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Effect of tunicamycin on the glycosylation of rhodopsin
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 201, No. 2, May, pp. 527-532, 1980
Effect of Tunicamycin JAMES
J. PLANTNER,
The Lorand V. Johnson Ophthalmology,
on the Glycosylation LOUIS
PONCZ,2
AND
of Rhodopsin
EDWARD
L. KEAN3
Laboratory for Research in Ophthalmology, Department of Surgery, Division and the Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106 Received
January
of
16, 1980
The incorporation of ~H]glucosamine, [3H]mannose, and [“%]methionine into rhodopsin was investigated in retinas which had been incubated in the presence and absence of the antibiotic, tunicamycin. In its presence, the incorporation of glucosamine was inhibited 70% and mannose, 96% compared to controls. In the presence of tunicamycin the attachment of glucosamine to core-region sites was virtually eliminated. The formation of unglycosylated rhodopsin was also indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concanavalin A-Sepharose chromatography. These findings are consistent with the participation of the lipid-linked pathway in the glycosylation of this wellcharacterized intrinsic glycoprotein of the membranes of the disk of the rod outer segment. As indicated by the incorporation of [%]methionine, the synthesis of rhodopsin apoprotein was inhibited by a much lesser amount. This suggests that the glycosylation of rhodopsin is not required for its insertion into the disk membrane.
The antibiotic tunicamycin inhibits the formation of N-acetylglucosaminyl-pyrophosphoryldolichol, thus blocking the process of core-region glycosylation of the asparagine-linked class of glycoproteins by preventing the assembly of the polyprenololigosaccharide precursor (1). In addition to its extensive use in cell-free studies, treatment of intact animal cells with tunicamycin has resulted in the formation of unglycosylated molecules by blocking de nova glycosylation (2-6). This has provided a means of examining the influence of the carbohydrate components of glycoproteins on transport, secretion, and biological function of these heteropolymers. The visual pigment, rhodopsin, is a glycoprotein containing 9 mol of mannose and 5 mol of glucosamine/mol (7). It has recently been shown (8, 9) that the oligosaccharide ’ This work was supported in part by Public Health Service Research Grant EY 00393 from the National Eye Institute, and by the Ohio Lions Eye Research Foundation. 2 Supported by National Research Service Award T32 EY 07006. 3 To whom reprint requests should be addressed at the Laboratory for Eye Research, Room 653 Wearn Research Building, Case Western Reserve University, Cleveland, Ohio 44106.
chains of rhodopsin are rather unique, being hybrids of the high mannose and complex types of asparagine-linked glycoproteins, in addition to containing a single nonreducing terminal N-acetylglucosamine residue. Rhodopsin has been observed exclusively as a membranous component, being present primarily in the membranes of the disks of the outer segment of the rod cell, and to a lesser extent in the plasma membrane (10). Rhodopsin is synthesized in the inner segment of the rod cell and is transported to the base of the outer segment where it is incorporated into the membrane of the disk by an invagination of the plasma membrane. It has been estimated to account for up to 80% of the protein of the disk membrane (11). We are thus dealing with a system which involves the process of biosynthesis, transport, and assembly of a major membranous constituent. MATERIALS
Tissue Preparation;
AND
METHODS
Incubation
Conditions
All procedures were performed in dim red light, except where indicated. Retinas were dissected from fresh bovine eyes, obtained from a local slaughterhouse, within 2 h after death of the animal. Incuba527
0003-9861/80/060527-06$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
528
PLANTNER,
PONCZ, AND KEAN
tions were performed at 37°C in Krebs-Ringer bicarbonate buffer containing casamino acids (DIFCO, Detroit, Mich.), 0.01 mg/ml; penicillin, 35 units/mI; streptomycin, 35 pg/ml (Gibco, Grand Island, N. Y.); glycerol, 0.14 M, as a carbon source (12, 13); and llc&retinal, 0.03 mM, added in ethanol (0.002 ml/ml medium). During dissection the retinas were maintained in the complete incubation medium, on ice, through which was bubbled a gas mixture of 95% OJ5% CO*. Individual retinas were bissected and one half was used for control incubations and the other half for incubations performed in the presence of the antibiotic, using a total of 10 retinas for each condition. The equivalent of 2 retinas per incubation flask containing 9 ml of incubation medium was preincubated for 1 h in the absence or presence of tunicamycin (0.02 mg/ml). Tunicamycin was dissolved in dimethylsulfoxide and then dispersed in the incubation medium. The same concentration of dimethylsulfoxide (0.014 M) was present in control incubations as well. The flasks were gassed with a mixture of 95% 02/5% CO* at a flow rate of 500 ml/min/flask. After preincubation, the radioactive compounds were added, dissolved in 1 ml of Krebs-Ringer medium. The following were the radioactive compounds which were used and their final concentrations: [6-3H(N)]glucosamine (18 Ci/ mmol), 0.014 mCi/ml; [2JH]mannose (17 Ci/mmol), 0.016 mCi/ml; [35S]methionine (540-930 Ciimmol), 0.01-0.02 mCi/ml. All were purchased from New England Nuclear Corporation. The incubation mixtures contained either [3H]glucosamine or [SHImannose. Doubly labeled experiments also contained [35S]methionine. After the addition of the radioactive compounds, the incubations were continued for an additional 3 h.
