426
Biochimica et Biophysica Acta, 538 (t978) 426--434 © Elsevier/North-Holland Biomedical Press
BBA 28426 A MEMBRANE-ASSOCIATED ISOZYME OF I N V E R T A S E IN YEAST P R E C U R S O R OF T H E E X T E R N A L G L Y C O P R O T E I N
PETER BABCZINSKI and WIDMAR TANNER Botanik L Fachbereich Biologie und Vorklinische Medizin, Universit~t Regensburg, Postfach 397, 8400 Regensburg (G.F.R.)
(Received July 21st, 1977) Summary
An enzymatic test is described which allows the localization of yeast invertase activity directly on sodium dodecyl sulfate gels. When crude membrane fractions are prepared from S a c c h a r o m y c e s cerevisiae cells which are actively synthesizing external invertase0 these membranes show an activity band on sodium d o d e c y l sulfate gels additional to the external and the internal invertase. In the soluble fraction this form was c ompl et el y absent, t t has a molecular weight of ap p r o x i m at el y 190 000 and was 50 000 smaller than the external invertase. It showed kinetic characteristics of a precursor of the external enzyme. Thus it appeared transiently, when yeast cells were shifted from a con° dition of non-synthesizing external invertase to one where the e n z y m e was synthesized. When the increase in the external e n z y m e slewed down after some time, the membrane-associated f or m almost com pl et el y disappeared. T h e addition of tunicamycin to yeast cells synthesizing external invertase inhibited f u r th er synthesis of the e n z y m e by 97%; also the form at i on of t he membrane-associated form was strongly inhibited. The a m o u n t of it present before the addition of tunicamycin com pl e t e l y disappeared in the presence of the antibiotic. The precursor form, therefore, seems to possess already those c a r b o h y d r a t e parts which contain N-acetylglucosamine and are transferred via dolichyl p h o s p h a t e - b o u n d intermediates. The membrane-associated precursor amounts to less than 5% of the total invertase activity of a yeast cell.
Introduction The biosynthesis of yeast m a n n o p r o t e i n s has been studied intensively in vivo [1--5] and in vitro [6--14]. T w o main features have emerged from the in vitro A b b r e v i a t i o n s : G l c N A c , N - a c e t y l g t u c o s a r a i n e ; SDS, s o d i u m d o d e c y t s u l f a t e .
427 studies: (1), the glycosylation reactions are catalyzed exclusively by membraneb o u n d enzymes; and (2), dolichyl phosphate b o u n d sugars [15,16] are intermediates for some of these reactions. Evidence for this latter statement has been obtained from the formation of O-glycosidic bonds [6,7,10,14,17] and the formation of N-glycosidic linkages [10,13,18,19]. To study the formation of a well-defined mannoprotein, the biosynthesis of external yeast invertase, reported to possess both types of glycosidic linkages [20,21], has been investigated more closely. MaterJials and Methods
Chemicals and instruments. Glucose oxidase test system and standard proteins for estimation of molecular weights were purchased from Boehringer, Mannheim, G.F.R.; bovine serum albumin was obtained from Behringwerke, Marburg/Lahn, G.F.R.; o-dianisidine and dithiothreitol from Sigma, St. Louis, Mo., U.S.A. Chemicals used for polyacrylamide gel electrophoresis were from Serva, Heidelberg, G.F.R. Tunicamycin was kindly provided by Prof. G. Tamura from the University of Tokyo. Spectrophotometric determinations were c o n d u c t e d in a Gilford S p e c t r o p h o t o m e t e r 240. For membrane preparations, cells were disrupted in a Bio X-Press at --25°C. Organism and culture. The haploid yeast strain Saccharomyces cerevisiae X 2180 a was used in all experiments and was obtained from Prof. C.E. Ballou, University of California, Berkeley, U.S.A. The culture medium consisted of 0.5% (w/v) bacto-peptone (Difco Labs., Detroit, Mich., U.S.A.), 1% yeast extract (Merck, Darmstadt, G.F.R.), and 2% glucose; the low sucrose medium contained 0.12% bacto-peptone, 0.25% yeast extract, and 0.5% sucrose. Yeast cells were grown at 30°C in 1-1 flasks containing 300 ml medium using a rotary water bath shaker. For invertase derepression, cells were washed twice with ice-cold water t o remove glucose and resuspended in an equal volume of sucrose medium. Growth was monitored turbidimetrically in an Eppendorf P h o t o m e t e r in 1-cm cuvettes by determining absorbance at 578 nm. Samples were diluted to absorbances n o t exceeding 0.2. Homogenization o f cells. Cells were harvested by centrifugation, washed once with 0.05 M Tris • HC1, pH 7.4 (containing 0.15 mM MgC12) and broken in a Bio X-Press. Whole cells and debris were spun down at 600 X g for 10 min, the supernatant was centrifuged at 48 000 X g for 20 min. Membrane aliquots corresponding to 1.2 mg protein were washed twice. The yield of membranes from 300 ml culture medium was a b o u t 6 mg protein. Extraction of invertase. Whole yeast cells (corresponding to 1 ml culture with absorbance 8.0; 8.3 mg wet wt.) were harvested, washed with ice-cold water and desiccated to dryness where indicated. Upon suspending in 0.1 ml of deoxycholate (concentrations as stated in the legends) samples were extracted overnight at room temperature. After centrifugation at 48 000 X g, supern~atants were used to separate invertases on SDS gels (see below). Membrane :preparations corresponding to 1.2 mg protein were extracted with deoxycholate in the same way. Estimation o f invertase activity. In 0.3 ml cell suspension invertase activity (referred to as external invertase) was determined according to Smith and
