Identification of 59 and 62 kDa plasma membrane proteins as putative glucose transporters in Saccharomyces cerevisiae

Journal of Biotechnology, 27 (1992) 59-73

59

© 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00

BIOTEC 00845

Identification of 59 and 62 kDa plasma membrane proteins as putative glucose transporters in Saccharomyces cerevisiae B. V61ker a, Ch. K r e u t z f e l d t ", J. L e h m a n n b, H. F a s o l d c a n d G.F. F u h r m a n n a a Department of Pharmacology and Toxicology, Philipps-Universita'tMarburg, Germany, b Institut fiir Organische Chemie und Biochemie derAlbert Ludwigs Universitiit, Freiburg, Germany, c Institutfiir Biochemie derJohann Wolfgang Goethe-Universita't, Frankfurt (Main), Germany

(Received 10 December 1991; accepted 15 December 1991)

Summary In this investigation two different glucose derivatives, the affinity label N-{O-([3o-glucopyranosyl)ethyl}bromoacetamide (GEBrA) and the photoaffinity label 4azi-4-deoxy-D-xylohexopyranose, were found to be effective inhibitors of glucose countertransport in plasma membrane vesicles prepared from Saccharomyces cerevisiae cells. Other common inhibitors of glucose transport for example in human red cells, cytochalasin B, the organic mercurial mersalyl, phlorizin and phloretin at low concentration were completely ineffective in inhibition. G E B r A and 4-azi-4-deoxy-o-xylohexopyranose were used radioactively to label membrane proteins of plasma membranes prepared from Saccharomyces cerevisiae cells. Both compounds labeled very distinctly two main integral membrane proteins at 59 and 62 kDa. These proteins are therefore the most probable candidates to represent the glucose carrier region in plasma membranes. By raising antibodies against these two membrane proteins at 59 and 62 kDa, the hypothesis that these proteins represent glucose carriers in the plasma membrane could further be supported. The polyvalent antibodies reacted only with plasma membrane proteins and not with proteins of other cellular membranes. Mainly the two proteins at 59 and 62 kDa were reactive. Reaction around 44 kDa is due to the deglycosylated forms of the double band. This SDS-PAGE region was Correspondence to: G.F. Fuhrmann, Department of Pharmacologyand Toxicology,Philipps-Universit~it

Marburg, Karl-von-Friseh-Strasse,3550 Marburg, Germany.

60 also slightly labeled by the two glucose derivatives. Other minor labeled proteins with lower molecular masses are highly probably proteolytic products of the glucose carrier. The proteins labeled at 120 kDa by antibodies and glucose derivatives are most likely dimers of the double band at 59 and 62 kDa.

Saccharomyces cerec'isiae; Glucose transporter; Plasma membrane proteins; Affinity labeling; Inhibitor

Introduction

The availability of structurally related protein sequences for glucose carriers in

Saccharomyces cerevisiae lends strong impetus to the analysis and location of these proteins in the plasma membrane. The first sequence shown was predicted from the SNF3 gene (Celenza et al., 1988) with 28% homology between amino acids 86-581 to the human H e p G 2 glucose transporter. The strain MCY638 used was originally isolated from decaying figs and is very dissimilar from its genetic background to industrial Saccharomyces strains such as DFY1, which also contains the SNF3 gene (Bisson, 1988). Further the molecular mass of 97 kDa is unusually high for a glucose carrier. In 1990 Kruckeberg and Bisson reported about isolation of a HXT2 gene from Saccharomyces cereuisiae strain MCY1407 (Kruckeberg and Bisson, 1990), which encodes a 60 kDa protein most similar to the galactose transporter (64 kDa) of Saccharomyces cerevisiae whereby both proteins are identical to a degree of homology of 65%. These results are suggestive for plasma membrane proteins of 60 kDa involved in glucose transport or in the case of the SNF3 transporter of the 100 kDa region. In this investigation we screened for chemical substances, which were able to inhibit glucose transport and could be used by their affinity to the glucose transporter as label for identification of plasma membrane proteins. Two effective inhibitors of glucose transport were found. N-{O-(/3-D-glucopyranosyl)ethyl}bromoacetamide (GEBrA) and 4-azi-4-deoxy-D-xylohexopyranose, which were able to serve as chemical probes for identification. It was possible to show with these different inhibitors, that protein bands at 59 and 62 kDa represent most likely the glucose carrier region in the plasma membrane. By the use of antibodies against these two proteins the hypothesis could further be corroborated.