Isolation and Purijieation
of Rhodopsin
After incubation, the retinas were recovered by centrifugation and washed twice by gentle resuspension in buffer. Rod outer segments were isolated and purified by flotation in dense sucrose, followed by purification using sucrose density centrifugation (14). Rhodopsin was extracted from the purified rod outer segments with 1% Emulphogene BC 720 (GAF Corp., New York) in 0.05 M Tris buffer, pH 7.0, as described previously (7). Rhodopsin was purified from the detergent extract by adsorption chromatography on columns of calcium phosphate-Celite (7) followed by preparative isoelectric focusing as described previously (15). Electrofocusing was performed on an LKB 110 ml Ampholine column over the pH range 5 to 7 stabilized by a sucrose gradient. The focusing column also contained 0.3% Emulphogene BC 720. Focusing was carried out at 4°C in the dark for 2 days. In these experiments, 2 mM DTT4 was present during the 4 Abbreviations used: ROS, rod outer segments; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; Con
extraction and chromatographic procedures. As described previously, three major isoelectric forms have been detected in bovine rhodopsin (15). When the isolation was performed in the presence of DTT, this multiplicity was prevented from being expressed5 and a single form dominated, thus simplifying the analyses. Con A-Sepharose chromatography was performed as described previously (7) on purified rhodopsin, recovered from the focusing column, after dialysis and concentration.
Enzyme Treatments Pronuse. Purified rhodopsin, recovered from the focusing column, after dialysis and concentration, was digested with Pronase (Calbiochem, LaJolla, Calif.) for 3 days in 0.15 M Tris buffer, pH 8.0, containing 0.1 M CaCl, under a toluene atmosphere (16). Three additions of 0.02 mg Pronase each were made over this period. The digest was then placed over a Bio-Gel P-4 column (1.5 x 80-cm), and the products were eluted with 0.1 N acetic acid. fi-Hexosaminidase. The peptide fraction from the Pronase digest was lyophilized, redissolved in 0.1 M citrate-phosphate buffer, pH 4.5, and incubated with 4.8 units of ezo-P-hexosaminidase (BoehringerMannheim, New York) for 16 h, as described by Struck and Lennarz (2). After digestion, the products were placed over a Bio-Gel P-4 column and the products eluted with 0.1 N acetic acid.
Analytical
Procedures
Radioactivity was determined by scintillation spectrometry using No. 963 counting solution obtained from New England Nuclear Corporation. In doubly labeled experiments, “H was counted with an efficiency of about 15%, and 35S, about 55%. The concentration of rhodopsin was calculated using an extinction coefficient of 40,600 at 498 nm (17). Strong acid hydrolysis was carried out at 100°C in sealed tubes using 3 N HCl for 4 h. After removal of the acid, the hydrolysate was re-l\i-aeetylated (18) and passed over columns of linked cation and anion exchange resins. The water eluate from these columns was examined by descending paper chromatography on Whatmann 3MM paper using as solvent system, 1-butanol:pyridine:water (6:4:3), and by high voltage paper electrophoresis in 1% sodium tetraborate buffer, as described previously (19). Carbohydrates were visualized by the alkaline silver nitrate procedure (20). Radioactivity on paper after chromatography and electrophoresis was measured by scintillation specA, concanavalin A; GlcNAc, Ir;-acetylglucosamine; HAc, acetic acid. J Plantner and Kean, manuscript in preparation.
TUNICAMYCIN
EFFECT
ON RHODOPSIN
GLYCOSYLATION
529
&Y- A.98 0 30 Man (-1)
0 25 t
i
0 20
,’
G,CNAC (+T)
/
:, /
0 I5 0 IO
GlcNAc
t-T)
‘4 ;‘.
,J v..
005
.i
o35m.-A, 0 00 55
---
”
=,.:...
10.000
-
8000
-4000
4=-.-.. . *.. +.p&.&;
60
12.000
-
ELECTROFOCUSING
-
.
,
65
0 Y . 3
- 6000
: Man UtT)
-
2000 0
r 5000 - 4000 - 3000
2 d,
- 2000
1 :: --0
1000
0 3_ 3 2 & 5.
70 (PHI
FIG. 1. Electrofocusing patterns of rhodopsin obtained from retinas incubated in the absence and presence of tunicamycin. Bovine retinas were incubated with [6-3H]GlcNHZ or [2-3H]mannose, and rhodopsin was isolated as indicated under Materials and Methods. After chromatography on columns of calcium phosphate-Celite, the rhodopsin-containing fractions were subjected to isoelectric focusing on sucrose-density stabilized columns. The dashed lines refer to the retinas incubated in the absence of tunicamycin, and the dotted lines refer to incubations performed in the presence of tunicamycin (0.018-0.02 mgiml). The solid line refers to the A 49Hnmwhich was the same under all circumstances. trometry after sectioning into l-cm strips. SDSpolyacrylamide gel electrophoresis analysis (as described previously (7)) was performed on the dialyzed, concentrated product after electrofocusing. Due to the low specific activity it was necessary to analyze relatively large amounts on multiple gels. Approximately 1 nmol of rhodopsin was applied to each of eight gels. After electrophoresis the gels were stained with Coomassie blue and scanned at 600 nm using a Varian Model 635 spectrophotometer gel scanner attachment (Varian, Sunnydale, Calif.). The gels were sliced into l.l-mm sections with a Bio-Rad gel slicer (Richmond, Calif.), and corresponding slices were pooled as indicated by reference to the stained bands. The gels were leached with 1 ml of Unisol (Isolab, Akron, Ohio) at room temperature for 1 day, after which the radioactivity in the extract was measured as described previously (21). Greater than 80% of the radioactivity applied to the gels was recovered. In addition to measuring radioactivity, the A 600nm of the scintillation fluid extract of the gels was obtained. This was then correlated to the patterns of Coomassie staining from the individual gel scans. RESULTS