428 Ballou [22] following the original procedure described by Bernfeld [_231o Cells were spun down, washed twice with 2 ml 0.I M sodium acetate/acetic acid buffer, pH 5.0, and resuspended in 0.5 m! 0.05 M sodium acetate/acetic acid buf~ fer, pH 5.0. Upon addition of 0.5 ml sucrose solution (7% w/v) cells were incuo bated for 15 rain at 37°C. The reaction was stopped by heating° After centrio fugation sugar was determined in the supernatant by using 1 rn] of basic 3fl° dinitrosalicylate reagent; absorbance was read at 540 nm. Extinctions of giu~ cose and fructose were identical in this assay. One unit of invertase was defined as i #tool of sucrose hydrolyzed per rain at 37 ° Co Other analytical procedures° Protein was assayed by the m e t h o d of Lowry et al. [24] using bovine serum albumin as standard~ Protein content of whole cells was determined by boiling yeast cells in 1 N NaOH for i h and by estimating the protein in the supematanto PolyacryIarnide gel electrophoresis of invertase. Separation of invertase species was carried out using 5.8% gels (pH 7.5, 0.25% SDS) prepared according to Maurer [--25] as described by Meyer and Matile [-5]. Electrophoresis was carried out at room temperature, applying i mA/tube for 15 rain, followed by a constant current of 2 mA/tube, until the tracking dye had reached the end of the gel. Samples for gel electrophoresis were prepared by mixing the 48 000 X g supernatant (80 #l) after deoxyeholate extraction with I0 #l of glycerol 5 ~d of 5% (w/v) sodium dodecyl sulfate and some Bromophenol blue. Gels were rinsed well with distilled water and stained overnight by placing in 20 ml medium containing glucose oxidase test system from Boehringer: 5 mg glucose oxidase~ 0.8 mg peroxidase, 1.3 mg o-dianisidine~ 0.I M phosphate buffer, pH 7.0. The reaction was started by addition of sucrose (I0 mM final concentration). Stained areas were recorded in a gel scanner at 438 nm. Quantitative determination of invertase activity was carried ou'~ by measuring the areas below the peaks obtained by scanning the gels. The areas were found to be proportional to the amount of enzyme from i0 to I00 #I of extract° Molecular weight estimation. 5% acrylamide gels were used for estimation of apparent molecular weights for the various invertase species. SDS get electrophoresis was carried out following the procedure of Segvest and Jackson [,26~. Standard proteins were dissolved in 5 #l 1% SDS (w/v), containing iG-2M dithiothreitol and incubated for 1 h at 37°C. Before electrophoresis an equal volume of 2 mM sodium phosphate buffer, pH 7.1 in 8 M urea was added for a final SDS concentration of 0.5%. 50 gl of the deoxycholate extract of yeast cells were treated as above and applied directly (without incubation) onto the gel. Localization of invertase activity was achieved by the :orocedure described above. Results
Derepression o[ external inuertase The synthesis of external invertase is repressed by the presence of glucose in the medium [27]. With the yeast strain and the conditions used in this work, the activity of external invertase increased by a factor of about 8 when glucose grown cells were harvested shortly before glucose was completely used up and
429 transferred to a low sucrose medium (Fig. 1). Since the fastest rate of synthesis of external invertase was observed about 1 h after the shift, cells were normally harvested 50--60 min after the shift and their invertase activity determined in various fractions. When the total membrane pellet obtained from yeast cells shortly before and 55 min after the shift was extracted with deoxycholate and the solubilized proteins were separated on SDS gels and tested for invertase activity, the bands shown in Fig. 2 were observed. Since only the appearance of the bands I, II and III depends on the presence of sucrose in the test system, the band designated X is n o t an invertase. The amounts of invertase I and II increase dramatically due to the shift of the cells, whereas the invertase III is n o t affected.
Distribution o f invertase I, H and III in the soluble and particulate fraction For Fig. 3 the deoxycholate extractable, membrane associated invertases were separated on sodium dodecyl sulfate gels (left) and compared with the invertases obtained from an aliquot of the soluble fraction from the same yeast cells. The two bands obtained with the soluble fraction were identical (by the criterJLa of gel separation) with invertase I and III from the membrane fraction. The fact t h a t invertase I and III from the soluble fraction are closer together on the gel than the corresponding bands obtained from membranes was f o u n d to be due to the presence of deoxycholate in the latter; co-separation clearly identified the slow-moving band of the soluble fraction as invertase I of the membranes. The data of Fig. 3 do n o t give a measure of the relative amounts of the various invertases in a yeast cell, since the sample size differed greatly. The total activity of all three membrane-associated invertase species observed is less than 5% of the total extractable invertase activity of the cells.
o8
~ c 0.6. °o4 ~E ~~
Q2.
Time (h) F i g . 1. K i n e t i c s o f d e r e p r e s s i o n o f e x t e r n a l i n v e r t a s e . Y e a s t cells w e r e s h i f t e d o n t o s u c r o s e m e d i u m at a n a b s o r b a n c e o f 9 . 2 ( f i l l e d signs). C o n t r o l y e a s t s c o n t i n u e d g r o w t h in g l u c o s e m e d i u m ( o p e n signs). • a n d D, cell m a s s ; • a n d o, i n v e r t a s e a c t i v i t y .
430
0
I E
X
TIT L
Fig. 2, S e p a r a t i o n o f i n v e r t a s e species b y SDS gel e ! e c t r o p h q r e s i s . M e m b r a ~ e s w e r e p r e p a r e d f r o m y e a s t cells g r o w n o n glucose m e d i u m u p to an a b s o r b a n c e o f 8.(} (left) a n d f r o m cells s u b s e q u e n t l y c u l t i v a t e d f o r 55 m i n on s u c r o s e m e d i u m (Aght). A f t e r d e s i c c a t i o n m e m b r a n e s w e r e e x t r a c t e d w i t h 0,7% deoxyo c h o l a t e ( t m g ] m g p r o t e i n ) , t h e s o l u b l l i z e d pro~eL~s s e p a r a t e d o n SDS gels a n d s t a i n e d f o r i n v e r t a s e activo ity as d e s c r i b e d in Materials a n d M e t h o d s . B a n d X is d u e t o a n u n k n o w n a c t i v i t y w h i c h is n o t an h~ve~ tase. O- origin. Fig. 3. S e p a r a t i o n o f m e m b r a n e - a s s o c i a t e d a n d soluble i n v e r t a s e species b y SDS gel eIectrophoresiso Y e a s t cells w e r e g r o w n o n glucose m e d i u m u p to a n a b s o r b a n e e o f 5.8. A f t e r c u l t i v a t i o n on s u c r o s e m e d i u m for 50 rain m e m b r a n e s w e r e p r e p a r e d a n d e x t r a c t e d w i t h 2% d e o x y c h o l a t e (2.8 r a g / r a g p r o t e l n ) (left), A n a l i q u o t of t h e c y t o p l a s m i c f r a c t i o n ( 4 8 0 0 0 × g s u p e r n a t a n t ) was a p p l i e d d i r e c t l y t o a l e c t r o p h o r e s i s (right). T h e h i g h e r m o b i l i t y o f t h e small i n v e r t a s e species in t h e left figure is d u e t e t h e p r e s e n c e of d e o x y c h o l a t e in this s a m p l e .
