Vol. 124, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, OCt. 1975, P. 127-133 Copyright 0 1975 American Society for Microbiology
Site of Initial Glycosylation of Mannoproteins from Saccharomyces cerevisiae J. RUIZ-HERRERA'* AND R. SENTANDREU Departmento de Microbiologia, C.S.LC., Facultad de Ciencias, Universidad de Salamanca, Spain
Received for publication 16 July 1975
The cellular site of initial glycosylation of proteins from Saccharomyces cerevisiae has been studied. Short pulses of [U- "C ]mannose label the ribosomal fraction of the yeast. Most of the label was associated with polysomes; monosomes contained only a small amount of radioactivity. All of the radioactivity present in the polysomal fraction was accounted by mannose and smaller amounts of glucose and glucosamine. Puromycin treatment detached more than 50% of the radioactivity from the polysomes; treatment of polysomes at pH 10.0 also caused the release of radioactivity. These results indicate that initial sugar binding occurs while the nascent polypeptide chains are still growing on the ribosomes. When the cells were preincubated with 2-deoxy-D-glucose, incorporation of [U -14CImannose into the polysomes and the cell wall was inhibited, whereas its incorporation into membrane fractions was unimpaired. It was concluded that 2-deoxy-D-glucose inhibited the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level. attachment of the most internal sugars is not well understood (16). A study of the cellular organelles involved in protein glycosylation may throw some light on the role of the carbohydrate moiety in the secretion process. In this paper we describe the site of initial glycosylation of proteins in Saccharomyces cerevisiae and the effect of 2-deoxyD-glucose on the process.
Mannoproteins constitute the major part of the matrix material of Saccharomyces cerevisiae wall (28). Some of these mannoproteins are known to have enzymatic activity (12). Alkaline treatment of the cell walls causes a partial degradation of these mannoproteins and the resulting product(s) which is enriched in the carbohydrate moiety is known as yeast mannan. It has been found that the glycosyl donor for yeast mannan biosynthesis is guanosine-diphosphate-mannose and that the mannan chains grow by stepwise addition of mannose units (2, 25). Transfer of mannose units occurs through a lipid intermediate identified as a phosphodiester of mannose and a polyprenol (10, 26, 27). Recent findings from Ballou's group (1) provide evidence that different specific enzymes are involved in the biosynthesis of the mannan backbone and its further branching. Despite the several studies on the mechanism of biosynthesis of yeast mannoproteins, there are no data about the subcellular site at which the first step(s) or protein glycosylation occurs. Most extracellular proteins produced by eucaryotic cells are glycoproteins, and whereas the majority of their sugar residues are incorporated while associated with the internal membrane systems of the cells, the subcellular site of 'Present address: Departamento de Microbiologia, Escuela Nacional de Ciencias Biologicas, I.P.N. Mexico 17, D.F. Mexico.
MATERIALS AND METHODS Strain and culture media. A thermosensitive strain requiring adenine and uracyl of Saccharomyces cerevisiae, ts-136 derived from strain A 364 by treatment with N-methyl-N-nitro-N-nitrose guanidine, was obtained from L. H. Hartwell, University of Washington, Seattle. It was maintained on slants of medium YM-1: yeast extract, 5 g; peptone, 1 g; nitrogen base (Difco), 6.7 g; adenine, 0.01 g; uracil, 0.01 g; succinic acid, 10 g; NaOH, 6 g; sodium lactate, 30 g; pH 6.8, solidified with 2% agar; and propagated in liquid YM-1 medium. Culture conditions. Erlenmeyer flasks containing 100 ml of medium were inoculated with an overnightgrown culture and incubated with shaking at 25 C for 9 to 10 h. Absorbancy of the culture was measured at 600 nm with a Bausch & Lomb Spectronic 20 photoelectric colorimeter. Number of cells was calculated using a calibration curve. Cells were harvested by centrifugation, washed once with 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.5; once with TKM buffer [6.05 g of tris(hydroxymethyl)aminomethane, 1.764 g of KCl, and 1.016 g of MgCl,-6H,O were dissolved in distilled water, pH
127
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RUIZ-HERRERA AND SENTANDREU
J. BACTERIOL.
was adjusted to 7.5 with 2 N HCl and volume was the latter case, protoplast lysates in TKM buffer taken to 1 liter], and resuspended in an appropriate containing 100 pg of cycloheximide per ml and 0.01% volume of this buffer. heparin were treated with 0.33% Triton X-100, made Preparation of protoplasts. Five milliliters of a 0.2 M with sucrose, and then centrifuged at 17,500 x yeast suspension containing 1 x 109 to 5 x 10' g for 10 min. The supernatant was layered on a 36-ml cells per ml in TKM buffer were incubated at 30 C linear gradient of sucrose (0.3 to 1.0 M) in TKM with 6 ml of 1.6 M KCI containing 0.02 M MgSO., placed over 10 ml of a 2.0 M sucrose solution in 0.033 M mercaptoethanol, 0.01% heparin, and 1.0 ml TKM. The tubes were centrifuged at 25,000 rpm in of the lytic complex from Helix pomatia (L'Industrie the SW25.2 rotor for 140 min. The gradient was Biologique Francaise, Gennevilliers, France) (previ- collected introducing a needle down to the 2.0 M ously centrifuged at 40,000 x g for 20 min). Formation sucrose cushion and pumping up the material. Conof protoplasts was almost complete at the end of 90 to tinuous measurement of the absorbancy at 254 em 120 min as determined by their sensitivity to osmotic was achieved by flowing the sample through the cell shock with hypotonic buffer. Protoplasts were cen- of an LKB Uvicord recording photometer. Samples trifuged at 6,000 rpm for 10 min, washed once with were recovered with a fraction collector, precipitated 0.8 M KCI in TKM buffer, and resuspended in the with trichloroacetic acid, filtered, washed, dried, same hypertonic buffer. and counted as described above. Incorporation of radioactive mannose, glucose, Detachment ot labeled peptides from the or threonine by protoplasts. Protoplasts were in- ribosomes. Treatment of whole cells with puromycin cubated at 25 C in YM-1 medium in TKM buffer was not attempted since the strain was not sensitive made hypertonic with 0.8 M KCI. Variable amounts to puromycin. Crude lysates of protoplasts (see above) of the radioactive compounds were added. At selected were treated with 2 mM puromycin and 2 mM times samples were withdrawn and mixed with an guanosine 5'-triphosphate (GTP) for 40 min at 0 C. equal volume of 10% trichloroacetic acid. Samples Untreated controls were incubated for the same time were kept on ice and after not less than 1 h they were at 0 C. Then Triton X-100 was added to both samples filtered through GF/C Whatman glass fiber disks and ribosomes and polysomes were separated by (2.4 cm diameter), washed with 50 ml of 5% tri- sucrose density centrifugation and their radioactivity chloroacetic acid and dried in an oven at 80 C. was assayed as described above. Bulk-isolated riboRadioactivity was measured by placing the dried somes were treated with 1 or 2 mM puromycin and 2 disks in small vials containing 2 ml of toluene-based mM GTP; after an appropriate period of time at liquid scintillation fluid. The small vials were placed either 