Materials and Methods

Synthesis of N-{O-([3-D-glucopyranosyl)ethyl}bromoacetamide (GEBrA).

The synthesis of G E B r A was similar to that of 10-N-(bromoacetyl)amino-t-decyl-fl-D-glucopyranoside (BADG) (Neeb et al., 1985). Instead of 10-amino-l-decanol in B A D G synthesis 2-aminoethanol was used. The amino group was protected by a benzyloxycarbonyl group (Graham and Neuberger, 1968). In order to form the

61

(GEBrA) CH2 OH H ~ O~-CH2--CH2--NH-CO-CH2-Br H 6H Fig. 1. Structural formula of N-{O-(/3-D-glucopyranosyl)ethyl}bromoacetamide

(GEBrA).

glycosidic bond 21.1 g of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide dissolved in 200 ml dry dichloromethane were added to a mixture of the protected aminoethanol (10.74 g), silver carbonate (5.54 g), iodine (700 mg) and molecular sieves 4 ,~ (10.74 g) in 250 ml dry dichloromethane and stirred for 1 day in the dark (Rosevear et al., 1980). After filtration through Celite the solvent was evaporated, the residue dissolved in chloroform/methanol (97/3, v/v) and purified with this solvent on a silicic acid column. The compound was deacetylated in dry methanol containing 0.2 N sodium methoxide (Graham and Neuberger, 1968). The benzyloxycarbonylaminoethyl-/3-D-glucopyranoside was purified on a silicic acid column with chloroform/methanol (9/1, v/v). To remove the benzyloxycarbonyl group the compound (700 mg) was hydrogenolysed in 20 ml 0.14 N HC1 in 60% (v/v) methanol containing 120 mg of palladium on carbon. The resulting aminoglycoside was chromatographically pure by TLC and paper electrophoresis after filtration and lyophilization. The bromoacetyl derivative GEBrA was synthesized by dissolution of 100 mg aminoglycoside in 2.25 ml dry dimethylformamide containing 62 ~i triethylamine and addition of 56 mg bromoacetic acid in 1.1 ml dry dimethylformamide. After starting the reaction with 166 mg N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinone (EEDQ) in 620 ~1 toluene the mixture was kept at room temperature for one day (Tserng et al., 1977). For 14C-labeled GEBrA the bromo[a4C]acetic acid was used. GEBrA was purified by paper electrophoresis on Whatman 3 MM paper in 0.1 M acetic acid-KOH (pH 4), eluted from the paper with water/ethanol (9/1, v/v), ethanol was evaporated and the final product GEBrA lyophilized (Fig. 1, GEBrA). Azido and diazirino derivatives of sugars. The following derivatives of sugars have been used in 1 mM or 3 mM concentrations for testing inhibition of glucose countertransport in plasma membrane vesicles: (1) methyl 6-azi-6-deoxy-/3-Dgalactopyranoside, (2) methyl 4-azi-4-deoxy-a-D-xylohexopyranoside, (3) 4-azi-4-deoxy-D-xylohexopyranose, (4) 4-azido-4-deoxy-D-galactose, (5) methyl 6-azi-6-deoxya-D-glucopyranoside, (6) 3,7-anhydro-2-azi-l,2-dideoxy-D-glycero-L-mannooctitol, (7) 3,7-anhydro-2-azi-l,2-dideoxy-D-glycero-D-gulo-octitol and (8) l'-methoxy-3'azibutyl-/3-D-glucopyranoside. The syntheses of these photolabile sugar analogues have been described by Lehmann and coworkers (Kurz et al., 1985; Midgley et al., 1985; Lehmann and Thieme, 1986; Kuhn and Lehmann, 1987; Lehmann et al., 1988).