Determination o f apparent molecular weights o f the three invertases Since only minute amounts of the m e m b r a n e associated inve~ase H have been obtained so far, its apparent molecular weight had t o be determined by the m e t h o d o f sodium d o d e c y l sulfate get electrophoresis [26]. In Fig. 4 a calibration run is shown and the apparent molecular weight o f the invertase I! which can be d ed uced from the position o f its band ls about 190 000. The distances the large and the small invertase migrate on the gel correspond co apparent molecular weights of approx. 240 000 and 100 000, respectively° Since it is k n o wn t hat the large isozyme of invertase is a glycoprotein [21] and this also seems to be the case for the membrane-associated invertase H (see below), the molecular weights from SDS gets must be treated with rese~ation~ Membrane-associated invertase H as precursor of the large invertase [ At zero time (Fig. 5) yeas~ cells were shifted from a glucose to a low sucrose m e d iu m and at the times indicated aliquots were withdrawn and the cells were centrifuged o f f and e x t r a c t e d with 0.7% d e o x y c h o l a t e (0.3 rag/1 mg d ~ weight) overnight. The various invertases of the extracts were separated as [n Fig. 3 and the e n z y m e activities expressed as area of the peaks° As m e n t i o n e d in Materials and Methods, the test has also been calibrated for quantitative determinations, using increasing amounts of ext ract or of commercial inver~ase (Serva); it was used for quantitative purposes only in the range where proportionality o f a m o u n t of e n z y m e used and peak area had been assured.
431
::: ~t
\
15-
serum o0umo
"~ne
9× 10.-~
serum albumin (dimer) ~ _
B-
_b 6 _e o
III
Ctr m e t ,
Bovine serum a[bumin'~Olaovalbumin~OS e
4
- Chymotrypsinogenl~
Mobility (cm) Fig. 4. Estimation of apparent molecular weights of mvez~ase species on BDS gels. Open ~gns refer to invez~ase species.
Shortly after the shift, both invertase I and II increased rapidly, whereas no significant change was observed in the amount of invertase III, i.e. the small form. The amount of the small form, however, was underestimated by the
Shift m
m
.nvertase III
.->m10t I'-
f
~-
0
I Time (h)
2
3
F i g . 5. K i n e t i c s o f d e r e p z e s s i o n e l i n v e r t a s e s p e c i e s . Y e a s t c e l l s w e r e g r o w n o n g l u c o s e m e d i u m u p t o an absorbance of 5.8, and subsequently shifted on to sucrose medium. Aliquots were taken and extracted, after desiccation, with 0.7% deoxyeholate (1 rag/rag protein) and the invertases sepaxated on SDS gels. A c t i v i t y is e x p r e s s e d a s a r e a o f p e a k s o b t a i n e d b y s c a n n i n g t h e g e l s a n d r e c o r d i n g t h e a b s o r b a n c e a t 436 nm.
432 extraction procedure applied here~ since in extracts from broken derepressed cells the small form always was present in larger quantity than the external enzyme (see also Fig. 3). To obtain additional information a b o u t the composition and the possible precursor role of invertase II, experiments with tunicamycin have been carried out. The antibiotic tunicamycin [28,29] has been shown to inhibit the syn~ thesis of external invertase [30]; it prevents the gtycosylation of dolichy~ m o n o p h o s p h a t e [ 31--33 ] and thereby the formation of N-glycosidical!y linked carbohydrate chains of glycoproteins. In a control experiment external invertase increased from 0.14 to 1.0 units/rag protein within 40 rain on sucrose medium. In the presence of tunicamycin (6 #g/rot) the increase was inhibited by 97% (from 0.14 to 0.16 units/rag protein)° In Fig. 6, the enzyme pattern o2 the membrane-associated invertases is shown at zero time (left) and 50 rain after the shift in presence (right) and absence (control, middle) of tunicamycin° Two interesting effects are obvious from this figure. For one~ the formation of inver~tase I and H, which normally takes place after the shift (Fig. 6 control, Fig. 2) is completely prevented in the presence of tunicamycin. Secondly, aI~heugh a small a m o u n t of enzyme H is present already at the time of the shift (left) this small band disappears completely in the presence of tunicamycino Normally enzyme II is processed to enzyme t which is released from the membrane. Therefore, the decrease of enzyme H observed in the presence of tunicamycin cannot be accounted for by an equal increase in membrane-associated enzyme I. The apparent increase in the membrane-bound internal enzyme in the presence of tunicamycin (right) is not considered significant; more than 20 times the amount found associated with the membranes is present in the soluble fraction. The results obtained indicate, therefore, that the specifically membraneassociated invertase II is a glycoprotein, the glycosylation of which requires dolichyl pyrophosphate-activated N-acetylglucosamine and that it is a precursor for the external invertase, the large invertase L
L~
/v t
Fig. 6. I n h i b i t i o n o f i n v e r t a s e f o r m a t i o n b y t u n i c a m y c i n . M e m b r a n e s w e r e p r e p a r e d f r o m y e a s t c e l l s g r o w n o n g l u c o s e m e d i u m u p t o a n a b s o r b a n c e o f 5.8 (left) and f r o m c e l l s s u b s e q u e n t l y c u l t i v a t e d o n s u c r o s e m e d i u m f o r 50 r a i n w i t h o u t t u n i c a m y c i n ( m i d d l e ) a n d i n t h e p r e s e n c e of 6 tLg tunicam:~ch~ frill (right), r e s p e c t i v e l y . M e m b r a n e s w e r e e x t r a c t e d w i t h 2% d e o x y c h o l a t e (2.8 r a g / r a g p r o t e i n ) a n d t h e inver~ t a s e s s e p a r a t e d o n S D S gels.