0 C or room temperature, 5 ml of pH 1.9 buffer into normal-sized vials and counted in a Packard (8.7% acetic acid and 2.5% formic acid [30]) were added and the samples were filtered through WhatTriCarb liquid scintillation spectrometer. Pulse-labeling of whole cells and protoplasts. man DE-81 diethylaminoethyl paper and treated as Whole cells were preincubated in hypertonic buffered described by Weber and De Moss (30). Detachrhent of YM-1 medium. After several minutes, the radioactive nascent polypeptide chains from isolated ribosomes compound was added and incubation was continued was also achieved by incubation with 0.2 M glycinefor 30 s to 2 min. Protein synthesis was stopped by sodium hydroxide buffer, pH 10.0, at 37 C for 1 to 3 h, addition of 5 volumes of ice cold TKM buffer con- and further treatment was as described for the putaining 100 pg of cycloheximide per ml. Cells were romycin reaction. Other methods. Protein was measured with the centrifuged and resuspended in the incubation mixture described above for the preparation of protoplasts Folin phenol reagent (15) and ribosomes as described plus cycloheximide and incubated further at 30 C to by Rich (24). For the detection of sugars, samples obtain protoplasts. For protoplasts, labeling was were mixed with carrier sugars, hydrolyzed with 2 N stopped by the addition of 5 volumes of ice cold 0.8 M HCl at 105 C for 2 h, neutralized with Dowex-2 resin KCI in TKM buffer containing 100 pig of cyclo- in the HCO,- form, and passed through a small heximide per ml. The protoplasts were centrifuged column of Dowex-50 in the H- form. Neutral sugars and washed once with hypertonic buffer containing eluted from this column were concentrated and chromatographed in paper for 36 h with ethyl acetatecycloheximide. Breakage of cells and protoplasts and obtention pyridine-water, 8:2:1 (vl/vol/vol). Sugars were visuof different subeellular fractions. Cells were broken alized in the chromatograms by spraying with amwith a Braun cell homogenizer for 30 s. Protoplasts monium-silver nitrate, whereas radioactive spots were were osmotically shocked with TKM buffer usually measured by cutting 1-cm sections from the chromacontaining 100 pg of cycloheximide per ml and 0.01% tography paper and counting as above. Hexosamines heparin. Lysates were fractionated by centrifugation and amino acids were eluted from the Dowex-50 colin discontinuous sucrose gradients by two differents umn with HCl, concentrated, and chromatographed methods. One of them was carried out essentially with the following solvent: ethyl acetate-pyridineas described by Melchers (18), except that no 2.0 M butanol-butyric acid-water, 10:10:5:1:5 (vol/vol). Rasucrose layer was placed at the bottom of the tubes dioactive spots were located as mentioned for neutral and the extracts were centrifuged in a Beckman sugars. SW25.2 rotor, and the other method used was that described by Lawford and Schachter (13). Bulk riboRESUILTS somes were isolated essentially as described by Munro Incorporation of mannose, glucose, and et al. (22), and polysomes by a slight modification of the method described by Preisler et al. (23). In threonine by protoplasts. Protoplasts incorpo-
SYNTHESIS OF MANNOPROTEINS IN S. CEREVISIAE
VOL. 124, 1975
rated radioactive [U- 14C Imannose and [LJl4 ]glucose into trichloroacetic acid-insoluble material (Fig. 1). Both mannose and glucose were found in the trichloroacetic acid-precipitable material independently of the sugar added to the incubation medium. The hexoses incorporated by the protoplasts were found associated mostly to the fractions which corresponded to those described as "smooth endoplasmic reticulum" by the methods of Melchers (18) and Lawford and Schachter (13). Almost no radioactivity appeared bound to the fraction corresponding to the "rough endoplasmic reticulum" in the above mentioned methods nor to ribosomes isolated as described by Munro et al. (22). This result appeared to be due to the poor biosynthetic capacities of protoplasts since ["IC ]threonine was incorporated at very low rates, Incorporation of [U- ICimannose by whole oeils. Growing cultures of Saccharomyces were labeled for 2 min with ["4C]mannose and cycloheximide was added to freeze the polysomes. It is known that this treatment stops the synthesis of mannose-acceptor proteins (25). When the cells were broken with a Braun cell homogenizer only a small amount of label appeared associated with the ribosomes obtained as described above. On the other hand when protoplasts were prepared from the labeled cells and
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then lysed, a significant amount of radioactivity Xappeared associated with the ribosomes. Ribosomal profiles from cells similarly labeled (see Materials and Methods) showed most of the radioactivity in the polysomal fraction (Fig. 2); monosomes contained much less radioactivity. The peculiar shape of the polysomal curve was attributed to "banding" of the heaviest polysomes over the 2 M sucrose cushion. Similar results were obtained when the labeling period was reduced to 30 s. Radioactive polysomes were precipitated with trichloroacetic acid, filtered, dried, and counted. Scintillation fluid was washed away with toluene and the samples present in the glass fiber disks were hydrolyzed and analyzed as described under Materials and Methods. The only radioactive components present were mannose, glucose, and glucosamine. Mannose accounted for more than 60% of the radioactivity, whereas glucosamine was present in the smaller amounts. Detachment of radioactive components from the ribosomal fractions. Incubation of bulk-isolated ribosomes (22) with puromycin and GTP caused the release of 20 to 30% of the radioactivity (Table 1). Also incubation of ribosomes with pH 10 buffer brought about the release of 50% of the radioactivity from the ribosomal material (Table 1). These low yields may be due to extensive polysomal breakage during handling, and that conditions for pu-
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FIG, 1. Incorporation of [U-_"4Cglucose and [U"4C]mannose by protoplasts of Saccharomyces cerevisiae. Protoplasts (6 x 108 per ml) were shaken in the presence of radioactive sugars. Total volume was 5 ml. At intervals 1-mI aliquots were withdrawn and received over 1 ml of ice-cold 10% trichloroacetic acid. Samples were left on ice for 2 h, filtered, washed, dried, and counted. (a) Protoplasts incubated with [U-"4CJglucose (1.25 mg and 0.1 uCi per ml). (b) Protoplasts incubated with [U-14Clmannose (1.25 mg and 0.1 jCi/ml). Symbols: (0) Protoplasts incubated in 0.8 M KCI alone; (0) protoplasts incubated in 0.8 MKCI in YM-1 medium.
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FIG. 2. Ribosomal pattern of cells labeled with [U-"1C]mannose. Yeasts (2.5 x 1010 cells) resuspended in a final volume of 4 ml of YM-1 medium buffered with TKM were incubated with shaking at 25 C for 15 min and labeled with [U-14C]mannose (0.6 mg and 1 gCi/ml) for 2 min. Protoplasts were prepared, lysed, and centrifuged as described. The supernatant (6 ml) was divided into two aliquots of 1 and 5 ml, respectively. Both aliquots were placed over sucrose gradients and centrifuged and treated as described in the text. Symbols: (0) data of absorbancy calculated from the Uvicord tracing of the 1-ml sample; (0) radioactivity data of the 5-ml sample.