62

Synthesis of 6-4-[ SH]-azi-4-deoxy-D-xylo-hexopyranose (Thieme, 1986). 250 mg (1.2 mmol) methyl 4-azi-4-deoxy-D-xylohexopyranoside (Kurz et al., 1985) were pertrimethylsilylated and subsequently the 6-trimethylsilyl (TMS) group selectively replaced by an acetyl group and the remaining TMS groups removed (Fuchs and Lehmann, 1974). The so obtained methyl 6-O-acetyl-4-azi-4-deoxy-D-xylohexopyranoside after differential blocking with hydropyranyl residues was deacetylated. The product with a free hydroxy group in 6-position was submitted to DMSO-oxidation and methyl 4-azi-4-deoxy-2,3-tetrahydropyranyl-a-D-xylohexodialdo-l,5-pyranoside was obtained in an overall yield of approx. 15%. 5 mg of this compound was dissolved in a solution of 200/zl dioxane and 20 ~1 1 N NaOH and immediately reduced with 100 mCi [3H]sodium borohydride (25 Ci mmol-1). The radioactive product after being treated with iodotrimethylsilane in CCI 4 at room temperature under nitrogen was purified by silica gel chromatography (yield 5.05 mCi, 5%) and identified by comparison with authentic, unlabeled 4-azi-4-deoxy-Dxylohexopyranose. 6-4-[3H]-azi-4-deoxy-D-xylohexopyranose is kept in methanolic solution at - 20°C.

Strain and growth conditions.

Saccharomyces cerer,isiae H 1022 (ETH ZiJrich) was grown in batch cultures under aeration at 30°C. The composition of the growth medium was 2% peptone, 1% yeast extract and 2% carbohydrate.

Preparation of plasma membrane L~esicles. Plasma membrane vesicles were prepared as described by Fuhrmann and co-workers (Fuhrmann et al., 1976; Witt et al., 1982; Kreutzfeldt and Fuhrmann, 1984).

Countertransport experiments. Countertransport experiments with 2% plasma membrane vesicles were carried out under ice bath conditions in 0.4 M KC1 solution adjusted to pH 4.5. As shown by Fuhrmann et al. (1976) our plasma membrane vesicles are only sealed between pH 4 and 5 and diffusion of glucose is negligibly small in countertransport experiments. The amount of glucose in vesicles was analysed by 14C labeled glucose. Separation of the vesicles was done by the Millipore filter technique (Fuhrmann et al., 1976; Kreutzfeldt and Fuhrmann, 1984). The techniques and mathematics of countertransport are described in detail by Fuhrmann et al. (1989).

Preparation of antibodies against 59 and 62 kDa plasma membrane proteins.

Antibodies were raised in rabbits by two s.c. injections of approximately 100 /~g of purified 59 and 62 kDa plasma membrane proteins. The first immunization was carried out with complete Freund's adjuvant and was followed 1 month later by a second immunization in incomplete Freund's adjuvant.

SDS-polyacrylamide gel electrophoresis (PAGE).

The stacking gel was 4% and the separating gel 10%. Plasma membrane samples contained 2% (w/v) SDS, 12.5% (w/v) glycerol or 10% sucrose, 2% (w/v) 2-mercaptoethanol, 0.00025% (w/v)

63

bromophenol blue and 62.5 mM Tris-HCl adjusted to pH 6.8. Denaturation was carried out at 100°C for 2 min.

Western blot. The Western-blot technique was applied as described by Towbin et al. (1979).

Immunofluorescence-labeling (FITC). For immunofluorescence-iabeling of cells the method of Wick et al. (1976) was applied.

Labeling of plasma membrane proteins. Plasma membranes of Saccharomyces cerevisiae cells were prepared as described for plasma membrane vesicles but finally resuspended in 50 mM sodium phosphate, pH 7.0. (i) Labeling with GEBrA. Plasma membranes in 0.1 ml 10 mM sodium phosphate, pH 7.0, at a protein concentration of 1 mg ml-1 were incubated with 200 /xM radioactively labeled GEBrA (approximately 1.3 /~Ci) in the presence or absence of 100 mM D-glucose for 4 h at room temperature with a 20 min preincubation before adding of GEBrA. After incubation with GEBrA the mixture was prepared for SDS-PAGE by addition of Tris-HC1 (pH 6.8), SDS, sucrose, 2-mercaptoethanol and bromophenol blue (final concentrations see SDS-PAGE) and 60 ~g portions of protein were subjected to SDS-PAGE. The gel was stained with Coomassie blue, treated with enhancer (AmplifyTM,Amersham Buchler, Braunschweig, FRG), dried and autoradiographed on Kodak X-ray film (X-Omat AR film, Eastman Kodak, Rochester, USA). (ii) Labeling with 6-4-[3H]-azi-4-deoxy-D-xylohexopyranose. Plasma membranes in 0.5 ml of 10 mM sodium phosphate, pH 7.0, at a protein concentration of 0.6 mg m1-1 were incubated at room temperature for 20 min with approx. 10 /~Ci

