Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3043-3046, April 1991
Biochemistry
Methylation and demethylation reactions of guanine nucleotidebinding proteins of retinal rod outer segments (methyltransferase/esterase/transducin/retina/S-farnesylcysteine)
DOLORES PIREZ-SALA, ENG Wui TAN, FRANCISCO J. CANADA, AND ROBERT R. RANDO* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115
Communicated by William von Eggers Doering, January 2, 1991 (received for review October 26, 1990)
ABSTRACT Retinal transducin was previously shown to be farnesylated on its y subunit. This farnesylation reaction on a cysteine residue near the carboxyl terminus is followed by peptidase cleavage at the cysteine. Thus the modified cysteine becomes the carboxyl terminus. It is shown here that the free carboxyl group can be methylated by an S-adenosyl-Lmethionine-dependent methyltransferase associated with the rod outer segment membranes. This process can be inhibited by S-adenosyl-L-homocysteine and sinefungin. Moreover, synthetic N-acetyl-S-farnesyl-L-cysteine, but not N-acetyl-Lcysteine, is a substrate for the enzyme. Rapid demethylation of N-acetyl-S-farnesyl-L-cysteine methyl ester can be observed in the membranes. Transducin is also enzymatically demethylated by the rod outer segment membranes. Moreover, the 23to 29-kDa small G proteins are methylated and demethylated in this system. These data suggest that methylation/demethylation may play a regulatory role in visual signal transduction.
The reversible methylation of enzymes at acidic residues can have important regulatory consequences (1). Recently, methylation processes have been shown to be combined with prenylation in the posttranslational modifications of "small" guanine nucleotide-binding proteins (G proteins), including Ras (2, 3), and the heterotrimeric G proteins (4, 5). The putative sequence of events here involves the initial prenylation of cysteine residues that are part of a CAAX motif (where C = cysteine, A = an aliphatic amino acid, and X = any amino acid) located at the carboxyl-terminal end of the protein (6). The prenylation process can involve either farnesylation (C15) (7) or geranylgeranylation (C20) (8). These modifications involve the enzymatic transfer of the prenyl group from farnesyl or geranylgeranyl pyrophosphate to the cysteine residue of the protein, forming a new thioether bond in the process. Recently, a farnesyl pyrophosphatedependent farnesyltransferase has been purified (9). Following prenylation, the AAX sequence is thought to be cleaved by a specific protease, resulting in the formation of a carboxyl-terminal prenylated cysteine residue (2). The free carboxyl terminus can be then methylated (10). It is thought that the function of these posttranslational modifications is to anchor the G protein to the target membrane, allowing the G protein to express its activity (6). This association may or may not be receptor-mediated. The overall process appears to be critical in cellular function, because interruption of the prenylation reaction via mutagenesis or with inhibitors leads to a decrease in cellular growth and function (11-13). Since the carboxyl methylation reaction is the only one of the three posttranslational modifications likely to be reversible, this process is apt to be important in controlling the activities of prenylated proteins. We previously showed that the y subunit of the heterotrimeric retinal G protein transThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3043
ducin (Ty) is farnesylated at cysteine (14). As expected, this farnesylcysteine residue is also methylated (15). Here we show that bovine retinal rod outer segments (ROS) contain an S-adenosyl-L-methionine (SAM)-linked methyltransferase activity that can methylate endogenous and added transducin, or synthetic substrates. In addition, the methylation of the a subunit of phosphodiesterase (16) and of a group of 23to 29-kDa membrane proteins (17) is also observed. The 23to 29-kDa polypeptides have been postulated to belong to a family of small G proteins (17). We previously demonstrated that these latter proteins could be prenylated in vitro (14). The terminal cysteine of TY is the site of methylation in this G protein. The methylating activity is inhibited by S-adenosylL-homocysteine (SAH) and sinefungin. In the presence of inhibitor, the membranes hydrolyze previously methylated transducin and the 23- to 29-kDa small G proteins as well as synthetic substrates. Thus, transducin and the small G proteins are shown to be reversibly methylated and demethylated by separate enzymatic activities. Since signal transduction is best understood in the vertebrate visual system, these studies pave the way for a quantitative understanding of the role of methylation in signal transduction.
MATERIALS AND METHODS Materials. Frozen bovine retinas were obtained from Wanda Lawson Co. (Lincoln, NE). [methyl-3HJSAM (85 Ci/mmol; 1 Ci = 37 GBq) and Amplify were from Amersham. Sinefungin, dithiothreitol, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, GTP, and N-acetyl-L-cysteine were from Sigma. SAH, endoproteinase Glu-C (Staphylococcus aureus V8 protease), leupeptin, pepstatin, and aprotinin were obtained from Boehringer Mannheim. trans,trans-Farnesyl bromide was from Aldrich. Preparation of ROS, Transducin, and Washed ROS Membranes. The protocol used was based on a published method (18). A fraction of the ROS collected at the interface of a step gradient from 25% to 35% (wt/wt) sucrose was resuspended in 50 mM Hepes Na, pH 7.4/100 mM NaCl/5 mM MgCl2/0.1 mM phenylmethylsulfonyl fluoride/0.1 mM dithiothreitol (buffer A) and stored in small aliquots at -80°C until used. The remaining ROS membranes were washed by a series of centrifugation steps and transducin was eluted from the membranes with 100 ,uM GTP. Washed ROS membranes were then resuspended in buffer A and stored at -80°C until used. Synthesis of N-Acetyl-S-trans,trans-Farnesyl-L-Cysteine (AFC) and Its Methyl Ester. AFC was prepared from N-acetyl-L-cysteine and trans,trans-farnesyl bromide by a method similar to that described previously (19). AFC was treated Abbreviations: SAM, S-adenosyl-L-methionine; SAH, S-adenosylL-homocysteine; AFC, N-acetyl-S-trans,trans-farnesyl-L-cysteine; ROS, rod outer segment(s); G protein, guanine nucleotide-binding protein; Ty, y subunit of transducin. *To whom reprint requests should be addressed.