130
RUIZ-HERRERA AND SENTANDREU
romycin reaction were not the most adequate because (i) puromycin added to bulk-isolated ribosomes labeled with [14C]threonine released a similar proportion of radioactive peptides than those in Table 1; and (ii) puromycin reaction carried out with crude lysates released more than 50% of the radioactivity (see below). Also, by incubation of a mannose-labeled polysomal fraction isolated from a sucrose gradient for 2 h at 38 C in glycine-sodium hydroxide buffer, pH 10, 80% of the radioactivity was released. In further experiments whole cells were labeled with ['IC Imannose and protoplasts were prepared and lysed as described. Crude lysates were incubated with puromycin and GTP either in the presence or in the absence of cycloheximide. Puromycin caused the release of radioactivity and the disaggregation of the polysomes. The effect was more pronounced when no cycloheximide was present during the puromycin treatment (Fig. 3). Data calculated from Fig. 3 showed that polysomes (fractions 1 to 13) lost 55% radioactivity and 49% of the absorbancy at 254 nm by treatment with puromycin. When the polysomal fraction isolated from a sucrose gradient was washed and incubated for 2 h at 38 C in glycine-sodium hydroxide buffer, pH 10, 89% of the radioactivity was released as measured by the method of Weber and De Moss (30). Effect of 2-deoxy-D-glucose on mannose incorporation. Mannose incorporation into mannan by whole cells was inhibited by 2-deoxy-D-glucose. We could not detect any incorporation of [1-_ H]-2-deoxy-glucose (50 jgCi) into polysomes, membranes, or cell walls of S. cerevisiae even though it was transformed into an unidentified component (2-deoxy-Dglucose 6-phosphate?). TABLE 1. Release of radioactivity from ribosomes by puromycin and alkaline pH
Expta 1 2 3
Treatment
None
Counts/min ribosomes in % Released
Puromycin
206 164
21
None Puromycin
218 157
28
None pH 10
126 64
49
aFor each experiment a different batch of ribosomes was used. 0.3 ml of ribosomes (0.22 to 0.4 mg) were incubated for 40 min at room temperature with and without 1 mM puromycin and 2 mM GTP, or for 1 h at 37 C with and without 0.1 M glycine-sodium
hydroxide buffer.
J. BACTERIOL.
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FIG. 3. Release of labeled peptides from polysomes by puromycin. Yeasts (4.8 x 1010 cells) were labeled as described for Fig. 2. Protoplasts were prepared in the presence of cycloheximide, centrifuged, and resuspended in hypotonic buffer without cycloheximide. The lysate was divided into two equal samples of 3.0 ml, one received puromycin and GTP, and both were incubated at 0 C for 30 min. Samples were processed as described in Materials and Methods. Upper graph: radioactivity. Lower graph: Absorbancy at 254 nm. Symbols: (0) control; (0) sample treated with puromycin.
Protoplasts were labeled with [U- I4C Imannose for 3 min and 1 aliquot was removed and treated with cycloheximide. The remaining was divided into two samples and one was treated with 2-deoxy-D-glucose (125 gg/ml). Both samples were further incubated for 6 min. Protoplasts were lysed and fractionated as described by Lawford and Schachter (13). 2-Deoxy-Dglucose inhibited the incorporation of [U14C]mannose in the ribosomal fraction but did not affect the incorporation into the membrane fractions (Table 2). Whole cells were preincubated with and without 2-deoxy-D-glucose for 10 min and then labeled for 2 min with [U- "C]mannose. The incubation was stopped as described and the cells were centrifuged and washed. Aliquots were withdrawn and precipitated with 10% trichloroacetic acid to measure total incorporation of [U- 14C ]mannose.
VOL. 124, 1975
SYNTHESIS OF MANNOPROTEINS IN S. CEREVISIAE
131
TABLE 2. Effect of 2-deoxy-D-glucose on the incorporation of mannose into several cellular fractions of yeast protoplastsa Radioactivity (counts/min) Fraction
Soluble Membranes Ribosomes
Control (3 min)
484 2,609 132
Control (9 min)
2-deoxy-Dglucose
875 5,396 327
1,176 5,742 123
a Protoplasts (1.2 x 109 per ml) were labeled for 3 min with 0.5 yCi of [U- 14C ]mannose (specific activity 2.7 mCi per mmol) and treated as described in the text.
Cells were incubated with snail enzyme to obtain protoplasts, centrifuged, washed, and lysed, all in the presence of cycloheximide. Aliquots of lysates were removed to measure radioactivity precipitable with trichloroacetic acid protein. Lysates were treated with Triton X-100 and centrifuged in a linear sucrose gradient as described for the separation of polysomes. 2Deoxy-D-glucose inhibited incorporation of mannose into polysomes (Fig. 4). Comparison of the data of whole cells, protoplast lysates, and ribosomes indicated that incorporation of mannose into the cell wall, but not in membrane fractions, was also inhibited (Table 3). DISCUSSION It is generally accepted that animal glycoproteins are synthesized by the stepwise addition of sugar residues to the polypeptide chains. This occurs during passage from the rough endoplasmic reticulum to the smooth endoplasmic reticulum and the Golgi apparatus (16). However the mechanism of the initial steps of glycosylation is still subject to debate. Some authors have presented evidences that the primary glycosylation occurs while the polypeptides are still bound to the polyribosomes (13, 20, 21, 29). Heretofore no data were available on the mechanism of the initiation of glycoprotein synthesis in microbial eukaryotes. We present evidences that the initial glycosylation of mannan from S. cerevisiae occurs at the ribosomal level. Firstly, radioactive sugars appeared bound to polysomes when cells were labeled with [U- 14C ]mannose for 30 s to 2 min. The low amount of radioactivity associated with manosomes, which were more abundant than polysomes, argues against the possibility that sugars were merely adsorbed on to the ribosomes. Furthermore, radioactivity bound to polysomes was detached by treatment with puromycin; this is
00
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12
15
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21
24
30
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FIG. 4. Effect of 2-deoxy-D-glucose on the incorporation of [U-14C]mannose into polysomes. Yeasts (2.7 101° cells) were resuspended in 5 ml of YM-1 medium in TKM and then two 2.5-ml aliquots were separated. One received I mg of 2-deoxy-D-glucose per ml. After 10 min of preincubation at 25 C both samples received [U-14CJmannose (1.0 mg and 0.8 gCi per ml each) and were incubated for 2 min. Protoplasts were prepared, lysed and fractionated as described. Symbols: (0) control; (0) sample incubated with 2-deoxy-D-glucose. x
TABLE 3. Effect of 2-deoxy-D-glucose on the incorporation of mannose by intact yeastsa Radioactivity (counts/min)
% Fraction... Fraction 2-Deoxy-D- Inhibition Control glucose
Whole cells Lysed protoplasts Polysomes Monosomes Membrane and soluble Whole gradient
64,360 19,626 4,001 1,126 8,999
40,960 17,940 2,931 895 9,413
36.4 8.6 26.8 20.6 None
14,126
13,239
6.3
a Conditions
of the experiment described in the text and in the legend for Fig. 4. Data are corrected for protein content in the fractions of both samples (the difference was less than 6%). Polysomes are fractions 1 to 14, monosomes: 15 to 21 and membrane and soluble fractions: 22 to 31.