433 Discussion The test used in this work for demonstrating invertase activity directly on SDS gels is based on the observation of Meyer and Matile [5] that invertase is still active after SDS gel separation. The glucose oxidase used has to be very low in invertase, however; otherwise no clear banding on the gel will appear. Unfortunately many commercial batches of glucose oxidase contain levels of invertase which are t o o high. Besides the large external invertase, the mannoprotein [21] corresponding to the invertase I described herein, and the small carbohydrate-free internal one [34], enzyme III of this paper, the occurrence of invertase species of intermediate sizes has been r e p o r t e d before [35,36]. However, only that described by Holbein et al. [36] is also membrane-associated and could, as far as its :molecular size is concerned, correspond to the invertase II of this report. The data of this paper cannot really contribute to the lengthily-discussed problem of whether the small internal invertase is a precursor of the external mannoprotein [27,35]: From the data of Fig. 5 t h e internal invertase does not .increase significantly after the shift, while the external enzyme, as well as the membrane-associated form, increase rapidly. This could be taken as an indication, at best, that the internal form is not a precursor for the external one. The data both of Fig. 5 and Fig. 6 are in agreement, however, with the assumption that the membrane-associated form is an intermediate in the formation of the external invertase. Since its formation also is completely inhibited b y tunicamycin it most likely is already a glycosylated form of invertase. Nakajima and BaUou [37] have presented evidence for the existence of core units consisting of a b o u t 16 sugars in the cell wall mannoprotein; due to the great similalities between the external invertase and the cell wall mannan [22] it seems likely that similar core units exist in external invertase. These units are expected to be transferred en bloc via dolichol (ref. 10 and Lehle and Tanner, unpublished), and their transfer should be inhibited by tunicamycin. Since external invertase contains a b o u t 40 glucosamine residues per molecule, whereby t w o GlcNAc molecules initiate a N-glycosidically linked carbohydrate unit [38,39], it is tempting to speculate that the external invertase might possess 20 carbohydrate chains with a core region of a b o u t 16 sugars units (2 GlcNAc and 14 mannoses) to which further branched mannan chains would have to be linked [22,38,39]. If the membrane-associated invertase II consisted of a protein moiety of 135 000, as that of the external enzyme [38], and, in addition, of all the core carbohydrate units as described above, it would have a molecular weight of 186 000, which is close to the one determined. It is postulated, therefore, that the invertase II represents a pool of membrane-associated molecules possessing the complete protein moiety as well as the total a m o u n t of the core carbohydrate units of the external invertase. The core units are possibly transferred en bloc from a dolichyl pyrophosphateoligosaccharide, whereas further mannosylation of the core units proceeds w i t h o u t dolichyl phosphate-activated sugars. This agrees with the observation that only the formation b u t n o t the further processing of invertase II is inhibited by tunicamycin.
434
Acknowledgements Thanks are due to Dr. Hasilik for a small amount of tunicamycin kindly sent to him by Dr. Tamura. This work has been supported by the Deutsche Forschungsgemeinschaft.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
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( 1 9 7 3 ) B i o c h h n . B i o p h y s . A c t a 3 0 4 , 7 3 6 - - 7 4 7 L e h l e , L. a n d T a n n e r , W. ( 1 9 7 5 ) B i o c h i r n . B i o p h y s . A c t a 3 9 9 , 3 6 4 - - 3 7 4 N a k a j i m a . T. a n d B a l l o u , C.E. ( 1 9 7 5 ) P r o c . N a t l . A c a d . Sci, U.S, 7 2 , 3 9 1 2 - - 3 9 1 6 F a r k a s , V., V a g a b o v , V.M. a n d B a u e r , S0 ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 2 8 , 5 7 3 5 8 2 R e u v e r s , F., H a b e t s - W i l l e m s , C., R e i n t d n g , A. a n d B o e r , P. ( 1 9 7 7 ) B i o c h i m . B i e p h y s . A c t a 4 8 6 , 5 4 1 552 L a r r i b a , G.~ E l o r z a , M.V., V i l l a n u e v a , J.B,. a n d S e n t a n d r e u , I~. ( 1 9 7 6 ) F E B S Lett. 71~ 3 1 6 3 2 0 J u n g , P, a n d T a n n e r , W. ( 1 9 7 3 ) Eur. J. B i o e h e m . 3 7 , 1 - - 6 P a l a m a r c z y k . G. a n d C h o j n a c k i , T. ( 1 9 7 3 ) F E B S L e t t . 34, 2 0 1 - - 2 0 3 B r e t t h a u e r , [%.K, a n d W u , S. ( 1 9 7 5 ) A r c h , B i o c h e m . B i o p h y s . 167~ 1 5 1 - - 1 6 0 P a r o d i , A . J . ( 1 9 7 6 ) F E B S L e t t , 71, 2 8 3 - - 2 8 6 N a k a y a m a , K . , A r a k i , J. a n d I t o , E. ( 1 9 7 6 ) F E B S L e t t . 7 2 , 2 8 7 - - 2 9 0 Greiling~ H., VSgele, P., K i s t e r s , R. a n d O h l e n b u s c h , H.-D. ( 1 9 6 9 ) H o p p e S e y t e r ' s Z. P h y s i o L C h e m . 