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with methanolic HCl (0.05 M) to afford AFC methyl ester. NMR spectroscopic and mass spectrometric data for both compounds were in complete accordance with the assigned structures. In Vitro Methylation Reactions. The basic reaction mixture contained 20 GCi of [methyl-3H]SAM (2.34 ,uM) and an aliquot of ROS (120 gg of total protein) or of washed ROS membranes (80 ,ug of total protein), as the source of methyltransferase, in 100 ,ul of buffer A. Purified transducin was added to this mixture at 5 uM final concentration. AFC, N-acetylcysteine, or AFC methyl ester was added in 2 ,ul of dimethyl sulfoxide to give a final concentration of 20 p.M. Incubations were carried out at 370C. SDS/PAGE and Fluorography. For SDS/PAGE, aliquots of the reaction mixture were processed as described (14) and run in 15% gels. To improve the resolution of low molecular weight polypeptides, 0.1 M sodium acetate was included in the anode buffer (20). Radioactive polypeptides were visualized by fluorography (14). Exposure was at -70'C for 3-6 days. Proteolysis of [3H]Methylated T. Purified [3H]methylated dpm) was freeze-dried and redissolved in 300 Ty of(300.1Lg,M10-5ammonium bicarbonate containing 1.5 pug of S. ttl aureus V8 protease. Proteolysis was carried out at 37°C for 4 hr.
When the radioactively labeled 6-kDa protein was analyzed by HPLC, the radioactivity was coeluted with T. (Fig. 2A). When the radioactive polypeptide was cleaved with V8 protease and analyzed by HPLC, two main peaks of radioactivity were detected, one at 3 min, in the position expected for radioactive methanol, and a second peak at 42 min (Fig. 2B). Analysis of this peptide by Edman degradation gave the sequence Leu-Lys-Gly-Gly-Xaa, which corresponds to the carboxyl-terminal fragment of T.,, confirming that methylation occurs at the cysteine residue, as has been recently reported (15). Nature of the Methylation Process. The results described above show that the in vitro methylation of T., occurs at the terminal cysteine residue. The nature of the methylation process was investigated next. The enzymatic activity was destroyed by heat and inhibited by two well-characterized inhibitors of SAM-dependent methyltransferases, SAH (22) and sinefungin (23) (Fig. 1B). In addition to SAH and sinefungin, AFC also inhibited the incorporation of label into proteins (Fig. 1B). Importantly, AFC also serves as a substrate for the methyltransferase (Fig. 3). The identity of the product as AFC [3H]methyl ester was demonstrated by coelution with authentic standard by TLC and HPLC criteria.
1.0
RESULTS In Vitro Methylation of T.,. Incubation of bovine ROS with [methyl-3H]SAM results in the radioactive labeling of several polypeptides with apparent molecular masses of 88, 60, 23-29, and 6 kDa (Fig. 1A). The methylation of the 88-kDa protein (a subunit of retinal phosphodiesterase) and of the 23to 29-kDa polypeptides has already been reported (16, 17). The 6-kDa polypeptide coincides with TY in SDS/polyacrylamide gels (Fig. 1C). Moreover, incubation of purified transducin with extensively washed ROS and [3H]SAM results in the labeling of the 6-kDa polypeptide and the membrane-associated 23- to 29-kDa proteins (Fig. 1B). These latter proteins are almost certainly the retinal analogs of the prenylated and carboxyl-methylated small G proteins observed in cultured cells (3) and the 23-kDa G protein purified
0.5-
0.01,
10 ° x
E 5 CL
B
from brain (21). This conclusion is based on our previous observation that they are prenylated (14), the fact that they are methylated, their molecular masses, and their ability to bind GTP on nitrocellulose membranes (data not shown). A
B
4
0.8-
II
LKGGC*
LO
C 3
t15
K 0.4
5 -
_
V8
I
106 80 50
0.0 2'x
k*
I
33 -
28
_ 19
14
-2.5 FIG. 1. Methylation of ROS proteins and puirified transducin. In vitro methylation reactions were carried out uinder the conditions specified in Materials and Methods. After 3 hr. aliquots of the reaction mixtures were processed for SDS/PAG;E and fluorography. (A) Methylation of ROS. (B) Methylation of piurified transducin (5 ,uM) under standard conditions (lane 1), when RI OS membranes were heated at 100NC for 5 min before the incubatio)n (lane 2), or in the presence of 200 .M sinefungin (lane 3), 2 mM 'SAH (lane 4), or 250 ,uM AFC (lane 5). (C) Coomassie blue staining offpurified transducin.
0
5
20
25
30
35
40
45
50
Retention time, min
Ty.