known to release nascent polypeptide chains serving as polypeptide acceptor. Release of radioactive peptides in our experiments was higher when incubation was carried out in the absence of cycloheximide, and under the same conditions a larger destruction of polysomes occurred. Puromycin is known to destroy polysome integrity. Besides mannose we detected the presence of glucose bound to polysomes, a result indicative that there are glucose-containing proteins in Saccaromyces. It is possible that mannoproteins contain a small amount of glucose buried into the mannan tufts as has been reported by Falcone and Nickerson (7) or that this glucose might represent the bridges be-
132
RUIZ-HERRERA AND SENTANDREU
tween the two main polymers of the wall (glucan and mannoproteins). An alternative (and attractive) hypothesis would be that this glycosylation represents the first step in the synthesis of yeast glucan. Further experiments are necessary to discern between these alternatives. It is known that the presence of hexosamine constitutes the link of the mannan to the protein components (1). Biosynthesis of glycoproteins in animals cells (9), yeasts (8, 11, 14), and fungi (17; J. Ruiz-Herrera, unpublished data) is very sensitive to 2-deoxy-D-glucose. Biely et al. (3, 4) have described that 2-deoxy-D-glucose is incorporated into mannan, but its incorporation by different species is not related to their corresponding susceptibility to the compound, and since it only affects mannan branching, this effect seems to be a later one and not the primary cause of inhibition of glycoprotein biosynthesis. We could not detect incorporation of the analogue by the strain used. 2-Deoxy-Dglucose is known to inhibit phosphoglucose isomerase (31) and Kuo and Lampen (11) found that it also inhibits phosphomannose isomerase. According to these authors availability of mannose to the transglycosidases is thus prevented. Since we used mannose as starting material, the inhibition we found suggests that there is a further and more specific step blocked. We found that 2-deoxy-D-glucose inhibited glycosylation of polysome-bound polypeptides. These results are in agreement with Melchers (19) who described that 2-deoxy-D-glucose inhibited the migration of immunoglobulin Gl (produced by the plasma cell tumor MOPC 21 transplanted to mice) from the membranebound polyribosomes to the cisternae of the rough endoplasmic reticulum. Marzluf (17) described that regeneration of invertase in Neurospora required de novo synthesis of proteins and that the process was completely inhibited by 2-deoxy-D-glucose. We found that 2-deoxy-D-glucose also inhibited incorporation of mannose into the cell wall, but had no effect on the synthesis of membranebound glycoproteins. This suggests that secreted and membrane-bound glycoproteins are synthesized by a different mechanism. Bretscher (5) has sustained the same hypothesis. Preliminary results from Forni and Melchers (19) also point in the same direction. Altogether, our results agree with Eylar's hypothesis (6) that glycosylation is necessary for protein secretion in eukaryotes. As a speculation it is possible to think that glycosylation of the nascent polypeptides permits them to transverse the membrane of the rough endoplasmic reticu-
J. BACtERIDL.
lum, or that glycosylation of the growing protein chain in the lumen side of the cisternae of the endoplasmic reticulum is the mechanism which pulls the polypeptide through the membrane or avoids its recoil into the cytoplasmic space. ACKNOWLEDGMENTS The stay of J. Ruiz-Herrera (Professor Fellow from the Comisi6n de Operaci6n y Fomento de las Actividades Academicas, M6xico) at the University of Salamanca was made possible through funds from grant no. 020 of the Consejo Nacional de Ciencia y Tecnologia, M6xico, and financial help from the Instituto de Cultura Hispanica, Spain. J.R.H. is indebted to the facilities given by Julio R. Villanueva during his stay at the University of Salamanca. We are grateful to Professors Julio R. Villanueva and S. Bartnicki-Garcria for critical reading of the manuscript. LITERATURE CITED 1. Ballou, C. E., and W. C. Raschke. 1974. Polymorphism of the somatic antigen of yeast. Science 184:127-134. 2. Behrens, N. H., and E. Cabib. 1968. The biosynthesis of mannan in Saccharomyces carlsbergensis. J. Biol. Chem. 243:502-509. 3. Biely, P., S. Kratky, and S. Bauer. 1972. Metabolism of 2-deoxy-D-glucose by bakers yeasts. IV. Incorporation of 2-deoxy-D-glucose into cell-wall mannan. Biochim. Biophys. Acta 255:631-639. 4. Biely, P., Z. Kratky, J. Rovarik, and S. Bauer. 1971. Effect of 2-deoxy-D-glucose on cell wall formation in Saccharomyces cerevisiae and its relation to cell growth inhibition. J. Bacteriol. 107:121-129. 5. Bretscher, M. S. 1973. Membrane structure: some general principles. Science 181:622-629. 6. Eylar, E. H. 1966. On the biological role of glycoproteins. J. Theor. Biol. 10:89-113. 7. Falcone, G., and W. J. Nickerson. 1956. Cell-wall mannan-protein of bakers' yeast. Science 124:272-273. 8. Farkas, V., A. Svoboda, and S. Bauer. 1970. Secretion of cell-wall glycoproteins by yeast protoplasts. Effect of 2-deoxy-D-glucose and cycloheximide. Biochem. J. 118:755-758. 9. Gandhi, S. S., P. Stanly, J. M. Taylor, and D. 0. White. 1972. Inhibition of influenza glycoprotein synthesis by sugars. Microbios 5:41-50. 10. Jung, P., and W. Tanner. 1973. Identification of the lipid intermediate in yeast mannan biosynthesis. Eur. J. Biochem. 37:1-6. 11. Kuo, S. C., and J. 0. Lampen. 1972. Inhibition by 2-deoxy-D-glucose of synthesis of glycoprotein enzymes by protoplasts of Saccharomyces: relation to inhibition of sugar uptake and metabolism. J. Bacteriol. 111:419429. 12. Lampen, J. 0. 1968. External enzymes of yeast. Their nature and formation. Antonie van Leeuwenhoek J. Microbiol. Serol. 34:1-18. 13. Lawford, G. R., and H. Schachter. 1966. Biosynthesis of glycoprotein by liver. The incorporation "in vivo" of "C-glucosamine into protein-bound hexosamine and sialic acid of rat liver subcellular fractions. J. Biol. Chem. 241:5408-5418. 14. Liras, P., and S. Gascon. 1971. Biosynthesis and secretion of yeast invertase. Effect of cycloheximide and 2-deoxy-D-glucose. Eur. J. Biochem. 23:160-165. 15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 198:265-275.
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16. Marshall, R. D. 1972. Glycoproteins. Annu. Rev. Biochem. 41:673-702. 17. Marzluf, G. A. 1973. Regeneration of invertase in Neurospora crassa. J. Bacteriol. 115:146-152. 18. Melchers, F. 1971. Biosynthesis of the carbohydrate portion of immunoglobulin. Radiochemical and chemical analysis of the carbohydrate moieties of 2 myeloma proteins purified from different subcellular fractions of plasma cells. Biochemistry 10:653-659. 19. Melchers, F. 1973. Biosynthesis, intracellular transport and secretion of immunoglobulins. Effect of 2-deoxy-Dglucose in tumor-plasma cells producing and secreting immunoglobulin Gl. Biochemistry 12:1471-1476. 20. Molnar, J., and D. Sy. 1967. Attachment of glucosamine to protein at the ribosomal site of rat liver. Biochemistry 6:1941-1947. 21. Molnar, J., G. B. Robinson, and R. Winzler. 1965. Biosynthesis of glycoproteins. IV. The subcellular site of incorporation of glucosamine-1-"IC into glycoprotein in rat liver. J. Biol. Chem. 240:1882-1888. 22. Munro, A. J., R. J. Jackson, and A. Korner. 1964. Studies on the nature of polysomes. Biochem. J. 92:289-299. 23. Preisler, H. D., W. Scher, and C. Friend. 1973. Polyribosome profiles and polyribosome-associated RNA in Friend leukemia cells following DMSO-induced differentiation. Differentiation 1:27-37. 24. Rich, A. 1967. Preparation of polysomes from mammalian reticulocytes, lymph nodes, and cells grown in tissue
25. 26.
27.
28.
29.
30.
31.
133
culture, p. 481-491. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 12, part A. Academic Press Inc., New York. Sentandreu, R., and J. 0. Lampex. 1970. Biosynthesis of yeast mannan: inhibition of synthesis of mannose acceptor by cycloheximide. FEBS Lett. 11:95-99. Sentandreu, R., and J. 0. Lampen. 1971. Participation of a lipid intermediate in the biosynthesis of Saccharomyces cerevisiae LK,G,, mannan. FEBS Lett. 14:109-113. Sentandreu, R., and J. 0. Lampen. 1972. Biosynthesis of mannan in Saccharomyces cerevisiae. Isolation of a lipid intermediate and its identification as a mannosyl1-phosphorylpolyprenol. FEBS Lett. 27:331-334. Sentandreu, R., and D. H. Northcote. 1968. The structure of a glycopeptide isolated from the yeast cell wall. Biochem. J. 109:419-432. Sherr, C. J., and J. W. Uhr. 1969. Immunoglobulin synthesis and secretion. III. Incorporation of glucosamine into immunoglobulin on polyribosomes. Proc. Natl. Acad. Sci. U.S. 64:381-387. Weber, M. J., and J. A. De Moss. 1969. Inhibition of peptide bond synthesizing cycle by chloramphenicol. J. Bacteriol. 97:1099-1105. Wick, A. N., D. R. Drury, H. I. Nakada, and J. B. Wolfe. 1957. Localization of the primary metabolic block produced by 2-deoxy-glucose. J. Biol. Chem. 224:963-969.