Mannose and Glucosamine Labeling
As can be seen in Fig. 1, the labeling patterns of [3H]mannose and [3H]glucosamine after electrofocusing were identical to that of the 498nm absorption of rhodopsin. After strong acid hydrolysis and re-Nacetylation greater than 80% of the radioactivity was recovered after mixed bed resin
Paper chromatography and treatment. paper electrophoresis revealed that the label was due only to these sugars. The A 27x.,/ A 490,,,,, ratio in all of these studies was less than 2, indicative of rhodopsin in a relatively high state of purity. The isoelectric point at the peak was about pH 6.1 for both the control and rhodopsin isolated from retinas incubated in the presence of tunicamycin. Similar amounts of rhodopsin were obtained under both conditions and similar amounts were analyzed. As is evident from Fig. 1, the presence of tunicamycin in the incubation medium resulted in extensive inhibition of the incorporation of glucosamine into rhodopsin, and almost complete suppression of the incorporation of mannose. The 35S pattern (not shown) was identical to that of AJSR. Shown in Table I are the specific activities of glucosamine and mannose in the pool of rhodopsin recovered after electrofocusing and dialysis. The range in specific activities in three experiments using glucosamine was less than 7%, and in two experiments using mannose, less than 10%. Sites of Glycosylation
Since there was still some labeling with glucosamine in the presence of tunicamycin, it was of interest to examine the sites of the attachment of this amino sugar
530
PLANTNER. TABLE
EFFECT
PONCZ,
I
OF TUNICAMYCIN ON THE GLYCOSYLATION OF RHODOPSIN" cpm x IO-” (Km01 rhodopsin)
Substrate
Control
[“H]GlcNH, l”H]Mannose
935 468
Tunicamycin 299 19
Inhibition (8) 68 96
’ The data refer to rhodopsin from retinas which had been incubated in the absence and presence of tunicamycin and purified by calcium phosphate-Celite chromatography and preparative isoelectric focusing. The specific activity was calculated from the radioactivity in the pool of rhodopsin from the focusing column (pH 5.9 to 6.3, Fig. 1) using either tritiated mannose or GlcNH,, and the concentration of rhodopsin calculated from the differential optical density at 498 nm obtained before and after photobleaching.
in rhodopsin. Peptides were generated from rhodopsin by extensive Pronase treatment, as described under Materials and Methods. The peptide pool was then treated with exo-hexosaminidase. Peripherally exposed residues would be cleaved by this treatment while more internally located core residues would be resistant. In Fig. 2 are results of an experiment of this type. In the upper panel, dealing with rhodopsin isolated from control retinas, in addition to the release of GlcNAc from the Pronasegenerated peptides, considerable 3H-labeled material remained associated with the larger molecular weight peptide region. In contrast, virtually all of the tritium in the peptide derived from the tunicamycintreated retinas (lower panel) was converted to material eluting from the gel in a manner similar to GlcNAc. From experiments of this type, the incorporation of [“Hlglucosamine into control rhodopsin was shown to be about 60% into peripheral sites and 40% more internally located, i.e., resistant to cleavage by hexosaminidase. In rhodopsin from retinas incubated in the presence of tunicamycin, virtually all of the incorporated glucosamine (90 to 98% in different experiments) was located peripherally. Since internal or core-region [3H]GlcNAc would
AND
KEAN
result from newly synthesized carbohydrate chains, the relatively small amount of glucosamine which was incorporated into rhodopsin in the presence of tunicamycin would reflect its peripheral addition to sites already available on preformed molecules. Methionine
Labeling
While the incorporation of mannose and glucosamine into rhodopsin isolated from the ROS membrane was extensively inhibited by tunicamycin, the labeling of this glycoprotein by methionine was much less affected. Thus, in different experiments where tunicamycin blocked the incorporation of ]3H]glucosamine into rhodopsin 60 to 70%, labeling by [35S]methionine was inhibited only 8 to 30%. SDS-Electrophoresis;
Con A-Sepharose
The analysis by SDS-electrophoresis of rhodopsin doubly labled with [35S]methionine
FIG. 2. Elution pattern from Bio-Gel P-4 after Pronase and hexosaminidase treatment of rhodopsin. The rhodopsin-containing fraction recovered after electrofocusing (pH 5.9 to 6.3, Fig. 1) was dialyzed, concentrated, and incubated extensively with Pronase, after which the products were examined by gel filtration on Bio-Gel P-4. The fractions included within the bars were pooled and incubated with ezo-/3-hexosaminidase, and reapplied to the column. As with the pronase digests, elution was carried out with 0.1 N HAc. The arrows refer to the elution positions of blue dextran (left) and GlcNAc (right). (-) Pronase treated; (---) hexosaminidase treated.