FIG. 2. (A) HPLC purification of [3H]methylated [3H]Methylated transducin was extracted from the membranes by washing with 10 mM Tris HCI/0.1 mM EDTA/1 mM dithiothreitol/100 ,uM GTP. The concentrated extract was spiked with purified transducin and analyzed by reverse-phase HPLC on a C18 column (Dynamax 300 A, Rainin, Woburn, MA) with eluants A (10 mM trifluoroacetic acid in water) and B (10 mM trifluoroacetic acid in acetonitrile), using eluant Afor 5 min and then a0-95% gradient of B during 45 min. Flow rate was 0.75 ml/min. Fractions (0.5 ml) were collected and mixed with 7 ml of Hydrofluor (National Diagnostics, Manville, NJ) for scintillation counting. The identity of the T. peak was confirmed by
amino acid analysis. (B) Analysis of digested [3H~methylated TY with the HPLC system described above. The labeled peptide was purified and its sequence was obtained by Edman degradation.
Biochemistry: Pe'rez-Sala et al.
Proc. Natl. Acad. Sci. USA 88 (1991) 1
2
3
3045
KDa -106 -80 -50
o75x
-33
E
-
28
19 14 - 6 -2.5 -
-
25-
~0
60 120 180 Incubation time, min FIG. 3. Time-dependent formation of AFC [3H]methyl ester. AFC (o) or AFC methyl ester (s), at a concentration of 20 AM, was incubated with [3H]SAM and ROS membranes. At the indicated time points, 50-Al aliquots were withdrawn from the incubation mixture and the reaction was quenched with 500 ul of chloroform/methanol, 1:1 (vol/vol). AFC [3H]methyl ester was extracted by "vortexing" for 1 min in this mixture. Phase separation was achieved by adding 250 tl of water. The chloroform layer, containing 95% of the AFC [3H]methyl ester, was evaporated under argon, resuspended in 15% 2-propanol in hexane, and spiked with authentic AFC methyl ester standard for UV detection (210 nm). Samples were injected on a normal-phase HPLC column (Dynamax 60 A, Rainin) and elution was performed with the same solvent. Radioactivity was counted with an on-line Berthold (Nashua, NH) LB 506-C HPLC radioactivity monitor.
The enzymatic methyltransferase activity towards AFC was, as expected, destroyed by heat and again inhibited by SAH and sinefungin. The necessity of the farnesyl moiety for substrate activity was demonstrated by showing that N-acetylcysteine is not a substrate for the methyltransferase (data not shown). The Methylation Is a Reversible Process. Incubation of nonradioactive AFC methyl ester with [3H]SAM and washed ROS membranes resulted in a linear, time-dependent incorporation of radioactivity into AFC methyl ester for at least 2 hr (Fig. 3). To study the demethylation process directly, AFC [3H]methyl ester was incubated with ROS membranes in the presence of methylation inhibitors (Fig. 4A). The methyl
FIG. 5. Demethylation of the 23- to 29-kDa G proteins and transducin. Transducin was incubated with ROS membranes and [3H]SAM. After 2 hr of incubation, methylation was inhibited by addition of 200 uM sinefungin. Aliquots of the incubation mixture were taken 10 min (lane 1), 30 min (lane 2), and 2 hr (lane 3) after the addition of the inhibitor and processed for SDS/PAGE and fluorography. Fluorographic exposure was for 3 days. Quantitation of the fluorographic spots showed a decrease of 50% in the radioactivity associated with the 23- to 29-kDa proteins and of 20%o in the radioactivity associated with TY after 2 hr.
ester was rapidly hydrolyzed by the demethylase activity in the membranes. Heating of the membranes strongly diminished the demethylase activity, as is expected of an enzymatic activity. Under similar conditions, the catalyzed demethylation of T. (Fig. 4B) and the putative small G proteins (Fig. 5), was also demonstrated. It appears that the small G proteins are more rapidly demethylated than T.,
DISCUSSION The results show that T, can be enzymatically methylated by an SAM-dependent methyltransferase activity bound to the ROS membranes. It is interesting that as isolated here, TY appears to be largely (>80%) methylated, as deduced from HPLC analysis of control and base-treated transducin (results not shown). Other proteins are also methylated, including the small G proteins (17) and the a subunit of the retinal phosphodiesterase (16). The methylation site of TY was shown to be on the terminal cysteine residue, a result in accord with observations made by others using different
75 A
x 50
-
x
E
0.v
Incubation time, min FIG. 4. Demethylation of AFC [3H]methyl ester and [3H]methylated transducin. AFC [3H]methyl ester (20 nM, 85 Ci/mmol), obtained enzymatically from AFC and [3H]SAM (A), or [3H]methylated transducin (1 MM, -0.32 Ci/mmol) (B) was incubated with intact (o, e) or boiled (A, A) ROS membranes in the presence of 200 MuM sinefungin. (A) The amount of AFC [3H]methyl ester remaining (o, A) was determined by HPLC analysis as described in the legend of Fig. 3, and the radioactivity accumulated in the methanol phase (o, *) was measured by scintillation counting. (B) At the indicated times, protein was precipitated with chloroform/methanol and the protein pellets were solubilized with 5% SDS. The radioactivity bound to the protein (o, A) and accumulated in the methanol phase (e, A) was measured by scintillation counting.