JOURNAL OF BACTERIOLOGY, OCt. 1975, P. 127-133 Copyright 0 1975 American Society for Microbiology
Site of Initial Glycosylation of Mannoproteins from Saccharomyces cerevisiae J. RUIZ-HERRERA'* AND R. SENTANDREU Departmento de Microbiologia, C.S.LC., Facultad de Ciencias, Universidad de Salamanca, Spain
Received for publication 16 July 1975
The cellular site of initial glycosylation of proteins from Saccharomyces cerevisiae has been studied. Short pulses of [U- "C ]mannose label the ribosomal fraction of the yeast. Most of the label was associated with polysomes; monosomes contained only a small amount of radioactivity. All of the radioactivity present in the polysomal fraction was accounted by mannose and smaller amounts of glucose and glucosamine. Puromycin treatment detached more than 50% of the radioactivity from the polysomes; treatment of polysomes at pH 10.0 also caused the release of radioactivity. These results indicate that initial sugar binding occurs while the nascent polypeptide chains are still growing on the ribosomes. When the cells were preincubated with 2-deoxy-D-glucose, incorporation of [U -14CImannose into the polysomes and the cell wall was inhibited, whereas its incorporation into membrane fractions was unimpaired. It was concluded that 2-deoxy-D-glucose inhibited the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level. attachment of the most internal sugars is not well understood (16). A study of the cellular organelles involved in protein glycosylation may throw some light on the role of the carbohydrate moiety in the secretion process. In this paper we describe the site of initial glycosylation of proteins in Saccharomyces cerevisiae and the effect of 2-deoxyD-glucose on the process.
Mannoproteins constitute the major part of the matrix material of Saccharomyces cerevisiae wall (28). Some of these mannoproteins are known to have enzymatic activity (12). Alkaline treatment of the cell walls causes a partial degradation of these mannoproteins and the resulting product(s) which is enriched in the carbohydrate moiety is known as yeast mannan. It has been found that the glycosyl donor for yeast mannan biosynthesis is guanosine-diphosphate-mannose and that the mannan chains grow by stepwise addition of mannose units (2, 25). Transfer of mannose units occurs through a lipid intermediate identified as a phosphodiester of mannose and a polyprenol (10, 26, 27). Recent findings from Ballou's group (1) provide evidence that different specific enzymes are involved in the biosynthesis of the mannan backbone and its further branching. Despite the several studies on the mechanism of biosynthesis of yeast mannoproteins, there are no data about the subcellular site at which the first step(s) or protein glycosylation occurs. Most extracellular proteins produced by eucaryotic cells are glycoproteins, and whereas the majority of their sugar residues are incorporated while associated with the internal membrane systems of the cells, the subcellular site of 'Present address: Departamento de Microbiologia, Escuela Nacional de Ciencias Biologicas, I.P.N. Mexico 17, D.F. Mexico.
MATERIALS AND METHODS Strain and culture media. A thermosensitive strain requiring adenine and uracyl of Saccharomyces cerevisiae, ts-136 derived from strain A 364 by treatment with N-methyl-N-nitro-N-nitrose guanidine, was obtained from L. H. Hartwell, University of Washington, Seattle. It was maintained on slants of medium YM-1: yeast extract, 5 g; peptone, 1 g; nitrogen base (Difco), 6.7 g; adenine, 0.01 g; uracil, 0.01 g; succinic acid, 10 g; NaOH, 6 g; sodium lactate, 30 g; pH 6.8, solidified with 2% agar; and propagated in liquid YM-1 medium. Culture conditions. Erlenmeyer flasks containing 100 ml of medium were inoculated with an overnightgrown culture and incubated with shaking at 25 C for 9 to 10 h. Absorbancy of the culture was measured at 600 nm with a Bausch & Lomb Spectronic 20 photoelectric colorimeter. Number of cells was calculated using a calibration curve. Cells were harvested by centrifugation, washed once with 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.5; once with TKM buffer [6.05 g of tris(hydroxymethyl)aminomethane, 1.764 g of KCl, and 1.016 g of MgCl,-6H,O were dissolved in distilled water, pH
127
128
RUIZ-HERRERA AND SENTANDREU
J. BACTERIOL.
was adjusted to 7.5 with 2 N HCl and volume was the latter case, protoplast lysates in TKM buffer taken to 1 liter], and resuspended in an appropriate containing 100 pg of cycloheximide per ml and 0.01% volume of this buffer. heparin were treated with 0.33% Triton X-100, made Preparation of protoplasts. Five milliliters of a 0.2 M with sucrose, and then centrifuged at 17,500 x yeast suspension containing 1 x 109 to 5 x 10' g for 10 min. The supernatant was layered on a 36-ml cells per ml in TKM buffer were incubated at 30 C linear gradient of sucrose (0.3 to 1.0 M) in TKM with 6 ml of 1.6 M KCI containing 0.02 M MgSO., placed over 10 ml of a 2.0 M sucrose solution in 0.033 M mercaptoethanol, 0.01% heparin, and 1.0 ml TKM. The tubes were centrifuged at 25,000 rpm in of the lytic complex from Helix pomatia (L'Industrie the SW25.2 rotor for 140 min. The gradient was Biologique Francaise, Gennevilliers, France) (previ- collected introducing a needle down to the 2.0 M ously centrifuged at 40,000 x g for 20 min). Formation sucrose cushion and pumping up the material. Conof protoplasts was almost complete at the end of 90 to tinuous measurement of the absorbancy at 254 em 120 min as determined by their sensitivity to osmotic was achieved by flowing the sample through the cell shock with hypotonic buffer. Protoplasts were cen- of an LKB Uvicord recording photometer. Samples trifuged at 6,000 rpm for 10 min, washed once with were recovered with a fraction collector, precipitated 0.8 M KCI in TKM buffer, and resuspended in the with trichloroacetic acid, filtered, washed, dried, same hypertonic buffer. and counted as described above. Incorporation of radioactive mannose, glucose, Detachment ot labeled peptides from the or threonine by protoplasts. Protoplasts were in- ribosomes. Treatment of whole cells with puromycin cubated at 25 C in YM-1 medium in TKM buffer was not attempted since the strain was not sensitive made hypertonic with 0.8 M KCI. Variable amounts to puromycin. Crude lysates of protoplasts (see above) of the radioactive compounds were added. At selected were treated with 2 mM puromycin and 2 mM times samples were withdrawn and mixed with an guanosine 5'-triphosphate (GTP) for 40 min at 0 C. equal volume of 10% trichloroacetic acid. Samples Untreated controls were incubated for the same time were kept on ice and after not less than 1 h they were at 0 C. Then Triton X-100 was added to both samples filtered through GF/C Whatman glass fiber disks and ribosomes and polysomes were separated by (2.4 cm diameter), washed with 50 ml of 5% tri- sucrose density centrifugation and their radioactivity chloroacetic acid and dried in an oven at 80 C. was assayed as described above. Bulk-isolated riboRadioactivity was measured by placing the dried somes were treated with 1 or 2 mM puromycin and 2 disks in small vials containing 2 ml of toluene-based mM GTP; after an appropriate period of time at liquid scintillation fluid. The small vials were placed either 0 C or room temperature, 5 ml of pH 1.9 buffer into