Ri /~ Ro

•=

con~rol

+= 0.1 mM GEBrA • = 0,5 mM GEBrA ::

I

mM GEBrA

x= L mM GEBrA

~b

'

2'0

'

3'o

minutes

Fig. 2. Glucose countertransport in plasma membrane vesicles under ice bath conditions at pH 4.5 with and without GEBrA. R i / R o is the relation of glucose tracer concentration inside to outside.

64

6-4-[3H]-azi-4-deoxy-D-xylohexopyranose (specific activity, 6.25 Ci mmol l) in the presence or absence of 100 mM D-glucose or D-galactose. Irradiation of the mixture was performed in a Rayonet RPR 100 reactor for 10 min at 350 nm and room temperature. Before irradiation the mixture had been flushed with nitrogen. After irradiation plasma membrane samples were diluted 50-fold with 10 mM sodium phosphate, pH 7.0, spun down by centrifugation at 100000 g and washed two times with the same buffer by resuspension of the pellets and subsequent centrifugation. The final pellets were resuspended in SDS sample buffer with

o t,n c:~ ~

otooa <:~c9~

"-,1 o~ c~ o

t.~ o

*'-- t,J Q t,n

¢.o c:~

~,a ol

~-a o

~n

Fig. 3. R a d i o a c t i v e l a b e l i n g o f p l a s m a m e m b r a n e s w i t h G E B r A . T h e t w o b o t t o m l a n e s d e p i c t the p r o t e i n p a t t e r n o f t h e p l a s m a m e m b r a n e s by S D S - P A G E . A b o v e the a u t o r a d i o g r a p h i e s o f l a b e l e d p r o t e i n s a n d t h e i r s c a n s a r e s h o w n . L o w e r l a n e a n d t h i n line o f t h e s c a n with 100 m M g l u c o s e d u r i n g reaction with GEBrA.

65 glycerol (see S D S - P A G E ) a n d 70 /zg p o r t i o n s of p r o t e i n w e r e s u b j e c t e d to S D S - P A G E . A u t o r a d i o g r a p h y of the gel was c a r r i e d o u t as d e s c r i b e d for G E B r A .

Materials.

P e p t o n e f r o m c a s e i n a n d y e a s t extract w e r e p u r c h a s e d f r o m E. M e r c k , D a r m s t a d t , F R G . D-[U-14C]glucose, specific activity 4 m C i m m o l - 1 , was o b t a i n e d f r o m A m e r s h a m Buchler, B r a u n s c h w e i g , F R G . All o t h e r c h e m i c a l s w e r e of analytical g r a d e quality.

Results

Countertransport experiments with GEBrA In Fig. 2 t h e effect o f G E B r A on glucose c o u n t e r t r a n s p o r t in p l a s m a m e m b r a n e vesicles is shown. T h e c o u n t e r t r a n s p o r t m a x i m u m is significantly r e d u c e d by

62 59

1

2

3

4

5

Fig. 4. Western-blot with antibodies against the double band of 59 and 62 kDa (lanes 1 and 3) and respective plasma membrane proteins in SDS-PAGE (lanes 2 and 4). In lanes 1 and 2 plasma membranes were obtained from glucose grown cells and in lanes 3 and 4 from galactose grown cells. In lanes 1-4 50 ,~g of protein were applied each. Lane 5 marker proteins (66 kDa, 45 kDa, 36 kDa, 29 kDa and 21 kDa).