3046
Biochemistry: PNrez-Sala et al.
analytical methods (15). Standard inhibitors of SAM methyltransferase enzymes, such as SAH and sinefungin, potently inhibited enzymatic activity when T7 was the substrate. Interestingly, AFC also inhibited TY methylation. This inhibition occurred because AFC is a substrate for the methylating enzyme. The farnesyl moiety is important for activity, since N-acetyl-L-cysteine is without activity as a substrate for the enzyme. Although we have not directly studied the issue, it appears likely that the local peptide structure is relatively unimportant as a substrate determinant. When the methylated T.,, small G proteins, and AFC methyl ester were incubated with ROS membranes in the presence of methyltransferase inhibitors, the methyl esters were hydrolyzed by an enzymatic activity in the membranes. It appears, however, that the turnover rate for the 23- to 29-kDa proteins is faster than that for transducin. The lower rate of TY methyl ester hydrolysis might suggest that this reaction is of little regulatory significance. However, it is possible that the rate is substantially greater under physiological conditions. By contrast, the rate of demethylation of the 23- to 29-kDa proteins seemed to be quite substantial, suggesting a possible regulatory role for methylation/ demethylation of these proteins in visual transduction. The notion that reversible methylation is important in regulating the G-protein-mediated signal-transduction cascade is attractive. As previously mentioned, the other posttranslational modifications linked to methylation (prenylation and peptidase cleavage) are almost certainly irreversible. This leaves the methylation step as the only one that is reversible, as is demonstrated here. The effect of methylation/demethylation is chemically significant because the neutral ester moiety is converted into a carboxylate anion. This modification would be expected to be important irrespective of whether prenylated proteins associate with membranes by partitioning or by a receptor-mediated event. Since transducin is the best understood of the signaltransducing heterotrimeric G proteins, it is also the system that should be most fruitfully investigated with respect to the posttranslational modifications discussed here. It will be especially interesting to determine what the role of methylation is in the control of the visual transduction process. There are several candidates possible. First, the interaction of methylated and demethylated transducin with photoactivated rhodopsin will need to be investigated. Other points of possible control include the rates at which Ta hydrolyzes GTP and then reassociates with Tp3y. Some, or all, of these mechanisms could be involved in visual adaptation mechanisms. The role(s) of the 23- to 29-kDa G proteins in visual transduction processes is currently obscure and will need to be investigated. Given that methylation/demethylation steps can be important elements in signal transduction, it will be of some interest
Proc. Natl. Acad. Sci. USA 88 (1991)
to invent specific inhibitors of these enzymes. The finding that AFC and AFC methyl ester are substrates for the respective enzymes augurs well for the potential design of mechanism-based inhibitors of the methyltransferase and esterase enzymes. This work was supported by U.S. Public Health Service Research Grant EY03624 from the National Institutes of Health. D.P.-S. and F.J.C. are recipients of fellowships from Consejo Superior de Investigaciones Cientificas (Spain).
1. Clarke, S. (1985) Annu. Rev. Biochem. 54, 479-506. 2. Gutierrez, L., Magee, A. I., Marshall, C. J. & Hancock, J. F. (1989) EMBO J. 8, 1093-1098. 3. Maltese, W. A., Sheridan, K. M., Repko, E. M. & Erdman, R. A. (1990) J. Biol. Chem. 265, 2148-2155. 4. Yamane, H. K., Farnsworth, C. C., Xie, H., Howald, W., Fung, B. K.-K., Clarke, S., Gelb, M. H. & Glomset, J. A. (1990) Proc. Nadl. Acad. Sci. USA 87, 5868-5872. 5. Mumby, S. M., Casey, P. J., Gilman, A. G., Gutowski, S. & Sternweis, P. C. (1990) Proc. Nadl. Acad. Sci. USA 87, 58735877. 6. Hancock, J. F., Magee, A. I., Childs, J. E. & Marshall, C. J. (1989) Cell 57, 1167-1177. 7. Casey, P. J., Solski, P. A., Der, C. J. & Buss, J. E. (1989) Proc. Nadl. Acad. Sci. USA 86, 8323-8327. 8. Farnsworth, C. C., Gelb, M. H. & Glomset, J. A. (1990) Science 247, 320-322. 9. Reiss, Y., Goldstein, J. L., Seabra, M. C., Casey, P. J. & Brown, M. S. (1990) Cell 62, 81-88. 10. Clarke, S., Vogel, J. P., Deschenes, R. J. & Stock, J. (1988) Proc. Natd. Acad. Sci. USA 85, 4643-4647. 11. Barbacid, M. (1987) Annu. Rev. Biochem. 56, 779-827. 12. Schafer, W. R., Kim, R., Sterne, R., Thorner, J., Kim, S.-H. & Rine, J. (1989) Science 245, 379-385. 13. Maltese, W. A. & Sheridan, K. M. (1987) J. Cell. Physiol. 133, 471-481. 14. Lai, R. K., Pdrez-Sala, D., Cafiada, F. J. & Rando, R. R. (1990) Proc. Nail. Acad. Sci. USA 87, 7673-7677. 15. Fukada, Y., Takao, T., Ohguro, H., Yoshizawa-, T., Akino, T. & Shimonishi, Y. (1990) Nature (London) 346, 658-660. 16. Swanson, R. J. & Applebury, M. L. (1983) J. Biol. Chem. 258, 10599-10605. 17. Ota, I. M. & Clarke, S. (1989) J. Biol. Chem. 264,12879-12884. 18. Wessling-Resnick, M. & Johnson, G. L. (1987) J. Biol. Chem. 262, 3697-3705. 19. Kamiya, Y., Sakurai, A., Tamura, S., Takahashi, N., Tsuchiya, E., Abe, K. & Fukui, S. (1979) Agric. Biol. Chem. 43, 363-369. 20. Christy, K. G., LaTart, D. B. & Osterhoudt, W. (1989) BioTechniques 7, 692-693. 21. Yamane, H. K. & Fung, B. K.-K. (1989) J. Biol. Chem. 264, 20100-20105. 22. Barber, J. R. & Clarke, S. (1984) J. Biol. Chem. 259, 71157122. 23. Pugh, C. S. G., Borchardt, R. T. & Stone, H. O. (1978) J. Biol. Chem. 253, 4075-4077.