normal-sized vials and counted in a Packard (8.7% acetic acid and 2.5% formic acid [30]) were added and the samples were filtered through WhatTriCarb liquid scintillation spectrometer. Pulse-labeling of whole cells and protoplasts. man DE-81 diethylaminoethyl paper and treated as Whole cells were preincubated in hypertonic buffered described by Weber and De Moss (30). Detachrhent of YM-1 medium. After several minutes, the radioactive nascent polypeptide chains from isolated ribosomes compound was added and incubation was continued was also achieved by incubation with 0.2 M glycinefor 30 s to 2 min. Protein synthesis was stopped by sodium hydroxide buffer, pH 10.0, at 37 C for 1 to 3 h, addition of 5 volumes of ice cold TKM buffer con- and further treatment was as described for the putaining 100 pg of cycloheximide per ml. Cells were romycin reaction. Other methods. Protein was measured with the centrifuged and resuspended in the incubation mixture described above for the preparation of protoplasts Folin phenol reagent (15) and ribosomes as described plus cycloheximide and incubated further at 30 C to by Rich (24). For the detection of sugars, samples obtain protoplasts. For protoplasts, labeling was were mixed with carrier sugars, hydrolyzed with 2 N stopped by the addition of 5 volumes of ice cold 0.8 M HCl at 105 C for 2 h, neutralized with Dowex-2 resin KCI in TKM buffer containing 100 pig of cyclo- in the HCO,- form, and passed through a small heximide per ml. The protoplasts were centrifuged column of Dowex-50 in the H- form. Neutral sugars and washed once with hypertonic buffer containing eluted from this column were concentrated and chromatographed in paper for 36 h with ethyl acetatecycloheximide. Breakage of cells and protoplasts and obtention pyridine-water, 8:2:1 (vl/vol/vol). Sugars were visuof different subeellular fractions. Cells were broken alized in the chromatograms by spraying with amwith a Braun cell homogenizer for 30 s. Protoplasts monium-silver nitrate, whereas radioactive spots were were osmotically shocked with TKM buffer usually measured by cutting 1-cm sections from the chromacontaining 100 pg of cycloheximide per ml and 0.01% tography paper and counting as above. Hexosamines heparin. Lysates were fractionated by centrifugation and amino acids were eluted from the Dowex-50 colin discontinuous sucrose gradients by two differents umn with HCl, concentrated, and chromatographed methods. One of them was carried out essentially with the following solvent: ethyl acetate-pyridineas described by Melchers (18), except that no 2.0 M butanol-butyric acid-water, 10:10:5:1:5 (vol/vol). Rasucrose layer was placed at the bottom of the tubes dioactive spots were located as mentioned for neutral and the extracts were centrifuged in a Beckman sugars. SW25.2 rotor, and the other method used was that described by Lawford and Schachter (13). Bulk riboRESUILTS somes were isolated essentially as described by Munro Incorporation of mannose, glucose, and et al. (22), and polysomes by a slight modification of the method described by Preisler et al. (23). In threonine by protoplasts. Protoplasts incorpo-
SYNTHESIS OF MANNOPROTEINS IN S. CEREVISIAE
VOL. 124, 1975
rated radioactive [U- 14C Imannose and [LJl4 ]glucose into trichloroacetic acid-insoluble material (Fig. 1). Both mannose and glucose were found in the trichloroacetic acid-precipitable material independently of the sugar added to the incubation medium. The hexoses incorporated by the protoplasts were found associated mostly to the fractions which corresponded to those described as "smooth endoplasmic reticulum" by the methods of Melchers (18) and Lawford and Schachter (13). Almost no radioactivity appeared bound to the fraction corresponding to the "rough endoplasmic reticulum" in the above mentioned methods nor to ribosomes isolated as described by Munro et al. (22). This result appeared to be due to the poor biosynthetic capacities of protoplasts since ["IC ]threonine was incorporated at very low rates, Incorporation of [U- ICimannose by whole oeils. Growing cultures of Saccharomyces were labeled for 2 min with ["4C]mannose and cycloheximide was added to freeze the polysomes. It is known that this treatment stops the synthesis of mannose-acceptor proteins (25). When the cells were broken with a Braun cell homogenizer only a small amount of label appeared associated with the ribosomes obtained as described above. On the other hand when protoplasts were prepared from the labeled cells and
F _~~~~~~~~~~~~~~~~~ X
Ma U
then lysed, a significant amount of radioactivity Xappeared associated with the ribosomes. Ribosomal profiles from cells similarly labeled (see Materials and Methods) showed most of the radioactivity in the polysomal fraction (Fig. 2); monosomes contained much less radioactivity. The peculiar shape of the polysomal curve was attributed to "banding" of the heaviest polysomes over the 2 M sucrose cushion. Similar results were obtained when the labeling period was reduced to 30 s. Radioactive polysomes were precipitated with trichloroacetic acid, filtered, dried, and counted. Scintillation fluid was washed away with toluene and the samples present in the glass fiber disks were hydrolyzed and analyzed as described under Materials and Methods. The only radioactive components present were mannose, glucose, and glucosamine. Mannose accounted for more than 60% of the radioactivity, whereas glucosamine was present in the smaller amounts. Detachment of radioactive components from the ribosomal fractions. Incubation of bulk-isolated ribosomes (22) with puromycin and GTP caused the release of 20 to 30% of the radioactivity (Table 1). Also incubation of ribosomes with pH 10 buffer brought about the release of 50% of the radioactivity from the ribosomal material (Table 1). These low yields may be due to extensive polysomal breakage during handling, and that conditions for pu-
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129
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FIG, 1. Incorporation of [U-_"4Cglucose and [U"4C]mannose by protoplasts of Saccharomyces cerevisiae. Protoplasts (6 x 108 per ml) were shaken in the presence of radioactive sugars. Total volume was 5 ml. At intervals 1-mI aliquots were withdrawn and received over 1 ml of ice-cold 10% trichloroacetic acid. Samples were left on ice for 2 h, filtered, washed, dried, and counted. (a) Protoplasts incubated with [U-"4CJglucose (1.25 mg and 0.1 uCi per ml). (b) Protoplasts incubated with [U-14Clmannose (1.25 mg and 0.1 jCi/ml). Symbols: (0) Protoplasts incubated in 0.8 M KCI alone; (0) protoplasts incubated in 0.8 MKCI in YM-1 medium.
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FIG. 2. Ribosomal pattern of cells labeled with [U-"1C]mannose. Yeasts (2.5 x 1010 cells) resuspended in a final volume of 4 ml of YM-1 medium buffered with TKM were incubated with shaking at 25 C for 15 min and labeled with [U-14C]mannose (0.6 mg and 1 gCi/ml) for 2 min. Protoplasts were prepared, lysed, and centrifuged as described. The supernatant (6 ml) was divided into two aliquots of 1 and 5 ml, respectively. Both aliquots were placed over sucrose gradients and centrifuged and treated as described in the text. Symbols: (0) data of absorbancy calculated from the Uvicord tracing of the 1-ml sample; (0) radioactivity data of the 5-ml sample.