66 increasing G E B r A concentrations. By computer simulation of countertransport (Fuhrmann et al., 1989) the corresponding K m values for the countertransport maxima can be calculated. The countertransport maximum in the control ( R i / R o = 3.4) corresponds to a K m value of 6.5 mM, the ones with 0.1, 0.5, 1 and 4 mM G E B r A concentrations are 8.1, 10.0, 11.5 and 13 raM. The type of inhibition is not compatible with a completely competitive one, and a mixed type of inhibition is assumed. Other common inhibitors of glucose transport like phloretin were only successful in inhibition of countertransport at excessively high concentrations of 500 ~M. Phlorizin at 500/xM, cytochalasin B at 100/xM, and the organic mercurial mersalyl at 50 ~ M were without effect (results not shown).

Affinity labeling of plasma membrane proteins with [14C]GEBrA Derivatives of bromoacide are known to react covalently with NH~- or SHgroups. Since G E B r A as a bromoacide derivative was able to inhibit the glucose countertransport effectively, we tried to label putative glucose carriers in the plasma membrane with [14C]GEBrA as prepared according to the description under Methods. Incubation of this radioactive compound with plasma membranes protected and not protected by 100 mM glucose and subsequent 10% SDS-PAGE led to separation of the proteins shown in Fig. 3. The patterns are representative for the protein pattern in these membranes and no distinct changes to controls without G E B r A could be detected. Autoradiography of these protein patterns demonstrated excessive labeling in the carbohydrate rich area at the beginning of the scan and two sharp lines at 59 and 62 kDa. The lower autoradiography is the sample with 100 mM glucose addition, which slightly protected labeling by G E B r A at the 59 kDa band. The scans of the autoradiographies are shown by the two lines, that with glucose protection by the light line. Raising antibodies in rabbits against the double band at 59 and 62 kDa By preparative SDS-PAGE protein bands of plasma membranes were separated and visualized by 4 M sodium acetate solution (Higgins and Dahmus, 1979). The double band was cut and eluted from the gel and purified in a second run. The protein fraction was injected into rabbits and antibodies raised. Only the antisera and the purified IgG-fraction were immunologically reactive. As depicted in the Western-blots of Fig. 4 the antibodies recognize in plasma membranes the double band at 59 and 62 kDa quite sharply. This is the case with plasma membranes prepared from 2% glucose or 2% galactose grown cells. The reaction with the antibodies seemed to be increased in plasma membranes prepared from galactose grown cells. Another sharp band is seen at 44 kDa, which has been shown to be the deglycosylated form of the double band (V61ker et al., 1990). Above the double band there are 4 minor reactive proteins of unknown properties and the fairly diffuse reactivity around 120 kDa, which might be the dimeric form of the double-band proteins. Immunoblots with the antibodies against 59 and 62 kDa proteins on membranes of the vacuole, endoplasmatic reticulum and mitochondria were without any

67

TABLE 1 Inhibition of countertransport of glucose in yeast plasma membrane vesicles Number

Formula

Percent inhibition

Number

N=N

1

Formula

N=N

HO~

~:H3

4

5

I~oHO/~ HO~

OH

2

CH2OH ~-, 0 !. ~[ tbCH3

3

6

OH

0~

N

0 OH

37

7

OH

4

CH2OH N3 H ~ ' OH OH

OCH3 OH

(~H2OH HOLo N=N ~ C H 3 OH

CH20H

3

Percent inhibition

CH2OH ~ON~/x.N=N .~ H6"~I~ CH3 OH

L°

N~N

22

B

C"H20H ~/ / J~Q--CHCH2C--CH3 u[ I O( ~ OCH3 H OH

The inhibitors were all used at 3 mM concentration. Compound 3 at 1 mM concentration showed a 12% inhibition. reaction (Mertsching, 1986). By F I T C labeling an i m m u n o f l u o r e s c e n c e could be s e e n in spheroplasts and in a c e t o n e fixed cells only at the surface and not in the interior (data not shown).