Biochemistry
Methylation and demethylation reactions of guanine nucleotidebinding proteins of retinal rod outer segments (methyltransferase/esterase/transducin/retina/S-farnesylcysteine)
DOLORES PIREZ-SALA, ENG Wui TAN, FRANCISCO J. CANADA, AND ROBERT R. RANDO* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115
Communicated by William von Eggers Doering, January 2, 1991 (received for review October 26, 1990)
ABSTRACT Retinal transducin was previously shown to be farnesylated on its y subunit. This farnesylation reaction on a cysteine residue near the carboxyl terminus is followed by peptidase cleavage at the cysteine. Thus the modified cysteine becomes the carboxyl terminus. It is shown here that the free carboxyl group can be methylated by an S-adenosyl-Lmethionine-dependent methyltransferase associated with the rod outer segment membranes. This process can be inhibited by S-adenosyl-L-homocysteine and sinefungin. Moreover, synthetic N-acetyl-S-farnesyl-L-cysteine, but not N-acetyl-Lcysteine, is a substrate for the enzyme. Rapid demethylation of N-acetyl-S-farnesyl-L-cysteine methyl ester can be observed in the membranes. Transducin is also enzymatically demethylated by the rod outer segment membranes. Moreover, the 23to 29-kDa small G proteins are methylated and demethylated in this system. These data suggest that methylation/demethylation may play a regulatory role in visual signal transduction.
The reversible methylation of enzymes at acidic residues can have important regulatory consequences (1). Recently, methylation processes have been shown to be combined with prenylation in the posttranslational modifications of "small" guanine nucleotide-binding proteins (G proteins), including Ras (2, 3), and the heterotrimeric G proteins (4, 5). The putative sequence of events here involves the initial prenylation of cysteine residues that are part of a CAAX motif (where C = cysteine, A = an aliphatic amino acid, and X = any amino acid) located at the carboxyl-terminal end of the protein (6). The prenylation process can involve either farnesylation (C15) (7) or geranylgeranylation (C20) (8). These modifications involve the enzymatic transfer of the prenyl group from farnesyl or geranylgeranyl pyrophosphate to the cysteine residue of the protein, forming a new thioether bond in the process. Recently, a farnesyl pyrophosphatedependent farnesyltransferase has been purified (9). Following prenylation, the AAX sequence is thought to be cleaved by a specific protease, resulting in the formation of a carboxyl-terminal prenylated cysteine residue (2). The free carboxyl terminus can be then methylated (10). It is thought that the function of these posttranslational modifications is to anchor the G protein to the target membrane, allowing the G protein to express its activity (6). This association may or may not be receptor-mediated. The overall process appears to be critical in cellular function, because interruption of the prenylation reaction via mutagenesis or with inhibitors leads to a decrease in cellular growth and function (11-13). Since the carboxyl methylation reaction is the only one of the three posttranslational modifications likely to be reversible, this process is apt to be important in controlling the activities of prenylated proteins. We previously showed that the y subunit of the heterotrimeric retinal G protein transThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3043
ducin (Ty) is farnesylated at cysteine (14). As expected, this farnesylcysteine residue is also methylated (15). Here we show that bovine retinal rod outer segments (ROS) contain an S-adenosyl-L-methionine (SAM)-linked methyltransferase activity that can methylate endogenous and added transducin, or synthetic substrates. In addition, the methylation of the a subunit of phosphodiesterase (16) and of a group of 23to 29-kDa membrane proteins (17) is also observed. The 23to 29-kDa polypeptides have been postulated to belong to a family of small G proteins (17). We previously demonstrated that these latter proteins could be prenylated in vitro (14). The terminal cysteine of TY is the site of methylation in this G protein. The methylating activity is inhibited by S-adenosylL-homocysteine (SAH) and sinefungin. In the presence of inhibitor, the membranes hydrolyze previously methylated transducin and the 23- to 29-kDa small G proteins as well as synthetic substrates. Thus, transducin and the small G proteins are shown to be reversibly methylated and demethylated by separate enzymatic activities. Since signal transduction is best understood in the vertebrate visual system, these studies pave the way for a quantitative understanding of the role of methylation in signal transduction.