130
RUIZ-HERRERA AND SENTANDREU
romycin reaction were not the most adequate because (i) puromycin added to bulk-isolated ribosomes labeled with [14C]threonine released a similar proportion of radioactive peptides than those in Table 1; and (ii) puromycin reaction carried out with crude lysates released more than 50% of the radioactivity (see below). Also, by incubation of a mannose-labeled polysomal fraction isolated from a sucrose gradient for 2 h at 38 C in glycine-sodium hydroxide buffer, pH 10, 80% of the radioactivity was released. In further experiments whole cells were labeled with ['IC Imannose and protoplasts were prepared and lysed as described. Crude lysates were incubated with puromycin and GTP either in the presence or in the absence of cycloheximide. Puromycin caused the release of radioactivity and the disaggregation of the polysomes. The effect was more pronounced when no cycloheximide was present during the puromycin treatment (Fig. 3). Data calculated from Fig. 3 showed that polysomes (fractions 1 to 13) lost 55% radioactivity and 49% of the absorbancy at 254 nm by treatment with puromycin. When the polysomal fraction isolated from a sucrose gradient was washed and incubated for 2 h at 38 C in glycine-sodium hydroxide buffer, pH 10, 89% of the radioactivity was released as measured by the method of Weber and De Moss (30). Effect of 2-deoxy-D-glucose on mannose incorporation. Mannose incorporation into mannan by whole cells was inhibited by 2-deoxy-D-glucose. We could not detect any incorporation of [1-_ H]-2-deoxy-glucose (50 jgCi) into polysomes, membranes, or cell walls of S. cerevisiae even though it was transformed into an unidentified component (2-deoxy-Dglucose 6-phosphate?). TABLE 1. Release of radioactivity from ribosomes by puromycin and alkaline pH
Expta 1 2 3
Treatment
None
Counts/min ribosomes in % Released
Puromycin
206 164
21
None Puromycin
218 157
28
None pH 10
126 64
49
aFor each experiment a different batch of ribosomes was used. 0.3 ml of ribosomes (0.22 to 0.4 mg) were incubated for 40 min at room temperature with and without 1 mM puromycin and 2 mM GTP, or for 1 h at 37 C with and without 0.1 M glycine-sodium
hydroxide buffer.
J. BACTERIOL.
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FIG. 3. Release of labeled peptides from polysomes by puromycin. Yeasts (4.8 x 1010 cells) were labeled as described for Fig. 2. Protoplasts were prepared in the presence of cycloheximide, centrifuged, and resuspended in hypotonic buffer without cycloheximide. The lysate was divided into two equal samples of 3.0 ml, one received puromycin and GTP, and both were incubated at 0 C for 30 min. Samples were processed as described in Materials and Methods. Upper graph: radioactivity. Lower graph: Absorbancy at 254 nm. Symbols: (0) control; (0) sample treated with puromycin.
Protoplasts were labeled with [U- I4C Imannose for 3 min and 1 aliquot was removed and treated with cycloheximide. The remaining was divided into two samples and one was treated with 2-deoxy-D-glucose (125 gg/ml). Both samples were further incubated for 6 min. Protoplasts were lysed and fractionated as described by Lawford and Schachter (13). 2-Deoxy-Dglucose inhibited the incorporation of [U14C]mannose in the ribosomal fraction but did not affect the incorporation into the membrane fractions (Table 2). Whole cells were preincubated with and without 2-deoxy-D-glucose for 10 min and then labeled for 2 min with [U- "C]mannose. The incubation was stopped as described and the cells were centrifuged and washed. Aliquots were withdrawn and precipitated with 10% trichloroacetic acid to measure total incorporation of [U- 14C ]mannose.
VOL. 124, 1975
SYNTHESIS OF MANNOPROTEINS IN S. CEREVISIAE
131
TABLE 2. Effect of 2-deoxy-D-glucose on the incorporation of mannose into several cellular fractions of yeast protoplastsa Radioactivity (counts/min) Fraction
Soluble Membranes Ribosomes
Control (3 min)
484 2,609 132
Control (9 min)
2-deoxy-Dglucose
875 5,396 327
1,176 5,742 123
a Protoplasts (1.2 x 109 per ml) were labeled for 3 min with 0.5 yCi of [U- 14C ]mannose (specific activity 2.7 mCi per mmol) and treated as described in the text.
Cells were incubated with snail enzyme to obtain protoplasts, centrifuged, washed, and lysed, all in the presence of cycloheximide. Aliquots of lysates were removed to measure radioactivity precipitable with trichloroacetic acid protein. Lysates were treated with Triton X-100 and centrifuged in a linear sucrose gradient as described for the separation of polysomes. 2Deoxy-D-glucose inhibited incorporation of mannose into polysomes (Fig. 4). Comparison of the data of whole cells, protoplast lysates, and ribosomes indicated that incorporation of mannose into the cell wall, but not in membrane fractions, was also inhibited (Table 3). DISCUSSION It is generally accepted that animal glycoproteins are synthesized by the stepwise addition of sugar residues to the polypeptide chains. This occurs during passage from the rough endoplasmic reticulum to the smooth endoplasmic reticulum and the Golgi apparatus (16). However the mechanism of the initial steps of glycosylation is still subject to debate. Some authors have presented evidences that the primary glycosylation occurs while the polypeptides are still bound to the polyribosomes (13, 20, 21, 29). Heretofore no data were available on the mechanism of the initiation of glycoprotein synthesis in microbial eukaryotes. We present evidences that the initial glycosylation of mannan from S. cerevisiae occurs at the ribosomal level. Firstly, radioactive sugars appeared bound to polysomes when cells were labeled with [U- 14C ]mannose for 30 s to 2 min. The low amount of radioactivity associated with manosomes, which were more abundant than polysomes, argues against the possibility that sugars were merely adsorbed on to the ribosomes. Furthermore, radioactivity bound to polysomes was detached by treatment with puromycin; this is
00
Q
oo 200
0
:3
S
9
12
15
18
21
24
30
27
FRACTION
FIG. 4. Effect of 2-deoxy-D-glucose on the incorporation of [U-14C]mannose into polysomes. Yeasts (2.7 101° cells) were resuspended in 5 ml of YM-1 medium in TKM and then two 2.5-ml aliquots were separated. One received I mg of 2-deoxy-D-glucose per ml. After 10 min of preincubation at 25 C both samples received [U-14CJmannose (1.0 mg and 0.8 gCi per ml each) and were incubated for 2 min. Protoplasts were prepared, lysed and fractionated as described. Symbols: (0) control; (0) sample incubated with 2-deoxy-D-glucose. x
TABLE 3. Effect of 2-deoxy-D-glucose on the incorporation of mannose by intact yeastsa Radioactivity (counts/min)
% Fraction... Fraction 2-Deoxy-D- Inhibition Control glucose
Whole cells Lysed protoplasts Polysomes Monosomes Membrane and soluble Whole gradient
64,360 19,626 4,001 1,126 8,999
40,960 17,940 2,931 895 9,413
36.4 8.6 26.8 20.6 None
14,126
13,239
6.3
a Conditions
of the experiment described in the text and in the legend for Fig. 4. Data are corrected for protein content in the fractions of both samples (the difference was less than 6%). Polysomes are fractions 1 to 14, monosomes: 15 to 21 and membrane and soluble fractions: 22 to 31.