TUNICAMYCIN
EFFECT
ON RHODOPSIN
and [3H]glucosamine purified from retinas incubated in the presence of tunicamycin is seen in Fig. 3. As indicated above, limited labeling with glucosamine of endogenous rhodopsin was obtained in the presence of the antibiotic. The Coomassie pattern obtained from rhodopsin present in the preparation (serving as an internal standard) coincided with the 3H from glucosamine. The major peaks correspond to monomer and dimer forms of rhodopsin. On the other hand, the 35S-labeled product, reflecting newly formed aporhodopsin, migrated more rapidly, as would be expected for an unglycosylated species. The difference in molecular weights between the 35S and “H products of about 2500 would be consistent with this suggestion. A further indication that unglycosylated species were formed in the presence of tunicamycin was affinity chromatography on Con A-Sepharose of the products recovered after electrofocusing. Rhodopsin synthesized by control retinas labeled with [“YS]methionine and either [3H]mannose or [“Hlglucosamine was retained by the lectin (89 to 93%) and was released by cu-methylmannoside. In contrast, while 89% of the small amount of the 3H label in the rhodopsin formed in the presence of tunicamycin was bound by Con A-Sepharose, 54% of the 3sS was not retained by the lectin and had little or no 3H labeling, in agreement with the results from SDS-electrophoresis. DISCUSSION
In previous studies tunicamycin was shown to inhibit the incorporation of glucosamine into core-region saccharides in cellfree preparations of the retina (22), while it had no effect on the synthesis of dolichol phosphate-mannose. This is similar to observations made with other tissues (1, 2). The present studies demonstrated the extensive inhibition of glycosylation of rhodopsin by tunicamycin using the intact retina, in vitro. Thus, not only is the metabolic apparatus of the dolichol pathway present in the retina (21-25), but it is involved in the glycosylation of rhodopsin. Although the enzymes of this pathway have been shown in a variety of tissues to cata-
GLYCOSYLATION
531
FIG. 3. SDS-electrophoresis of rhodopsin from tunicamycin-treated retinas. Rhodopsin was purified by chromatography and isoelectric focusing as described under Materials and Methods. Approximately 1 nmol of purified rhodopsin containing 500 cpm of :lH and of “% was applied to each of eight gels. Electrophoresis was carried out at 8 mA per gel. After staining with Coomassie blue and scanning, the entire gels were sectioned and corresponding slices were pooled by reference to the stained bands. The radioactivity was determined and the A 600 nm of the counting solution extract was measured. The latter values were matched with the gel scans, a representative sample of which is shown in the Figure, (---); net cpmislice-pool: 35s (---A--); RH (- - n - -).
lyze the glycosylation of uncharacterized endogenous membrane proteins, this is the first demonstration of the participation of the lipid-linked pathway in the glycosylation of a specific, well-characterized intrinsic membranous glycoprotein in animal tissues. The labeling of rhodopsin by P4S]methionine reflected newly synthesized rhodopsin-protein. While tunicamycin almost completely blocked the glycosylation of these newly formed molecules by inhibiting core-region glycosylation, the incorporation of 35Sinto rhodopsin was affected relatively little. Since the rhodopsin was isolated from purified rod outer segments, it can be inferred that glycosylation was not required for the transport of this molecule from the site of its synthesis in the inner segment to the outer segment, nor for the insertion of aporhodopsin into the disk membranes of the outer segment of bovine retinal rods. Whether these molecules are
532
PLANTNER,
PONCZ,
present in the disk membrane in a correct orientation or are biologically active cannot be evaluated from the6 stu&es. ACKNOWLEDGMENTS The authors express their Robert L. Hamill and the Eli tories, Indianapolis, Indiana, of tunicamycin. We are grateful Nutley, New Jersey, for their
appreciation to Dr. Lilly Research Laborafor their generous gift to Hoffman-LaRoche, gift of II-&-retinal.
REFERENCES 1. TKACZ, J. S., AND LAMPEN, J. 0. (1975) Biochem. Biophys. Res. Commun. 65, 248257. 2. STRUCK, D. K., AND LENNARZ, W. J. (1977) J. Biol. Chem. 252, 1007-1013. 3. OLDEN, K., PRATT, R. M., AND YAMADA, K. M. (1978) Cell 13, 461-473. 4. HICKMAN, S., KULCZYCKI, A., LYNCH, R. G., AND KORNFELD, S. (1977) J. Biol. Chem. 252, 4402-4408. 5. DAMSKY, C. H., LEVY-BENSHIMOL, A., BUCK, C. A., AND WARREN, L. (1979) Exp. Cell Res. 119, 1-13. 6. STRUCK, D. K., SUITA, P. B., LANE, M. D., AND LENNARZ, W. J. (1978) J. Biol. Chem. 253, 5332-5337. 7. PLANTNER, J. J. AND KEAN, E. L. (1976) J. Biol. Chem. 251, 1548-1552. K., MUELLEN8. LIANG, C. -J., YAMASHITA, BERG, C. G., SHICHI, H., AND KOBATA, A. (1979) J. Biol. Chem. 254, 6414-6418.
AND
KEAN
9. FUKUDA, M. N., PAPERMASTER, D. S., AND HARGRAVE, P. A. (19’79) J. Biol. Chem. 254, 8201-8207. 10. DAEMAN, F. J. M. (19’73) Biochim. Biophys. Acta 300, 255-288. 11. HEITZMAN, H. (1972) Nature New Biol. 235, 114. 12. O’BRIEN, P. J. (1977) Exp. Eye Res. 24, 449-458. 13. O’BRIEN, P. J., AND MUELLENBERG, C. G. (1973) Arch. Biochem. Biophys. 158, 36-42. 14. PAPERMASTER, D. S., AND DREYER, W. J. (1974) Biochemistml 13, 2438-2444. 15. PLANTNER, J. J., AND KEAN, E. L. (1976) Exp. Eye Res. 23, 28-284. 16. WAECHTER, C. J., LUCAS, J. J., AND LENNARZ, W. J. (1973) J. Biol. Chem. 248, 75707579. 17. WALD, G., AND BROWN, P. K. (1953-1954) J. Gen. Physiol. 38, 189-200. 18. CARLSON, D. M. (1967) Anal. Biochem. 20, 195-198. 19. KEAN, E. L. (1970) J. Biol. Chem. 245, 23012308. 20. TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S. (1950) Nature (London) 166, 444-445. 21. KEAN, E. L. (1977) J. Biol. Chem. 252, 5622- 5629. 22. KEAN, E. L. (198O)J. Biol. Chem. 255,1921-1927. 23. KEAN, E. L. (1977) Exp. Eye Res. 25, 405417. 24. KEAN, E. L., AND BRUNER, W. E. (1977) Exp. Eye Res. 25, 419-426. 25. KEAN, E. L. (1977) J. Supramol. Struct. 7, 381-395.