MATERIALS AND METHODS Materials. Frozen bovine retinas were obtained from Wanda Lawson Co. (Lincoln, NE). [methyl-3HJSAM (85 Ci/mmol; 1 Ci = 37 GBq) and Amplify were from Amersham. Sinefungin, dithiothreitol, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, GTP, and N-acetyl-L-cysteine were from Sigma. SAH, endoproteinase Glu-C (Staphylococcus aureus V8 protease), leupeptin, pepstatin, and aprotinin were obtained from Boehringer Mannheim. trans,trans-Farnesyl bromide was from Aldrich. Preparation of ROS, Transducin, and Washed ROS Membranes. The protocol used was based on a published method (18). A fraction of the ROS collected at the interface of a step gradient from 25% to 35% (wt/wt) sucrose was resuspended in 50 mM Hepes Na, pH 7.4/100 mM NaCl/5 mM MgCl2/0.1 mM phenylmethylsulfonyl fluoride/0.1 mM dithiothreitol (buffer A) and stored in small aliquots at -80°C until used. The remaining ROS membranes were washed by a series of centrifugation steps and transducin was eluted from the membranes with 100 ,uM GTP. Washed ROS membranes were then resuspended in buffer A and stored at -80°C until used. Synthesis of N-Acetyl-S-trans,trans-Farnesyl-L-Cysteine (AFC) and Its Methyl Ester. AFC was prepared from N-acetyl-L-cysteine and trans,trans-farnesyl bromide by a method similar to that described previously (19). AFC was treated Abbreviations: SAM, S-adenosyl-L-methionine; SAH, S-adenosylL-homocysteine; AFC, N-acetyl-S-trans,trans-farnesyl-L-cysteine; ROS, rod outer segment(s); G protein, guanine nucleotide-binding protein; Ty, y subunit of transducin. *To whom reprint requests should be addressed.
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Proc. Natl. Acad. Sci. USA 88 (1991)
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with methanolic HCl (0.05 M) to afford AFC methyl ester. NMR spectroscopic and mass spectrometric data for both compounds were in complete accordance with the assigned structures. In Vitro Methylation Reactions. The basic reaction mixture contained 20 GCi of [methyl-3H]SAM (2.34 ,uM) and an aliquot of ROS (120 gg of total protein) or of washed ROS membranes (80 ,ug of total protein), as the source of methyltransferase, in 100 ,ul of buffer A. Purified transducin was added to this mixture at 5 uM final concentration. AFC, N-acetylcysteine, or AFC methyl ester was added in 2 ,ul of dimethyl sulfoxide to give a final concentration of 20 p.M. Incubations were carried out at 370C. SDS/PAGE and Fluorography. For SDS/PAGE, aliquots of the reaction mixture were processed as described (14) and run in 15% gels. To improve the resolution of low molecular weight polypeptides, 0.1 M sodium acetate was included in the anode buffer (20). Radioactive polypeptides were visualized by fluorography (14). Exposure was at -70'C for 3-6 days. Proteolysis of [3H]Methylated T. Purified [3H]methylated dpm) was freeze-dried and redissolved in 300 Ty of(300.1Lg,M10-5ammonium bicarbonate containing 1.5 pug of S. ttl aureus V8 protease. Proteolysis was carried out at 37°C for 4 hr.
When the radioactively labeled 6-kDa protein was analyzed by HPLC, the radioactivity was coeluted with T. (Fig. 2A). When the radioactive polypeptide was cleaved with V8 protease and analyzed by HPLC, two main peaks of radioactivity were detected, one at 3 min, in the position expected for radioactive methanol, and a second peak at 42 min (Fig. 2B). Analysis of this peptide by Edman degradation gave the sequence Leu-Lys-Gly-Gly-Xaa, which corresponds to the carboxyl-terminal fragment of T.,, confirming that methylation occurs at the cysteine residue, as has been recently reported (15). Nature of the Methylation Process. The results described above show that the in vitro methylation of T., occurs at the terminal cysteine residue. The nature of the methylation process was investigated next. The enzymatic activity was destroyed by heat and inhibited by two well-characterized inhibitors of SAM-dependent methyltransferases, SAH (22) and sinefungin (23) (Fig. 1B). In addition to SAH and sinefungin, AFC also inhibited the incorporation of label into proteins (Fig. 1B). Importantly, AFC also serves as a substrate for the methyltransferase (Fig. 3). The identity of the product as AFC [3H]methyl ester was demonstrated by coelution with authentic standard by TLC and HPLC criteria.
1.0
RESULTS In Vitro Methylation of T.,. Incubation of bovine ROS with [methyl-3H]SAM results in the radioactive labeling of several polypeptides with apparent molecular masses of 88, 60, 23-29, and 6 kDa (Fig. 1A). The methylation of the 88-kDa protein (a subunit of retinal phosphodiesterase) and of the 23to 29-kDa polypeptides has already been reported (16, 17). The 6-kDa polypeptide coincides with TY in SDS/polyacrylamide gels (Fig. 1C). Moreover, incubation of purified transducin with extensively washed ROS and [3H]SAM results in the labeling of the 6-kDa polypeptide and the membrane-associated 23- to 29-kDa proteins (Fig. 1B). These latter proteins are almost certainly the retinal analogs of the prenylated and carboxyl-methylated small G proteins observed in cultured cells (3) and the 23-kDa G protein purified
0.5-
0.01,
10 ° x
E 5 CL
B
from brain (21). This conclusion is based on our previous observation that they are prenylated (14), the fact that they are methylated, their molecular masses, and their ability to bind GTP on nitrocellulose membranes (data not shown). A
B
4
0.8-
II
LKGGC*
LO
C 3
t15
K 0.4
5 -
_
V8
I
106 80 50
0.0 2'x
k*
I
33 -
28
_ 19
14
-2.5 FIG. 1. Methylation of ROS proteins and puirified transducin. In vitro methylation reactions were carried out uinder the conditions specified in Materials and Methods. After 3 hr. aliquots of the reaction mixtures were processed for SDS/PAG;E and fluorography. (A) Methylation of ROS. (B) Methylation of piurified transducin (5 ,uM) under standard conditions (lane 1), when RI OS membranes were heated at 100NC for 5 min before the incubatio)n (lane 2), or in the presence of 200 .M sinefungin (lane 3), 2 mM 'SAH (lane 4), or 250 ,uM AFC (lane 5). (C) Coomassie blue staining offpurified transducin.