known to release nascent polypeptide chains serving as polypeptide acceptor. Release of radioactive peptides in our experiments was higher when incubation was carried out in the absence of cycloheximide, and under the same conditions a larger destruction of polysomes occurred. Puromycin is known to destroy polysome integrity. Besides mannose we detected the presence of glucose bound to polysomes, a result indicative that there are glucose-containing proteins in Saccaromyces. It is possible that mannoproteins contain a small amount of glucose buried into the mannan tufts as has been reported by Falcone and Nickerson (7) or that this glucose might represent the bridges be-
132
RUIZ-HERRERA AND SENTANDREU
tween the two main polymers of the wall (glucan and mannoproteins). An alternative (and attractive) hypothesis would be that this glycosylation represents the first step in the synthesis of yeast glucan. Further experiments are necessary to discern between these alternatives. It is known that the presence of hexosamine constitutes the link of the mannan to the protein components (1). Biosynthesis of glycoproteins in animals cells (9), yeasts (8, 11, 14), and fungi (17; J. Ruiz-Herrera, unpublished data) is very sensitive to 2-deoxy-D-glucose. Biely et al. (3, 4) have described that 2-deoxy-D-glucose is incorporated into mannan, but its incorporation by different species is not related to their corresponding susceptibility to the compound, and since it only affects mannan branching, this effect seems to be a later one and not the primary cause of inhibition of glycoprotein biosynthesis. We could not detect incorporation of the analogue by the strain used. 2-Deoxy-Dglucose is known to inhibit phosphoglucose isomerase (31) and Kuo and Lampen (11) found that it also inhibits phosphomannose isomerase. According to these authors availability of mannose to the transglycosidases is thus prevented. Since we used mannose as starting material, the inhibition we found suggests that there is a further and more specific step blocked. We found that 2-deoxy-D-glucose inhibited glycosylation of polysome-bound polypeptides. These results are in agreement with Melchers (19) who described that 2-deoxy-D-glucose inhibited the migration of immunoglobulin Gl (produced by the plasma cell tumor MOPC 21 transplanted to mice) from the membranebound polyribosomes to the cisternae of the rough endoplasmic reticulum. Marzluf (17) described that regeneration of invertase in Neurospora required de novo synthesis of proteins and that the process was completely inhibited by 2-deoxy-D-glucose. We found that 2-deoxy-D-glucose also inhibited incorporation of mannose into the cell wall, but had no effect on the synthesis of membranebound glycoproteins. This suggests that secreted and membrane-bound glycoproteins are synthesized by a different mechanism. Bretscher (5) has sustained the same hypothesis. Preliminary results from Forni and Melchers (19) also point in the same direction. Altogether, our results agree with Eylar's hypothesis (6) that glycosylation is necessary for protein secretion in eukaryotes. As a speculation it is possible to think that glycosylation of the nascent polypeptides permits them to transverse the membrane of the rough endoplasmic reticu-
J. BACtERIDL.
lum, or that glycosylation of the growing protein chain in the lumen side of the cisternae of the endoplasmic reticulum is the mechanism which pulls the polypeptide through the membrane or avoids its recoil into the cytoplasmic space. ACKNOWLEDGMENTS The stay of J. Ruiz-Herrera (Professor Fellow from the Comisi6n de Operaci6n y Fomento de las Actividades Academicas, M6xico) at the University of Salamanca was made possible through funds from grant no. 020 of the Consejo Nacional de Ciencia y Tecnologia, M6xico, and financial help from the Instituto de Cultura Hispanica, Spain. J.R.H. is indebted to the facilities given by Julio R. Villanueva during his stay at the University of Salamanca. We are grateful to Professors Julio R. Villanueva and S. Bartnicki-Garcria for critical reading of the manuscript. LITERATURE CITED 1. Ballou, C. E., and W. C. Raschke. 1974. Polymorphism of the somatic antigen of yeast. Science 184:127-134. 2. Behrens, N. H., and E. Cabib. 1968. The biosynthesis of mannan in Saccharomyces carlsbergensis. J. Biol. Chem. 243:502-509. 3. Biely, P., S. Kratky, and S. Bauer. 1972. Metabolism of 2-deoxy-D-glucose by bakers yeasts. IV. Incorporation of 2-deoxy-D-glucose into cell-wall mannan. Biochim. Biophys. Acta 255:631-639. 4. Biely, P., Z. Kratky, J. Rovarik, and S. Bauer. 1971. Effect of 2-deoxy-D-glucose on cell wall formation in Saccharomyces cerevisiae and its relation to cell growth inhibition. J. Bacteriol. 107:121-129. 5. Bretscher, M. S. 1973. Membrane structure: some general principles. Science 181:622-629. 6. Eylar, E. H. 1966. On the biological role of glycoproteins. J. Theor. Biol. 10:89-113. 7. Falcone, G., and W. J. Nickerson. 1956. Cell-wall mannan-protein of bakers' yeast. Science 124:272-273. 8. Farkas, V., A. Svoboda, and S. Bauer. 1970. Secretion of cell-wall glycoproteins by yeast protoplasts. Effect of 2-deoxy-D-glucose and cycloheximide. Biochem. J. 118:755-758. 9. Gandhi, S. S., P. Stanly, J. M. Taylor, and D. 0. White. 1972. Inhibition of influenza glycoprotein synthesis by sugars. Microbios 5:41-50. 10. Jung, P., and W. Tanner. 1973. Identification of the lipid intermediate in yeast mannan biosynthesis. Eur. J. Biochem. 37:1-6. 11. Kuo, S. C., and J. 0. Lampen. 1972. Inhibition by 2-deoxy-D-glucose of synthesis of glycoprotein enzymes by protoplasts of Saccharomyces: relation to inhibition of sugar uptake and metabolism. J. Bacteriol. 111:419429. 12. Lampen, J. 0. 1968. External enzymes of yeast. Their nature and formation. Antonie van Leeuwenhoek J. Microbiol. Serol. 34:1-18. 13. Lawford, G. R., and H. Schachter. 1966. Biosynthesis of glycoprotein by liver. The incorporation "in vivo" of "C-glucosamine into protein-bound hexosamine and sialic acid of rat liver subcellular fractions. J. Biol. Chem. 241:5408-5418. 14. Liras, P., and S. Gascon. 1971. Biosynthesis and secretion of yeast invertase. Effect of cycloheximide and 2-deoxy-D-glucose. Eur. J. Biochem. 23:160-165. 15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 198:265-275.
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SYNTHESIS OF MANNOPROTEINS IN S. CEREVISIAE
16. Marshall, R. D. 1972. Glycoproteins. Annu. Rev. Biochem. 41:673-702. 17. Marzluf, G. A. 1973. Regeneration of invertase in Neurospora crassa. J. Bacteriol. 115:146-152. 18. Melchers, F. 1971. Biosynthesis of the carbohydrate portion of immunoglobulin. Radiochemical and chemical analysis of the carbohydrate moieties of 2 myeloma proteins purified from different subcellular fractions of plasma cells. Biochemistry 10:653-659. 19. Melchers, F. 1973. Biosynthesis, intracellular transport and secretion of immunoglobulins. Effect of 2-deoxy-Dglucose in tumor-plasma cells producing and secreting immunoglobulin Gl. Biochemistry 12:1471-1476. 20. Molnar, J., and D. Sy. 1967. Attachment of glucosamine to protein at the ribosomal site of rat liver. Biochemistry 6:1941-1947. 21. Molnar, J., G. B. Robinson, and R. Winzler. 1965. Biosynthesis of glycoproteins. IV. The subcellular site of incorporation of glucosamine-1-"IC into glycoprotein in rat liver. J. Biol. Chem. 240:1882-1888. 22. Munro, A. J., R. J. Jackson, and A. Korner. 1964. Studies on the nature of polysomes. Biochem. J. 92:289-299. 23. Preisler, H. D., W. Scher, and C. Friend. 1973. Polyribosome profiles and polyribosome-associated RNA in Friend leukemia cells following DMSO-induced differentiation. Differentiation 1:27-37. 24. Rich, A. 1967. Preparation of polysomes from mammalian reticulocytes, lymph nodes, and cells grown in tissue
25. 26.
27.
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