350, 517--518 N e u m a n n , N.P. a n d L a m p e n , J . O . ( 1 9 6 9 ) B i o c h e m i s t r y 8, 3 5 5 2 - - 3 5 5 6 S m i t h , W.L. a n d B a U o u , CoE. ( 1 9 7 4 ) B i o c h e m i s t r y 13, 3 5 5 - - 3 6 1 B e r n f e l d , P, ( 1 9 5 1 ) i n A d v a n c e s in E n z y m o l o g y ( N o r d , F . F . . ed.), VoL 1 2 . p p . 3 7 9 - - 4 2 8 . I n t e r scmnce, New York L o w r y , O . H . , R o s e b r o u g h , N , H . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J, Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 M a u r e r , H . R . ( 1 9 6 8 ) in D i s k - E l e k t r o p h o r e s e - T h e o r i e u n d P r a x i s d e r d i s k o n t i n u i e r l i c h e n P o l y a c r y l a m i d g e l - E l e k t r o p h o r e s e ( F i s c h b e c k , K . , ed.), p p . 2 5 - - 1 0 3 , W. d e G r u y t e r a n d Co.~ Berlin S e g r e s t , J , P . a n d J a c k s o n , R . L . ( 1 9 7 2 ) i n M e t h o d s in E n z y m o l o g y ( C o l o w i c k 0 S.P. a n d K a p l a n , N,©.. eds.), V o l . 2 8 B , p p . 5 4 - - 6 3 , A c a d e m i c Press, N e w Y o r k L a m p e n , J . O . , K u o , S.-C., C a n o , F . R . a n d T k a c z , J . S . ( 1 9 7 2 ) in F e ~ m e n t a Z i o n T e c h n o l o g y T o d a y ( T e r u i , G., ed.), P r o c . IV, p p . 8 1 8 - - 8 2 4 7 $ o c . F e r m e n t . T e c h n o L , J a p a n T a k a t s u k i , A., A r i m a , K, a n d T a m u r a ~ G. ( 1 9 7 1 ) J. A n t i b i o t . 24~ 2 ! 5 - - 2 2 3 T a k a t s u k i , A. a n d T a m u r a , G. ( 1 9 7 1 ) J. A n t i b i o t . 2 4 , 2 2 4 - - 2 3 1 K u o , S.-C. a n d L a m p e n ~ J . O . ( 1 9 7 4 ) B i o c h e m . B i o p h y s , Res. C o m m u n . 58~ 2 8 7 - - 2 9 5 T k a c z , J . S . a n d L a m p e n , J . O . ( 1 9 7 5 ) B i o e h e m . B i o p h y s . l%es. C o m m u n . 6 5 . 2 4 8 - - 2 5 7 T a k a t s u l d , A . , K h o n o , K . a n d T a m u r a , G. (:~975) A g r i c . Biol. C h e m . 3 9 . 2 0 8 9 - - 2 0 9 1 L e h l e , L. a n d T a n n e r , W. ( 1 9 7 6 ) F E B S L e t t . 71~ 1 6 7 - - 1 7 0 G a s c o n , S. a n d L a m p e n , J . O . ( 1 9 6 8 ) J. Biol. C h e m . 2 4 3 , 1 5 6 7 - - 1 5 7 2 M o r e n o , F., O c h o a , A . G . , G a s c o n , S. a n d V i B a n u e v a , J . R . ( 1 9 7 5 ) E u r . J. B i o c h e m . 50, 5 7 1 - - 5 7 9 H o l b e i n , B . E . , F o r s b e r g , C.W. a n d K i d b y , D,K, ( 1 9 7 6 ) C a n . J, M i c r o b i o L 2 2 , 9 8 9 - - 9 9 5 N a k a j i m a , T. a n d B a l l o u , C.E. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 . 7 6 8 5 - - 7 6 9 4 N e u m a n n , N.P. a n d L a m p e n , J . O . ( 1 9 6 7 ) B i o c h e m i s t r y 6 , 4 6 8 - - 4 7 5 T a r e n t i n o . A . L . , P i n m m e r , J r . , T . H . a n d M a l e y , F. ( 1 9 7 4 ) J. BioL C h e m . 2 4 9 , 8 1 8 8 2 4
Biochimica et Biophysica Acta, 538 (t978) 426--434 © Elsevier/North-Holland Biomedical Press
BBA 28426 A MEMBRANE-ASSOCIATED ISOZYME OF I N V E R T A S E IN YEAST P R E C U R S O R OF T H E E X T E R N A L G L Y C O P R O T E I N
PETER BABCZINSKI and WIDMAR TANNER Botanik L Fachbereich Biologie und Vorklinische Medizin, Universit~t Regensburg, Postfach 397, 8400 Regensburg (G.F.R.)
(Received July 21st, 1977) Summary
An enzymatic test is described which allows the localization of yeast invertase activity directly on sodium dodecyl sulfate gels. When crude membrane fractions are prepared from S a c c h a r o m y c e s cerevisiae cells which are actively synthesizing external invertase0 these membranes show an activity band on sodium d o d e c y l sulfate gels additional to the external and the internal invertase. In the soluble fraction this form was c ompl et el y absent, t t has a molecular weight of ap p r o x i m at el y 190 000 and was 50 000 smaller than the external invertase. It showed kinetic characteristics of a precursor of the external enzyme. Thus it appeared transiently, when yeast cells were shifted from a con° dition of non-synthesizing external invertase to one where the e n z y m e was synthesized. When the increase in the external e n z y m e slewed down after some time, the membrane-associated f or m almost com pl et el y disappeared. T h e addition of tunicamycin to yeast cells synthesizing external invertase inhibited f u r th er synthesis of the e n z y m e by 97%; also the form at i on of t he membrane-associated form was strongly inhibited. The a m o u n t of it present before the addition of tunicamycin com pl e t e l y disappeared in the presence of the antibiotic. The precursor form, therefore, seems to possess already those c a r b o h y d r a t e parts which contain N-acetylglucosamine and are transferred via dolichyl p h o s p h a t e - b o u n d intermediates. The membrane-associated precursor amounts to less than 5% of the total invertase activity of a yeast cell.
Introduction The biosynthesis of yeast m a n n o p r o t e i n s has been studied intensively in vivo [1--5] and in vitro [6--14]. T w o main features have emerged from the in vitro A b b r e v i a t i o n s : G l c N A c , N - a c e t y l g t u c o s a r a i n e ; SDS, s o d i u m d o d e c y t s u l f a t e .