0
5
20
25
30
35
40
45
50
Retention time, min
Ty.
FIG. 2. (A) HPLC purification of [3H]methylated [3H]Methylated transducin was extracted from the membranes by washing with 10 mM Tris HCI/0.1 mM EDTA/1 mM dithiothreitol/100 ,uM GTP. The concentrated extract was spiked with purified transducin and analyzed by reverse-phase HPLC on a C18 column (Dynamax 300 A, Rainin, Woburn, MA) with eluants A (10 mM trifluoroacetic acid in water) and B (10 mM trifluoroacetic acid in acetonitrile), using eluant Afor 5 min and then a0-95% gradient of B during 45 min. Flow rate was 0.75 ml/min. Fractions (0.5 ml) were collected and mixed with 7 ml of Hydrofluor (National Diagnostics, Manville, NJ) for scintillation counting. The identity of the T. peak was confirmed by
amino acid analysis. (B) Analysis of digested [3H~methylated TY with the HPLC system described above. The labeled peptide was purified and its sequence was obtained by Edman degradation.
Biochemistry: Pe'rez-Sala et al.
Proc. Natl. Acad. Sci. USA 88 (1991) 1
2
3
3045
KDa -106 -80 -50
o75x
-33
E
-
28
19 14 - 6 -2.5 -
-
25-
~0
60 120 180 Incubation time, min FIG. 3. Time-dependent formation of AFC [3H]methyl ester. AFC (o) or AFC methyl ester (s), at a concentration of 20 AM, was incubated with [3H]SAM and ROS membranes. At the indicated time points, 50-Al aliquots were withdrawn from the incubation mixture and the reaction was quenched with 500 ul of chloroform/methanol, 1:1 (vol/vol). AFC [3H]methyl ester was extracted by "vortexing" for 1 min in this mixture. Phase separation was achieved by adding 250 tl of water. The chloroform layer, containing 95% of the AFC [3H]methyl ester, was evaporated under argon, resuspended in 15% 2-propanol in hexane, and spiked with authentic AFC methyl ester standard for UV detection (210 nm). Samples were injected on a normal-phase HPLC column (Dynamax 60 A, Rainin) and elution was performed with the same solvent. Radioactivity was counted with an on-line Berthold (Nashua, NH) LB 506-C HPLC radioactivity monitor.
The enzymatic methyltransferase activity towards AFC was, as expected, destroyed by heat and again inhibited by SAH and sinefungin. The necessity of the farnesyl moiety for substrate activity was demonstrated by showing that N-acetylcysteine is not a substrate for the methyltransferase (data not shown). The Methylation Is a Reversible Process. Incubation of nonradioactive AFC methyl ester with [3H]SAM and washed ROS membranes resulted in a linear, time-dependent incorporation of radioactivity into AFC methyl ester for at least 2 hr (Fig. 3). To study the demethylation process directly, AFC [3H]methyl ester was incubated with ROS membranes in the presence of methylation inhibitors (Fig. 4A). The methyl
FIG. 5. Demethylation of the 23- to 29-kDa G proteins and transducin. Transducin was incubated with ROS membranes and [3H]SAM. After 2 hr of incubation, methylation was inhibited by addition of 200 uM sinefungin. Aliquots of the incubation mixture were taken 10 min (lane 1), 30 min (lane 2), and 2 hr (lane 3) after the addition of the inhibitor and processed for SDS/PAGE and fluorography. Fluorographic exposure was for 3 days. Quantitation of the fluorographic spots showed a decrease of 50% in the radioactivity associated with the 23- to 29-kDa proteins and of 20%o in the radioactivity associated with TY after 2 hr.
ester was rapidly hydrolyzed by the demethylase activity in the membranes. Heating of the membranes strongly diminished the demethylase activity, as is expected of an enzymatic activity. Under similar conditions, the catalyzed demethylation of T. (Fig. 4B) and the putative small G proteins (Fig. 5), was also demonstrated. It appears that the small G proteins are more rapidly demethylated than T.,
DISCUSSION The results show that T, can be enzymatically methylated by an SAM-dependent methyltransferase activity bound to the ROS membranes. It is interesting that as isolated here, TY appears to be largely (>80%) methylated, as deduced from HPLC analysis of control and base-treated transducin (results not shown). Other proteins are also methylated, including the small G proteins (17) and the a subunit of the retinal phosphodiesterase (16). The methylation site of TY was shown to be on the terminal cysteine residue, a result in accord with observations made by others using different
75 A
x 50
-
x
E
0.v
Incubation time, min FIG. 4. Demethylation of AFC [3H]methyl ester and [3H]methylated transducin. AFC [3H]methyl ester (20 nM, 85 Ci/mmol), obtained enzymatically from AFC and [3H]SAM (A), or [3H]methylated transducin (1 MM, -0.32 Ci/mmol) (B) was incubated with intact (o, e) or boiled (A, A) ROS membranes in the presence of 200 MuM sinefungin. (A) The amount of AFC [3H]methyl ester remaining (o, A) was determined by HPLC analysis as described in the legend of Fig. 3, and the radioactivity accumulated in the methanol phase (o, *) was measured by scintillation counting. (B) At the indicated times, protein was precipitated with chloroform/methanol and the protein pellets were solubilized with 5% SDS. The radioactivity bound to the protein (o, A) and accumulated in the methanol phase (e, A) was measured by scintillation counting.