Effect of Tunicamycin JAMES
J. PLANTNER,
The Lorand V. Johnson Ophthalmology,
on the Glycosylation LOUIS
PONCZ,2
AND
of Rhodopsin
EDWARD
L. KEAN3
Laboratory for Research in Ophthalmology, Department of Surgery, Division and the Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106 Received
January
of
16, 1980
The incorporation of ~H]glucosamine, [3H]mannose, and [“%]methionine into rhodopsin was investigated in retinas which had been incubated in the presence and absence of the antibiotic, tunicamycin. In its presence, the incorporation of glucosamine was inhibited 70% and mannose, 96% compared to controls. In the presence of tunicamycin the attachment of glucosamine to core-region sites was virtually eliminated. The formation of unglycosylated rhodopsin was also indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concanavalin A-Sepharose chromatography. These findings are consistent with the participation of the lipid-linked pathway in the glycosylation of this wellcharacterized intrinsic glycoprotein of the membranes of the disk of the rod outer segment. As indicated by the incorporation of [%]methionine, the synthesis of rhodopsin apoprotein was inhibited by a much lesser amount. This suggests that the glycosylation of rhodopsin is not required for its insertion into the disk membrane.
The antibiotic tunicamycin inhibits the formation of N-acetylglucosaminyl-pyrophosphoryldolichol, thus blocking the process of core-region glycosylation of the asparagine-linked class of glycoproteins by preventing the assembly of the polyprenololigosaccharide precursor (1). In addition to its extensive use in cell-free studies, treatment of intact animal cells with tunicamycin has resulted in the formation of unglycosylated molecules by blocking de nova glycosylation (2-6). This has provided a means of examining the influence of the carbohydrate components of glycoproteins on transport, secretion, and biological function of these heteropolymers. The visual pigment, rhodopsin, is a glycoprotein containing 9 mol of mannose and 5 mol of glucosamine/mol (7). It has recently been shown (8, 9) that the oligosaccharide ’ This work was supported in part by Public Health Service Research Grant EY 00393 from the National Eye Institute, and by the Ohio Lions Eye Research Foundation. 2 Supported by National Research Service Award T32 EY 07006. 3 To whom reprint requests should be addressed at the Laboratory for Eye Research, Room 653 Wearn Research Building, Case Western Reserve University, Cleveland, Ohio 44106.
chains of rhodopsin are rather unique, being hybrids of the high mannose and complex types of asparagine-linked glycoproteins, in addition to containing a single nonreducing terminal N-acetylglucosamine residue. Rhodopsin has been observed exclusively as a membranous component, being present primarily in the membranes of the disks of the outer segment of the rod cell, and to a lesser extent in the plasma membrane (10). Rhodopsin is synthesized in the inner segment of the rod cell and is transported to the base of the outer segment where it is incorporated into the membrane of the disk by an invagination of the plasma membrane. It has been estimated to account for up to 80% of the protein of the disk membrane (11). We are thus dealing with a system which involves the process of biosynthesis, transport, and assembly of a major membranous constituent. MATERIALS
Tissue Preparation;
AND
METHODS
Incubation
Conditions
All procedures were performed in dim red light, except where indicated. Retinas were dissected from fresh bovine eyes, obtained from a local slaughterhouse, within 2 h after death of the animal. Incuba527
0003-9861/80/060527-06$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
528
PLANTNER,
PONCZ, AND KEAN
tions were performed at 37°C in Krebs-Ringer bicarbonate buffer containing casamino acids (DIFCO, Detroit, Mich.), 0.01 mg/ml; penicillin, 35 units/mI; streptomycin, 35 pg/ml (Gibco, Grand Island, N. Y.); glycerol, 0.14 M, as a carbon source (12, 13); and llc&retinal, 0.03 mM, added in ethanol (0.002 ml/ml medium). During dissection the retinas were maintained in the complete incubation medium, on ice, through which was bubbled a gas mixture of 95% OJ5% CO*. Individual retinas were bissected and one half was used for control incubations and the other half for incubations performed in the presence of the antibiotic, using a total of 10 retinas for each condition. The equivalent of 2 retinas per incubation flask containing 9 ml of incubation medium was preincubated for 1 h in the absence or presence of tunicamycin (0.02 mg/ml). Tunicamycin was dissolved in dimethylsulfoxide and then dispersed in the incubation medium. The same concentration of dimethylsulfoxide (0.014 M) was present in control incubations as well. The flasks were gassed with a mixture of 95% 02/5% CO* at a flow rate of 500 ml/min/flask. After preincubation, the radioactive compounds were added, dissolved in 1 ml of Krebs-Ringer medium. The following were the radioactive compounds which were used and their final concentrations: [6-3H(N)]glucosamine (18 Ci/ mmol), 0.014 mCi/ml; [2JH]mannose (17 Ci/mmol), 0.016 mCi/ml; [35S]methionine (540-930 Ciimmol), 0.01-0.02 mCi/ml. All were purchased from New England Nuclear Corporation. The incubation mixtures contained either [3H]glucosamine or [SHImannose. Doubly labeled experiments also contained [35S]methionine. After the addition of the radioactive compounds, the incubations were continued for an additional 3 h.