427 studies: (1), the glycosylation reactions are catalyzed exclusively by membraneb o u n d enzymes; and (2), dolichyl phosphate b o u n d sugars [15,16] are intermediates for some of these reactions. Evidence for this latter statement has been obtained from the formation of O-glycosidic bonds [6,7,10,14,17] and the formation of N-glycosidic linkages [10,13,18,19]. To study the formation of a well-defined mannoprotein, the biosynthesis of external yeast invertase, reported to possess both types of glycosidic linkages [20,21], has been investigated more closely. MaterJials and Methods
Chemicals and instruments. Glucose oxidase test system and standard proteins for estimation of molecular weights were purchased from Boehringer, Mannheim, G.F.R.; bovine serum albumin was obtained from Behringwerke, Marburg/Lahn, G.F.R.; o-dianisidine and dithiothreitol from Sigma, St. Louis, Mo., U.S.A. Chemicals used for polyacrylamide gel electrophoresis were from Serva, Heidelberg, G.F.R. Tunicamycin was kindly provided by Prof. G. Tamura from the University of Tokyo. Spectrophotometric determinations were c o n d u c t e d in a Gilford S p e c t r o p h o t o m e t e r 240. For membrane preparations, cells were disrupted in a Bio X-Press at --25°C. Organism and culture. The haploid yeast strain Saccharomyces cerevisiae X 2180 a was used in all experiments and was obtained from Prof. C.E. Ballou, University of California, Berkeley, U.S.A. The culture medium consisted of 0.5% (w/v) bacto-peptone (Difco Labs., Detroit, Mich., U.S.A.), 1% yeast extract (Merck, Darmstadt, G.F.R.), and 2% glucose; the low sucrose medium contained 0.12% bacto-peptone, 0.25% yeast extract, and 0.5% sucrose. Yeast cells were grown at 30°C in 1-1 flasks containing 300 ml medium using a rotary water bath shaker. For invertase derepression, cells were washed twice with ice-cold water t o remove glucose and resuspended in an equal volume of sucrose medium. Growth was monitored turbidimetrically in an Eppendorf P h o t o m e t e r in 1-cm cuvettes by determining absorbance at 578 nm. Samples were diluted to absorbances n o t exceeding 0.2. Homogenization o f cells. Cells were harvested by centrifugation, washed once with 0.05 M Tris • HC1, pH 7.4 (containing 0.15 mM MgC12) and broken in a Bio X-Press. Whole cells and debris were spun down at 600 X g for 10 min, the supernatant was centrifuged at 48 000 X g for 20 min. Membrane aliquots corresponding to 1.2 mg protein were washed twice. The yield of membranes from 300 ml culture medium was a b o u t 6 mg protein. Extraction of invertase. Whole yeast cells (corresponding to 1 ml culture with absorbance 8.0; 8.3 mg wet wt.) were harvested, washed with ice-cold water and desiccated to dryness where indicated. Upon suspending in 0.1 ml of deoxycholate (concentrations as stated in the legends) samples were extracted overnight at room temperature. After centrifugation at 48 000 X g, supern~atants were used to separate invertases on SDS gels (see below). Membrane :preparations corresponding to 1.2 mg protein were extracted with deoxycholate in the same way. Estimation o f invertase activity. In 0.3 ml cell suspension invertase activity (referred to as external invertase) was determined according to Smith and
428 Ballou [22] following the original procedure described by Bernfeld [_231o Cells were spun down, washed twice with 2 ml 0.I M sodium acetate/acetic acid buffer, pH 5.0, and resuspended in 0.5 m! 0.05 M sodium acetate/acetic acid buf~ fer, pH 5.0. Upon addition of 0.5 ml sucrose solution (7% w/v) cells were incuo bated for 15 rain at 37°C. The reaction was stopped by heating° After centrio fugation sugar was determined in the supernatant by using 1 rn] of basic 3fl° dinitrosalicylate reagent; absorbance was read at 540 nm. Extinctions of giu~ cose and fructose were identical in this assay. One unit of invertase was defined as i #tool of sucrose hydrolyzed per rain at 37 ° Co Other analytical procedures° Protein was assayed by the m e t h o d of Lowry et al. [24] using bovine serum albumin as standard~ Protein content of whole cells was determined by boiling yeast cells in 1 N NaOH for i h and by estimating the protein in the supematanto PolyacryIarnide gel electrophoresis of invertase. Separation of invertase species was carried out using 5.8% gels (pH 7.5, 0.25% SDS) prepared according to Maurer [--25] as described by Meyer and Matile [-5]. Electrophoresis was carried out at room temperature, applying i mA/tube for 15 rain, followed by a constant current of 2 mA/tube, until the tracking dye had reached the end of the gel. Samples for gel electrophoresis were prepared by mixing the 48 000 X g supernatant (80 #l) after deoxyeholate extraction with I0 #l of glycerol 5 ~d of 5% (w/v) sodium dodecyl sulfate and some Bromophenol blue. Gels were rinsed well with distilled water and stained overnight by placing in 20 ml medium containing glucose oxidase test system from Boehringer: 5 mg glucose oxidase~ 0.8 mg peroxidase, 1.3 mg o-dianisidine~ 0.I M phosphate buffer, pH 7.0. The reaction was started by addition of sucrose (I0 mM final concentration). Stained areas were recorded in a gel scanner at 438 nm. Quantitative determination of invertase activity was carried ou'~ by measuring the areas below the peaks obtained by scanning the gels. The areas were found to be proportional to the amount of enzyme from i0 to I00 #I of extract° Molecular weight estimation. 5% acrylamide gels were used for estimation of apparent molecular weights for the various invertase species. SDS get electrophoresis was carried out following the procedure of Segvest and Jackson [,26~. Standard proteins were dissolved in 5 #l 1% SDS (w/v), containing iG-2M dithiothreitol and incubated for 1 h at 37°C. Before electrophoresis an equal volume of 2 mM sodium phosphate buffer, pH 7.1 in 8 M urea was added for a final SDS concentration of 0.5%. 50 gl of the deoxycholate extract of yeast cells were treated as above and applied directly (without incubation) onto the gel. Localization of invertase activity was achieved by the :orocedure described above. Results
Derepression o[ external inuertase The synthesis of external invertase is repressed by the presence of glucose in the medium [27]. With the yeast strain and the conditions used in this work, the activity of external invertase increased by a factor of about 8 when glucose grown cells were harvested shortly before glucose was completely used up and
429 transferred to a low sucrose medium (Fig. 1). Since the fastest rate of synthesis of external invertase was observed about 1 h after the shift, cells were normally harvested 50--60 min after the shift and their invertase activity determined in various fractions. When the total membrane pellet obtained from yeast cells shortly before and 55 min after the shift was extracted with deoxycholate and the solubilized proteins were separated on SDS gels and tested for invertase activity, the bands shown in Fig. 2 were observed. Since only the appearance of the bands I, II and III depends on the presence of sucrose in the test system, the band designated X is n o t an invertase. The amounts of invertase I and II increase dramatically due to the shift of the cells, whereas the invertase III is n o t affected.