Countertransport experiments with azido and diazirino deriuatiues of sugars In Table 1 the c h e m i c a l structure of the sugar derivatives from the laboratory of L e h m a n n w e r e given and their inhibitory properties in glucose countertransport w e r e depicted. T h e table shows that only two derivatives, c o m p o u n d s (3) 4-azi-4-

68

4 ? c~

"~-. x

.....

mM

~

2

-

*

I

0 0

~0

5

10

15 20 minutes

25

3

30

Fig. 5. Glucose countertransport in plasma membrane vesicles under ice bath conditions and pH 4.5 with and without 4-azi-4-deoxy-D-xylohexopyranose.R i/Ro is the relation of glucose tracer concentration inside to outside. deoxy-D-xylohexopyranose and (4) 4-azido-4-deoxy-D-xylopyranose inhibited countertransport of glucose significantly. The countertransport experiment with compound (3) is shown in Fig. 5. The countertransport maximum of the control ( R i / R o = 4.5) corresponds to a K m value of 4.2 raM. With 1 m M 4-azi-4-deoxy-Dxylohexopyranose the K m value is 5 mM and at 3 mM inhibitor concentration the affinity is further reduced to 7.7 mM. The type of inhibition seems to be similar to that by GEBrA.

Photoaffinity labeling of plasma membranes with 6-4-[3H]-azi-4-deoxy-D-xylohexo pyranose In these experiments the 4-diazirino-compound was used for photoaffinity labeling of plasma m e m b r a n e s prepared from cells grown with 2% glucose (Fig. 6) and grown with 2% galactose (Fig. 7) as carbon source. The two lanes at the bottom are the Coomassie-stained proteins, the u p p e r one was with 100 mM glucose (Fig. 6) and with 100 m M galactose addition (Fig. 7) during photoaffinitylabeling. The observed protein patterns are identical to the protein patterns without photoaffinity labeling, and no significant changes can be detected. The corresponding autoradiographies above the protein patterns and their scans show a prominent labeling of the double bands with 59 and 62 kDa. No protection by 100 m M glucose or galactose could be observed. In addition the area of the deglycosylated double band at 44 kDa is labeled and further 2 to 3 minor bands at the low molecular weight region, which might be related to proteolytic products of the double band (V61ker et al., 1990). In Fig. 7 a further distinct labeling is seen at 120 kDa which could represent a dimer of the double band protein.

Discussion

Isolated plasma m e m b r a n e vesicles of Saccharomyces cereuisiae are of great advantage for studying m e m b r a n e proteins and transport function such as glucose

69

lll'l'lll 0o(~ O0

¢". r,J 0 0

' I I'l' I' i 0 0

co-..,1Cn O0 0

c.n 0

~ 0

'

I

'

]

~ (.n

co 0

~ on

I',J -.,'

Fig. 6. Radioactive labeling of plasma membranes with 6-4-[3H]-azi-4-deoxy-D-xylohexopyranose. The two bottom lanes depict the protein pattern of the plasma membranes by SDS-PAGE. Above the autoradiographies of labeled proteins and their scans are shown. Upper lane and thin line of scan with 100 mM glucose during photoaffinity labeling. Plasma membranes were prepared from glucose grown cells.

transport. From glucose transport kinetics, especially countertransport experiments in plasma membrane vesicles as shown in Figs. 2 and 5 (review Fuhrmann and V61ker, 1992, this issue) the translocation of glucose across the membrane occurs by carrier mediated facilitated diffusion. From genetic studies several carriers as described in the introduction were thought to be implicated in such transport mechanism. The aim of this investigation was to localize carriers in the plasma membrane. The human red cell glucose transporter, which behaves in some functional respects similar to Saccharomyces cereuisiae glucose transporters, has been local-

70

O 0

0

0

0

O 0

0

0

0

~n

0

(31

--~

Fig. 7. As in Fig. 6. U p p e r lane and thin line of scan with 100 m M galactose during photoaffinity labeling. Plasma m e m b r a n e s were prepared from galactose grown cells.

ized by the attachment of a potent inhibitor, cytochalasin B (Deziel and Rothstein, 1984). Cytochalasin B even in 100 /zM concentration did not inhibit glucose transport in Saccharomyces cerevisiae. Also organic mercurials, for example mersalyl, which inhibited the red cell glucose transport with a K i value of a few ~M, were without effect in Saccharomyces cerevisiae. Only phloretin at very high concentrations of 500 ~ M was successful in inhibition of countertransport in Saccharomyces cerevisiae. Both transport systems are therefore different. In the laboratory of Fasold several bromoacetamide derivatives of glucose have been synthesized in order to inhibit and label Na + coupled glucose transport in brush-border membranes of the kidney. In our countertransport experiments the compound G E B r A (Fig. 1) was of high inhibitory potency as can be seen in countertransport experiments by reducing the affinity of the transport from a K m

