain we call 4AL (16), which produces external invertase with 4-7 oligosaccha- ride chains. Our quantitative comparison of the stability of invertase oligomers from the mnnl mnn9 and 4AL strains provides support for the report (7) that glycosylation stabilizes oligomer formation. In addition, our electron micrograph of nonglycosylated invertase confirms that even the internal invertase will form octamers if sufficiently concentrated. The fact that protein glycosylation can enhance subunit interac- tion may have wide biological significance.

An interesting feature of the electron micrographs of the external invertases is the shape of the octamer, which uni- formly has one open side. This could result if the association sites on each dimer were oriented in such a manner that the angle between dimers is greater than 90". In contrast, the octamers formed by the nonglycosylated internal invertase appear to be more nearly symmetrical, so the unusual shape of the glycosylated invertase octamer might result from the presence of carbohydrate chains, which could, through steric interference, prevent formation of the closed conformation even though they stabilize oligomerization.

Of broader interest is the relevance of oligomerization of invertase in a test tube to that of invertase in the periplasm of the cell, a behavior that will be most importantly influenced by pH and concentration. Owing to the permeability of the cell wall to small molecules (%I), it is probable that the periplasmic pH is the same as that of the medium, about 5.5 in a culture growing in YEPD (21) and a pH that favors multimer formation (7). With regard to concentration, Arnold (22) has calculated the volume occupied by an unhydrated invertase dimer (2.8 X mm3) and the hydrated form (5 x mm3), which are close to what we calculate (3.4 x

mm3) from the apparent diameter (7 nm) of the invertase dimer seen in electron micrographs. From these volumes and the amount of invertase assayable in intact cells, Arnold (22) estimated the concentration of the invertase (20%) if the periplasmic space had a thickness of 15 nm. At this concen- tration, we expect from our data that the invertase would be completely associated into octamer and that even a t a peri- plasmic thickness of 50 nm the invertase would still be highly associated. Thus, there seems little question that, in wild-type cells induced to make and secrete invertase, the enzyme is present in the periplasm as large aggregates and might even appear to form a crystalline array such as is seen in published electron micrographs of fractured freeze-etched yeast cell envelopes that show organized particles of this same dimen- sion (23).

In the mnnl mnn9 and 4AL strains, which release 20% of the external invertase into the growth medium, we find about 40 mg/ml of invertase assuming a periplasmic space with a

thickness of 15 nm. This concentration of invertase would assure complete association of the mnnl mnn9 invertase, although the 4AL invertase might be significantly dissociated. This point is important in assessing whether the release of invertase into the medium in these mutants results primarily from changes in wall organization or from a reduced tendency of the invertase to form higher oligomers. In addition to its effect on invertase, the mnn9 mutation is known to decrease the size of the carbohydrate chains on the cell wall manno- proteins (19) and to alter the structural integrity of the cell wall itself (24). Our results showing that mnnl mnn9 and 4AL external invertases are released to similar extents, even though they differ in oligomeric stability, suggest that wall structural defects may be more responsible for enzyme release from the mutant than a reduced oligomer stability.

1.

2.

3.

4.

5.

6. 7.

8.

9.

10.

11.

12.

13.

14. 15.

16.

17.

18. 19.

20.

21.

22. 23.

24.

REFERENCES Gascon, S., and Lampen, J. 0. (1968) J. Bid. Chem. 243 , 1567-

Neumann, N. P., and Lampen, J. 0. (1967) Biochemistry 6,468-

Gascon, S., Neumann, N. P., and Lampen, J. 0. (1968) J. Bid.

Trimble, R. B., and Maley, F. (1977) J. Bwl. Chem. 252,4409-

Taussig, R., and Carlson, M. (1983) Nucleic Acids Res. 11, 1943-

Chu, F., and Maley, F. (1981) Fed. Proc. 40, 1556 Chu, F., Watorek, W., and Maley, F. (1983) Arch. Biochem.

Esmon, B., Novick, P., and Schekman, R. (1981) Cell 25 , 451-

Esmon, P. C., Esmon, B. E., Schauer, I. E., Taylor, A., and

Williams, R. S., Trumbly, R. J., MacColl, R., Trimble, R. B., and

Raschke, W. C., Kern, K. A., Antalis, C., and Ballou, C. E. (1973)

Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

Kirschner, M., Honig, L., and Williams, R. (1975) J. Mol. Biol.

Laemmli, U. K. (1970) Nature 227,680-685 Ballou, L., Cohen, R. E., and Ballou, C. E. (1980) J. Biol. Chem.

Ballou, L., Gopal, P., Krummel, B., Tammi, M., and Ballou, C.

Lehle, L., Cohen, R. E., and Ballou, C . E. (1979) J. Bwl. Chem.

Morrissey, J. H. (1981) Anal. Biochem. 117 , 307-310 Tsai, P.-K., Frevert, J., and Ballou, C. E. (1984) J. B i d . Chem.

259,3805-3811 Arnold, W. N. (1981) in Yeast Cell Enuelopes: Biochemistry,

Biophysics and Ultrastructure (Arnold, W. N., ed) Vol. 11, pp. 25-47, CRC Press, Boca Raton, FL

Suomalainen, H., and Oura, E. (1971) in The Yeasts (Rose, A. H., and Harrison, J. S., eds) Vol. 2, pp. 3-74, Academic Press, New York

1572

475

Chem. 2 4 3 , 1573-1577

4412

1954

Biophys. 223,543-555

460

Schekman, R. (1987) J. Bwl. Chem. 262,4387-4394

Maley, F. (1985) J. Biol. Chem. 2 6 0 , 13334-13341

J. Bid. Chem. 248,4660-4666

(1951) J. Biol. Chem. 193,265-275

99,263-276

255,5986-5991

E. (1986) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 3081-3085

254,12209-12218

Arnold, W. N. (1973) Physiol. Chem. Phys. 5, 117-123 Matile, P., Moor, H., and Robinow, C. F. (1969) in The Yeasts

(Rose, A., H., and Harrison, J. S., eds) Vol. 1, pp. 219-302, Academic Press, New York

Frevert, J., and Ballou, C. E. (1985) Biochemistry 24,753-759