Distribution o f invertase I, H and III in the soluble and particulate fraction For Fig. 3 the deoxycholate extractable, membrane associated invertases were separated on sodium dodecyl sulfate gels (left) and compared with the invertases obtained from an aliquot of the soluble fraction from the same yeast cells. The two bands obtained with the soluble fraction were identical (by the criterJLa of gel separation) with invertase I and III from the membrane fraction. The fact t h a t invertase I and III from the soluble fraction are closer together on the gel than the corresponding bands obtained from membranes was f o u n d to be due to the presence of deoxycholate in the latter; co-separation clearly identified the slow-moving band of the soluble fraction as invertase I of the membranes. The data of Fig. 3 do n o t give a measure of the relative amounts of the various invertases in a yeast cell, since the sample size differed greatly. The total activity of all three membrane-associated invertase species observed is less than 5% of the total extractable invertase activity of the cells.
o8
~ c 0.6. °o4 ~E ~~
Q2.
Time (h) F i g . 1. K i n e t i c s o f d e r e p r e s s i o n o f e x t e r n a l i n v e r t a s e . Y e a s t cells w e r e s h i f t e d o n t o s u c r o s e m e d i u m at a n a b s o r b a n c e o f 9 . 2 ( f i l l e d signs). C o n t r o l y e a s t s c o n t i n u e d g r o w t h in g l u c o s e m e d i u m ( o p e n signs). • a n d D, cell m a s s ; • a n d o, i n v e r t a s e a c t i v i t y .
430
0
I E
X
TIT L
Fig. 2, S e p a r a t i o n o f i n v e r t a s e species b y SDS gel e ! e c t r o p h q r e s i s . M e m b r a ~ e s w e r e p r e p a r e d f r o m y e a s t cells g r o w n o n glucose m e d i u m u p to an a b s o r b a n c e o f 8.(} (left) a n d f r o m cells s u b s e q u e n t l y c u l t i v a t e d f o r 55 m i n on s u c r o s e m e d i u m (Aght). A f t e r d e s i c c a t i o n m e m b r a n e s w e r e e x t r a c t e d w i t h 0,7% deoxyo c h o l a t e ( t m g ] m g p r o t e i n ) , t h e s o l u b l l i z e d pro~eL~s s e p a r a t e d o n SDS gels a n d s t a i n e d f o r i n v e r t a s e activo ity as d e s c r i b e d in Materials a n d M e t h o d s . B a n d X is d u e t o a n u n k n o w n a c t i v i t y w h i c h is n o t an h~ve~ tase. O- origin. Fig. 3. S e p a r a t i o n o f m e m b r a n e - a s s o c i a t e d a n d soluble i n v e r t a s e species b y SDS gel eIectrophoresiso Y e a s t cells w e r e g r o w n o n glucose m e d i u m u p to a n a b s o r b a n e e o f 5.8. A f t e r c u l t i v a t i o n on s u c r o s e m e d i u m for 50 rain m e m b r a n e s w e r e p r e p a r e d a n d e x t r a c t e d w i t h 2% d e o x y c h o l a t e (2.8 r a g / r a g p r o t e l n ) (left), A n a l i q u o t of t h e c y t o p l a s m i c f r a c t i o n ( 4 8 0 0 0 × g s u p e r n a t a n t ) was a p p l i e d d i r e c t l y t o a l e c t r o p h o r e s i s (right). T h e h i g h e r m o b i l i t y o f t h e small i n v e r t a s e species in t h e left figure is d u e t e t h e p r e s e n c e of d e o x y c h o l a t e in this s a m p l e .
Determination o f apparent molecular weights o f the three invertases Since only minute amounts of the m e m b r a n e associated inve~ase H have been obtained so far, its apparent molecular weight had t o be determined by the m e t h o d o f sodium d o d e c y l sulfate get electrophoresis [26]. In Fig. 4 a calibration run is shown and the apparent molecular weight o f the invertase I! which can be d ed uced from the position o f its band ls about 190 000. The distances the large and the small invertase migrate on the gel correspond co apparent molecular weights of approx. 240 000 and 100 000, respectively° Since it is k n o wn t hat the large isozyme of invertase is a glycoprotein [21] and this also seems to be the case for the membrane-associated invertase H (see below), the molecular weights from SDS gets must be treated with rese~ation~ Membrane-associated invertase H as precursor of the large invertase [ At zero time (Fig. 5) yeas~ cells were shifted from a glucose to a low sucrose m e d iu m and at the times indicated aliquots were withdrawn and the cells were centrifuged o f f and e x t r a c t e d with 0.7% d e o x y c h o l a t e (0.3 rag/1 mg d ~ weight) overnight. The various invertases of the extracts were separated as [n Fig. 3 and the e n z y m e activities expressed as area of the peaks° As m e n t i o n e d in Materials and Methods, the test has also been calibrated for quantitative determinations, using increasing amounts of ext ract or of commercial inver~ase (Serva); it was used for quantitative purposes only in the range where proportionality o f a m o u n t of e n z y m e used and peak area had been assured.
431
::: ~t
\
15-
serum o0umo
"~ne
9× 10.-~
serum albumin (dimer) ~ _
B-
_b 6 _e o
III
Ctr m e t ,
Bovine serum a[bumin'~Olaovalbumin~OS e
4
- Chymotrypsinogenl~
Mobility (cm) Fig. 4. Estimation of apparent molecular weights of mvez~ase species on BDS gels. Open ~gns refer to invez~ase species.
Shortly after the shift, both invertase I and II increased rapidly, whereas no significant change was observed in the amount of invertase III, i.e. the small form. The amount of the small form, however, was underestimated by the
Shift m
m
.nvertase III
.->m10t I'-
f
~-
0
I Time (h)
2
3
F i g . 5. K i n e t i c s o f d e r e p z e s s i o n e l i n v e r t a s e s p e c i e s . Y e a s t c e l l s w e r e g r o w n o n g l u c o s e m e d i u m u p t o an absorbance of 5.8, and subsequently shifted on to sucrose medium. Aliquots were taken and extracted, after desiccation, with 0.7% deoxyeholate (1 rag/rag protein) and the invertases sepaxated on SDS gels. A c t i v i t y is e x p r e s s e d a s a r e a o f p e a k s o b t a i n e d b y s c a n n i n g t h e g e l s a n d r e c o r d i n g t h e a b s o r b a n c e a t 436 nm.