Isolation and Purijieation
of Rhodopsin
After incubation, the retinas were recovered by centrifugation and washed twice by gentle resuspension in buffer. Rod outer segments were isolated and purified by flotation in dense sucrose, followed by purification using sucrose density centrifugation (14). Rhodopsin was extracted from the purified rod outer segments with 1% Emulphogene BC 720 (GAF Corp., New York) in 0.05 M Tris buffer, pH 7.0, as described previously (7). Rhodopsin was purified from the detergent extract by adsorption chromatography on columns of calcium phosphate-Celite (7) followed by preparative isoelectric focusing as described previously (15). Electrofocusing was performed on an LKB 110 ml Ampholine column over the pH range 5 to 7 stabilized by a sucrose gradient. The focusing column also contained 0.3% Emulphogene BC 720. Focusing was carried out at 4°C in the dark for 2 days. In these experiments, 2 mM DTT4 was present during the 4 Abbreviations used: ROS, rod outer segments; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; Con
extraction and chromatographic procedures. As described previously, three major isoelectric forms have been detected in bovine rhodopsin (15). When the isolation was performed in the presence of DTT, this multiplicity was prevented from being expressed5 and a single form dominated, thus simplifying the analyses. Con A-Sepharose chromatography was performed as described previously (7) on purified rhodopsin, recovered from the focusing column, after dialysis and concentration.
Enzyme Treatments Pronuse. Purified rhodopsin, recovered from the focusing column, after dialysis and concentration, was digested with Pronase (Calbiochem, LaJolla, Calif.) for 3 days in 0.15 M Tris buffer, pH 8.0, containing 0.1 M CaCl, under a toluene atmosphere (16). Three additions of 0.02 mg Pronase each were made over this period. The digest was then placed over a Bio-Gel P-4 column (1.5 x 80-cm), and the products were eluted with 0.1 N acetic acid. fi-Hexosaminidase. The peptide fraction from the Pronase digest was lyophilized, redissolved in 0.1 M citrate-phosphate buffer, pH 4.5, and incubated with 4.8 units of ezo-P-hexosaminidase (BoehringerMannheim, New York) for 16 h, as described by Struck and Lennarz (2). After digestion, the products were placed over a Bio-Gel P-4 column and the products eluted with 0.1 N acetic acid.
Analytical
Procedures
Radioactivity was determined by scintillation spectrometry using No. 963 counting solution obtained from New England Nuclear Corporation. In doubly labeled experiments, “H was counted with an efficiency of about 15%, and 35S, about 55%. The concentration of rhodopsin was calculated using an extinction coefficient of 40,600 at 498 nm (17). Strong acid hydrolysis was carried out at 100°C in sealed tubes using 3 N HCl for 4 h. After removal of the acid, the hydrolysate was re-l\i-aeetylated (18) and passed over columns of linked cation and anion exchange resins. The water eluate from these columns was examined by descending paper chromatography on Whatmann 3MM paper using as solvent system, 1-butanol:pyridine:water (6:4:3), and by high voltage paper electrophoresis in 1% sodium tetraborate buffer, as described previously (19). Carbohydrates were visualized by the alkaline silver nitrate procedure (20). Radioactivity on paper after chromatography and electrophoresis was measured by scintillation specA, concanavalin A; GlcNAc, Ir;-acetylglucosamine; HAc, acetic acid. J Plantner and Kean, manuscript in preparation.
TUNICAMYCIN
EFFECT
ON RHODOPSIN
GLYCOSYLATION
529
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FIG. 1. Electrofocusing patterns of rhodopsin obtained from retinas incubated in the absence and presence of tunicamycin. Bovine retinas were incubated with [6-3H]GlcNHZ or [2-3H]mannose, and rhodopsin was isolated as indicated under Materials and Methods. After chromatography on columns of calcium phosphate-Celite, the rhodopsin-containing fractions were subjected to isoelectric focusing on sucrose-density stabilized columns. The dashed lines refer to the retinas incubated in the absence of tunicamycin, and the dotted lines refer to incubations performed in the presence of tunicamycin (0.018-0.02 mgiml). The solid line refers to the A 49Hnmwhich was the same under all circumstances. trometry after sectioning into l-cm strips. SDSpolyacrylamide gel electrophoresis analysis (as described previously (7)) was performed on the dialyzed, concentrated product after electrofocusing. Due to the low specific activity it was necessary to analyze relatively large amounts on multiple gels. Approximately 1 nmol of rhodopsin was applied to each of eight gels. After electrophoresis the gels were stained with Coomassie blue and scanned at 600 nm using a Varian Model 635 spectrophotometer gel scanner attachment (Varian, Sunnydale, Calif.). The gels were sliced into l.l-mm sections with a Bio-Rad gel slicer (Richmond, Calif.), and corresponding slices were pooled as indicated by reference to the stained bands. The gels were leached with 1 ml of Unisol (Isolab, Akron, Ohio) at room temperature for 1 day, after which the radioactivity in the extract was measured as described previously (21). Greater than 80% of the radioactivity applied to the gels was recovered. In addition to measuring radioactivity, the A 600nm of the scintillation fluid extract of the gels was obtained. This was then correlated to the patterns of Coomassie staining from the individual gel scans. RESULTS