71 value of 6.5 mM in the control to 13 mM at 4 mM GEBrA (Fig. 2). This effective inhibitor, which is known to react covalently with SH- and NH2-groups, was available 14C-labeled. Incubation of isolated plasma membranes with [14C]GEBrA resulted in radioactive labeling of two prominent membrane proteins of 59 and 62 kDa (Fig. 3). Labeling of the 59 kDa protein was slightly protected by 100 mM glucose. These results with the specific inhibitor GEBrA are in contrast to the seemingly unspecific radioactive labeling of the plasma membrane proteins with N-[lnc]-ethylmaleimide. The latter labeled all membrane proteins with no indication of glucose protection (results not shown). Thus, it seems very probable that the 59 a n d / o r the 62 kDa proteins, which are in the region of the expected glucose carrier molecular masses, are indeed the glucose carriers detected by GEBrA labeling. The highly putative glucose carrier proteins at 59 and 62 kDa were purified by preparative gel electrophoresis and injected SDS-solubilized into rabbits in order to raise antibodies. The antibodies received were selective for the plasma membrane. The double band at 59 and 62 kDa in the plasma membrane was the main target for the antibodies (Fig. 3). In this way a highly valuable tool to identify glucose carriers was developed. This instrument has been used to characterize several metabolic influences on the expression of the transport proteins. If the cells were grown on galactose as carbon source a derepression of the glucose transport takes place and the number of glucose carriers is increased (Fuhrmann and V61ker, 1992). This increase is clearly demonstrated by the broadening and intensification of the double band in lane 3 (Fig. 4). The antibodies themselves produced, however, no significant inhibition of glucose transport. This indicates, that the immunogenic sites of the transport molecules are not identical to the active glucose transport site. The reaction of antibodies with membrane proteins in the 44 kDa region seemed to be related to the deglycosylated forms of the double band (V61ker et al., 1990). Further experiments with the specific antibodies on intracellular membranes have shown their specificity for only the plasma membrane: Other membranes like those of mitochondria, vacuoles and endoplasmatic reticulum did not react with the antibodies. Consequently also FITC-labeled antibodies showed photoluminescence not with the intracellular membranes but only at the surface of the cells. The laboratory of Lehmann has synthesized a variety of photo-labile sugar analogues; some of them proved to be very effective in inhibition of sugar transport in human red cells (Midgley et al., 1985). As shown in Table 1 two of these compounds were also highly effective in inhibition of glucose countertransport in plasma membrane vesicles prepared from Saccharomyces cerevisiae cells. The compound 4-azi-4-deoxy-D-xylohexopyranose, which was very effective in inhibiting glucose countertransport in Saccharomyces cerevisiae was used in 6-3H labeled form for photoaffinity labeling. Irradiation of plasma membranes at 350 nm prior to electrophoresis on SDS-polyacrylamide gels caused covalent binding to proteins. Autoradiography of the labeled proteins showed a clearly visible reaction with the double band at 59 and 62 kDa in the plasma membrane. This was the case when the plasma membranes were prepared from cells grown on glucose (Fig. 6) or

72

if the plasma membranes were prepared from those grown on galactose (Fig. 7). Protection of the double band by 100 mM glucose or galactose could not be found. In addition to the double band some reactivity was seen at the region of the deglycosylated proteins around 44 kDa and at other lower molecular mass regions, which might correspond to proteolytic breakdown products of the double band (V61ker et al., 1990). Thus, similar to GEBrA, the diazirino compound labeled also the 59 and 62 kDa transport proteins. These latter experiments with compounds of a different class have suggested that the putative glucose carrier can be characterized also by its susceptibility against a photosensitive diazirino derivative. In summarizing, the conspicuous labeling of the plasma membrane protein bands at 59 and 62 kDa with two different potent inhibitors of the glucose transport, as well as the fact that these bands are integral main membrane proteins are in accordance with the assumption that the described electrophoretic bands are the glucose carriers in the plasma membrane of Saccharomyces cereL,isiae cells.

Acknowledgements Supported by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie. We are very thankful to Dipl. Chem. C.-St. Kuhn for photolabeling plasma m e m b r a n e proteins. It is a great pleasure to thank Professor K.J. Netter for his most valuable comments and suggestions.

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