432 extraction procedure applied here~ since in extracts from broken derepressed cells the small form always was present in larger quantity than the external enzyme (see also Fig. 3). To obtain additional information a b o u t the composition and the possible precursor role of invertase II, experiments with tunicamycin have been carried out. The antibiotic tunicamycin [28,29] has been shown to inhibit the syn~ thesis of external invertase [30]; it prevents the gtycosylation of dolichy~ m o n o p h o s p h a t e [ 31--33 ] and thereby the formation of N-glycosidical!y linked carbohydrate chains of glycoproteins. In a control experiment external invertase increased from 0.14 to 1.0 units/rag protein within 40 rain on sucrose medium. In the presence of tunicamycin (6 #g/rot) the increase was inhibited by 97% (from 0.14 to 0.16 units/rag protein)° In Fig. 6, the enzyme pattern o2 the membrane-associated invertases is shown at zero time (left) and 50 rain after the shift in presence (right) and absence (control, middle) of tunicamycin° Two interesting effects are obvious from this figure. For one~ the formation of inver~tase I and H, which normally takes place after the shift (Fig. 6 control, Fig. 2) is completely prevented in the presence of tunicamycin. Secondly, aI~heugh a small a m o u n t of enzyme H is present already at the time of the shift (left) this small band disappears completely in the presence of tunicamycino Normally enzyme II is processed to enzyme t which is released from the membrane. Therefore, the decrease of enzyme H observed in the presence of tunicamycin cannot be accounted for by an equal increase in membrane-associated enzyme I. The apparent increase in the membrane-bound internal enzyme in the presence of tunicamycin (right) is not considered significant; more than 20 times the amount found associated with the membranes is present in the soluble fraction. The results obtained indicate, therefore, that the specifically membraneassociated invertase II is a glycoprotein, the glycosylation of which requires dolichyl pyrophosphate-activated N-acetylglucosamine and that it is a precursor for the external invertase, the large invertase L
L~
/v t
Fig. 6. I n h i b i t i o n o f i n v e r t a s e f o r m a t i o n b y t u n i c a m y c i n . M e m b r a n e s w e r e p r e p a r e d f r o m y e a s t c e l l s g r o w n o n g l u c o s e m e d i u m u p t o a n a b s o r b a n c e o f 5.8 (left) and f r o m c e l l s s u b s e q u e n t l y c u l t i v a t e d o n s u c r o s e m e d i u m f o r 50 r a i n w i t h o u t t u n i c a m y c i n ( m i d d l e ) a n d i n t h e p r e s e n c e of 6 tLg tunicam:~ch~ frill (right), r e s p e c t i v e l y . M e m b r a n e s w e r e e x t r a c t e d w i t h 2% d e o x y c h o l a t e (2.8 r a g / r a g p r o t e i n ) a n d t h e inver~ t a s e s s e p a r a t e d o n S D S gels.
433 Discussion The test used in this work for demonstrating invertase activity directly on SDS gels is based on the observation of Meyer and Matile [5] that invertase is still active after SDS gel separation. The glucose oxidase used has to be very low in invertase, however; otherwise no clear banding on the gel will appear. Unfortunately many commercial batches of glucose oxidase contain levels of invertase which are t o o high. Besides the large external invertase, the mannoprotein [21] corresponding to the invertase I described herein, and the small carbohydrate-free internal one [34], enzyme III of this paper, the occurrence of invertase species of intermediate sizes has been r e p o r t e d before [35,36]. However, only that described by Holbein et al. [36] is also membrane-associated and could, as far as its :molecular size is concerned, correspond to the invertase II of this report. The data of this paper cannot really contribute to the lengthily-discussed problem of whether the small internal invertase is a precursor of the external mannoprotein [27,35]: From the data of Fig. 5 t h e internal invertase does not .increase significantly after the shift, while the external enzyme, as well as the membrane-associated form, increase rapidly. This could be taken as an indication, at best, that the internal form is not a precursor for the external one. The data both of Fig. 5 and Fig. 6 are in agreement, however, with the assumption that the membrane-associated form is an intermediate in the formation of the external invertase. Since its formation also is completely inhibited b y tunicamycin it most likely is already a glycosylated form of invertase. Nakajima and BaUou [37] have presented evidence for the existence of core units consisting of a b o u t 16 sugars in the cell wall mannoprotein; due to the great similalities between the external invertase and the cell wall mannan [22] it seems likely that similar core units exist in external invertase. These units are expected to be transferred en bloc via dolichol (ref. 10 and Lehle and Tanner, unpublished), and their transfer should be inhibited by tunicamycin. Since external invertase contains a b o u t 40 glucosamine residues per molecule, whereby t w o GlcNAc molecules initiate a N-glycosidically linked carbohydrate unit [38,39], it is tempting to speculate that the external invertase might possess 20 carbohydrate chains with a core region of a b o u t 16 sugars units (2 GlcNAc and 14 mannoses) to which further branched mannan chains would have to be linked [22,38,39]. If the membrane-associated invertase II consisted of a protein moiety of 135 000, as that of the external enzyme [38], and, in addition, of all the core carbohydrate units as described above, it would have a molecular weight of 186 000, which is close to the one determined. It is postulated, therefore, that the invertase II represents a pool of membrane-associated molecules possessing the complete protein moiety as well as the total a m o u n t of the core carbohydrate units of the external invertase. The core units are possibly transferred en bloc from a dolichyl pyrophosphateoligosaccharide, whereas further mannosylation of the core units proceeds w i t h o u t dolichyl phosphate-activated sugars. This agrees with the observation that only the formation b u t n o t the further processing of invertase II is inhibited by tunicamycin.
434
Acknowledgements Thanks are due to Dr. Hasilik for a small amount of tunicamycin kindly sent to him by Dr. Tamura. This work has been supported by the Deutsche Forschungsgemeinschaft.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
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