Mannose and Glucosamine Labeling
As can be seen in Fig. 1, the labeling patterns of [3H]mannose and [3H]glucosamine after electrofocusing were identical to that of the 498nm absorption of rhodopsin. After strong acid hydrolysis and re-Nacetylation greater than 80% of the radioactivity was recovered after mixed bed resin
Paper chromatography and treatment. paper electrophoresis revealed that the label was due only to these sugars. The A 27x.,/ A 490,,,,, ratio in all of these studies was less than 2, indicative of rhodopsin in a relatively high state of purity. The isoelectric point at the peak was about pH 6.1 for both the control and rhodopsin isolated from retinas incubated in the presence of tunicamycin. Similar amounts of rhodopsin were obtained under both conditions and similar amounts were analyzed. As is evident from Fig. 1, the presence of tunicamycin in the incubation medium resulted in extensive inhibition of the incorporation of glucosamine into rhodopsin, and almost complete suppression of the incorporation of mannose. The 35S pattern (not shown) was identical to that of AJSR. Shown in Table I are the specific activities of glucosamine and mannose in the pool of rhodopsin recovered after electrofocusing and dialysis. The range in specific activities in three experiments using glucosamine was less than 7%, and in two experiments using mannose, less than 10%. Sites of Glycosylation
Since there was still some labeling with glucosamine in the presence of tunicamycin, it was of interest to examine the sites of the attachment of this amino sugar
530
PLANTNER. TABLE
EFFECT
PONCZ,
I
OF TUNICAMYCIN ON THE GLYCOSYLATION OF RHODOPSIN" cpm x IO-” (Km01 rhodopsin)
Substrate
Control
[“H]GlcNH, l”H]Mannose
935 468
Tunicamycin 299 19
Inhibition (8) 68 96
’ The data refer to rhodopsin from retinas which had been incubated in the absence and presence of tunicamycin and purified by calcium phosphate-Celite chromatography and preparative isoelectric focusing. The specific activity was calculated from the radioactivity in the pool of rhodopsin from the focusing column (pH 5.9 to 6.3, Fig. 1) using either tritiated mannose or GlcNH,, and the concentration of rhodopsin calculated from the differential optical density at 498 nm obtained before and after photobleaching.
in rhodopsin. Peptides were generated from rhodopsin by extensive Pronase treatment, as described under Materials and Methods. The peptide pool was then treated with exo-hexosaminidase. Peripherally exposed residues would be cleaved by this treatment while more internally located core residues would be resistant. In Fig. 2 are results of an experiment of this type. In the upper panel, dealing with rhodopsin isolated from control retinas, in addition to the release of GlcNAc from the Pronasegenerated peptides, considerable 3H-labeled material remained associated with the larger molecular weight peptide region. In contrast, virtually all of the tritium in the peptide derived from the tunicamycintreated retinas (lower panel) was converted to material eluting from the gel in a manner similar to GlcNAc. From experiments of this type, the incorporation of [“Hlglucosamine into control rhodopsin was shown to be about 60% into peripheral sites and 40% more internally located, i.e., resistant to cleavage by hexosaminidase. In rhodopsin from retinas incubated in the presence of tunicamycin, virtually all of the incorporated glucosamine (90 to 98% in different experiments) was located peripherally. Since internal or core-region [3H]GlcNAc would
AND
KEAN
result from newly synthesized carbohydrate chains, the relatively small amount of glucosamine which was incorporated into rhodopsin in the presence of tunicamycin would reflect its peripheral addition to sites already available on preformed molecules. Methionine
Labeling
While the incorporation of mannose and glucosamine into rhodopsin isolated from the ROS membrane was extensively inhibited by tunicamycin, the labeling of this glycoprotein by methionine was much less affected. Thus, in different experiments where tunicamycin blocked the incorporation of ]3H]glucosamine into rhodopsin 60 to 70%, labeling by [35S]methionine was inhibited only 8 to 30%. SDS-Electrophoresis;
Con A-Sepharose
The analysis by SDS-electrophoresis of rhodopsin doubly labled with [35S]methionine
FIG. 2. Elution pattern from Bio-Gel P-4 after Pronase and hexosaminidase treatment of rhodopsin. The rhodopsin-containing fraction recovered after electrofocusing (pH 5.9 to 6.3, Fig. 1) was dialyzed, concentrated, and incubated extensively with Pronase, after which the products were examined by gel filtration on Bio-Gel P-4. The fractions included within the bars were pooled and incubated with ezo-/3-hexosaminidase, and reapplied to the column. As with the pronase digests, elution was carried out with 0.1 N HAc. The arrows refer to the elution positions of blue dextran (left) and GlcNAc (right). (-) Pronase treated; (---) hexosaminidase treated.
TUNICAMYCIN
EFFECT
ON RHODOPSIN
and [3H]glucosamine purified from retinas incubated in the presence of tunicamycin is seen in Fig. 3. As indicated above, limited labeling with glucosamine of endogenous rhodopsin was obtained in the presence of the antibiotic. The Coomassie pattern obtained from rhodopsin present in the preparation (serving as an internal standard) coincided with the 3H from glucosamine. The major peaks correspond to monomer and dimer forms of rhodopsin. On the other hand, the 35S-labeled product, reflecting newly formed aporhodopsin, migrated more rapidly, as would be expected for an unglycosylated species. The difference in molecular weights between the 35S and “H products of about 2500 would be consistent with this suggestion. A further indication that unglycosylated species were formed in the presence of tunicamycin was affinity chromatography on Con A-Sepharose of the products recovered after electrofocusing. Rhodopsin synthesized by control retinas labeled with [“YS]methionine and either [3H]mannose or [“Hlglucosamine was retained by the lectin (89 to 93%) and was released by cu-methylmannoside. In contrast, while 89% of the small amount of the 3H label in the rhodopsin formed in the presence of tunicamycin was bound by Con A-Sepharose, 54% of the 3sS was not retained by the lectin and had little or no 3H labeling, in agreement with the results from SDS-electrophoresis. DISCUSSION
In previous studies tunicamycin was shown to inhibit the incorporation of glucosamine into core-region saccharides in cellfree preparations of the retina (22), while it had no effect on the synthesis of dolichol phosphate-mannose. This is similar to observations made with other tissues (1, 2). The present studies demonstrated the extensive inhibition of glycosylation of rhodopsin by tunicamycin using the intact retina, in vitro. Thus, not only is the metabolic apparatus of the dolichol pathway present in the retina (21-25), but it is involved in the glycosylation of rhodopsin. Although the enzymes of this pathway have been shown in a variety of tissues to cata-
GLYCOSYLATION
531
FIG. 3. SDS-electrophoresis of rhodopsin from tunicamycin-treated retinas. Rhodopsin was purified by chromatography and isoelectric focusing as described under Materials and Methods. Approximately 1 nmol of purified rhodopsin containing 500 cpm of :lH and of “% was applied to each of eight gels. Electrophoresis was carried out at 8 mA per gel. After staining with Coomassie blue and scanning, the entire gels were sectioned and corresponding slices were pooled by reference to the stained bands. The radioactivity was determined and the A 600 nm of the counting solution extract was measured. The latter values were matched with the gel scans, a representative sample of which is shown in the Figure, (---); net cpmislice-pool: 35s (---A--); RH (- - n - -).
lyze the glycosylation of uncharacterized endogenous membrane proteins, this is the first demonstration of the participation of the lipid-linked pathway in the glycosylation of a specific, well-characterized intrinsic membranous glycoprotein in animal tissues. The labeling of rhodopsin by P4S]methionine reflected newly synthesized rhodopsin-protein. While tunicamycin almost completely blocked the glycosylation of these newly formed molecules by inhibiting core-region glycosylation, the incorporation of 35Sinto rhodopsin was affected relatively little. Since the rhodopsin was isolated from purified rod outer segments, it can be inferred that glycosylation was not required for the transport of this molecule from the site of its synthesis in the inner segment to the outer segment, nor for the insertion of aporhodopsin into the disk membranes of the outer segment of bovine retinal rods. Whether these molecules are
532
PLANTNER,
PONCZ,
present in the disk membrane in a correct orientation or are biologically active cannot be evaluated from the6 stu&es. ACKNOWLEDGMENTS The authors express their Robert L. Hamill and the Eli tories, Indianapolis, Indiana, of tunicamycin. We are grateful Nutley, New Jersey, for their
appreciation to Dr. Lilly Research Laborafor their generous gift to Hoffman-LaRoche, gift of II-&-retinal.
REFERENCES 1. TKACZ, J. S., AND LAMPEN, J. 0. (1975) Biochem. Biophys. Res. Commun. 65, 248257. 2. STRUCK, D. K., AND LENNARZ, W. J. (1977) J. Biol. Chem. 252, 1007-1013. 3. OLDEN, K., PRATT, R. M., AND YAMADA, K. M. (1978) Cell 13, 461-473. 4. HICKMAN, S., KULCZYCKI, A., LYNCH, R. G., AND KORNFELD, S. (1977) J. Biol. Chem. 252, 4402-4408. 5. DAMSKY, C. H., LEVY-BENSHIMOL, A., BUCK, C. A., AND WARREN, L. (1979) Exp. Cell Res. 119, 1-13. 6. STRUCK, D. K., SUITA, P. B., LANE, M. D., AND LENNARZ, W. J. (1978) J. Biol. Chem. 253, 5332-5337. 7. PLANTNER, J. J. AND KEAN, E. L. (1976) J. Biol. Chem. 251, 1548-1552. K., MUELLEN8. LIANG, C. -J., YAMASHITA, BERG, C. G., SHICHI, H., AND KOBATA, A. (1979) J. Biol. Chem. 254, 6414-6418.
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KEAN
9. FUKUDA, M. N., PAPERMASTER, D. S., AND HARGRAVE, P. A. (19’79) J. Biol. Chem. 254, 8201-8207. 10. DAEMAN, F. J. M. (19’73) Biochim. Biophys. Acta 300, 255-288. 11. HEITZMAN, H. (1972) Nature New Biol. 235, 114. 12. O’BRIEN, P. J. (1977) Exp. Eye Res. 24, 449-458. 13. O’BRIEN, P. J., AND MUELLENBERG, C. G. (1973) Arch. Biochem. Biophys. 158, 36-42. 14. PAPERMASTER, D. S., AND DREYER, W. J. (1974) Biochemistml 13, 2438-2444. 15. PLANTNER, J. J., AND KEAN, E. L. (1976) Exp. Eye Res. 23, 28-284. 16. WAECHTER, C. J., LUCAS, J. J., AND LENNARZ, W. J. (1973) J. Biol. Chem. 248, 75707579. 17. WALD, G., AND BROWN, P. K. (1953-1954) J. Gen. Physiol. 38, 189-200. 18. CARLSON, D. M. (1967) Anal. Biochem. 20, 195-198. 19. KEAN, E. L. (1970) J. Biol. Chem. 245, 23012308. 20. TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S. (1950) Nature (London) 166, 444-445. 21. KEAN, E. L. (1977) J. Biol. Chem. 252, 5622- 5629. 22. KEAN, E. L. (198O)J. Biol. Chem. 255,1921-1927. 23. KEAN, E. L. (1977) Exp. Eye Res. 25, 405417. 24. KEAN, E. L., AND BRUNER, W. E. (1977) Exp. Eye Res. 25, 419-426. 25. KEAN, E. L. (1977) J. Supramol. Struct. 7, 381-395.