3046
Biochemistry: PNrez-Sala et al.
analytical methods (15). Standard inhibitors of SAM methyltransferase enzymes, such as SAH and sinefungin, potently inhibited enzymatic activity when T7 was the substrate. Interestingly, AFC also inhibited TY methylation. This inhibition occurred because AFC is a substrate for the methylating enzyme. The farnesyl moiety is important for activity, since N-acetyl-L-cysteine is without activity as a substrate for the enzyme. Although we have not directly studied the issue, it appears likely that the local peptide structure is relatively unimportant as a substrate determinant. When the methylated T.,, small G proteins, and AFC methyl ester were incubated with ROS membranes in the presence of methyltransferase inhibitors, the methyl esters were hydrolyzed by an enzymatic activity in the membranes. It appears, however, that the turnover rate for the 23- to 29-kDa proteins is faster than that for transducin. The lower rate of TY methyl ester hydrolysis might suggest that this reaction is of little regulatory significance. However, it is possible that the rate is substantially greater under physiological conditions. By contrast, the rate of demethylation of the 23- to 29-kDa proteins seemed to be quite substantial, suggesting a possible regulatory role for methylation/ demethylation of these proteins in visual transduction. The notion that reversible methylation is important in regulating the G-protein-mediated signal-transduction cascade is attractive. As previously mentioned, the other posttranslational modifications linked to methylation (prenylation and peptidase cleavage) are almost certainly irreversible. This leaves the methylation step as the only one that is reversible, as is demonstrated here. The effect of methylation/demethylation is chemically significant because the neutral ester moiety is converted into a carboxylate anion. This modification would be expected to be important irrespective of whether prenylated proteins associate with membranes by partitioning or by a receptor-mediated event. Since transducin is the best understood of the signaltransducing heterotrimeric G proteins, it is also the system that should be most fruitfully investigated with respect to the posttranslational modifications discussed here. It will be especially interesting to determine what the role of methylation is in the control of the visual transduction process. There are several candidates possible. First, the interaction of methylated and demethylated transducin with photoactivated rhodopsin will need to be investigated. Other points of possible control include the rates at which Ta hydrolyzes GTP and then reassociates with Tp3y. Some, or all, of these mechanisms could be involved in visual adaptation mechanisms. The role(s) of the 23- to 29-kDa G proteins in visual transduction processes is currently obscure and will need to be investigated. Given that methylation/demethylation steps can be important elements in signal transduction, it will be of some interest
Proc. Natl. Acad. Sci. USA 88 (1991)
to invent specific inhibitors of these enzymes. The finding that AFC and AFC methyl ester are substrates for the respective enzymes augurs well for the potential design of mechanism-based inhibitors of the methyltransferase and esterase enzymes. This work was supported by U.S. Public Health Service Research Grant EY03624 from the National Institutes of Health. D.P.-S. and F.J.C. are recipients of fellowships from Consejo Superior de Investigaciones Cientificas (Spain).
1. Clarke, S. (1985) Annu. Rev. Biochem. 54, 479-506. 2. Gutierrez, L., Magee, A. I., Marshall, C. J. & Hancock, J. F. (1989) EMBO J. 8, 1093-1098. 3. Maltese, W. A., Sheridan, K. M., Repko, E. M. & Erdman, R. A. (1990) J. Biol. Chem. 265, 2148-2155. 4. Yamane, H. K., Farnsworth, C. C., Xie, H., Howald, W., Fung, B. K.-K., Clarke, S., Gelb, M. H. & Glomset, J. A. (1990) Proc. Nadl. Acad. Sci. USA 87, 5868-5872. 5. Mumby, S. M., Casey, P. J., Gilman, A. G., Gutowski, S. & Sternweis, P. C. (1990) Proc. Nadl. Acad. Sci. USA 87, 58735877. 6. Hancock, J. F., Magee, A. I., Childs, J. E. & Marshall, C. J. (1989) Cell 57, 1167-1177. 7. Casey, P. J., Solski, P. A., Der, C. J. & Buss, J. E. (1989) Proc. Nadl. Acad. Sci. USA 86, 8323-8327. 8. Farnsworth, C. C., Gelb, M. H. & Glomset, J. A. (1990) Science 247, 320-322. 9. Reiss, Y., Goldstein, J. L., Seabra, M. C., Casey, P. J. & Brown, M. S. (1990) Cell 62, 81-88. 10. Clarke, S., Vogel, J. P., Deschenes, R. J. & Stock, J. (1988) Proc. Natd. Acad. Sci. USA 85, 4643-4647. 11. Barbacid, M. (1987) Annu. Rev. Biochem. 56, 779-827. 12. Schafer, W. R., Kim, R., Sterne, R., Thorner, J., Kim, S.-H. & Rine, J. (1989) Science 245, 379-385. 13. Maltese, W. A. & Sheridan, K. M. (1987) J. Cell. Physiol. 133, 471-481. 14. Lai, R. K., Pdrez-Sala, D., Cafiada, F. J. & Rando, R. R. (1990) Proc. Nail. Acad. Sci. USA 87, 7673-7677. 15. Fukada, Y., Takao, T., Ohguro, H., Yoshizawa-, T., Akino, T. & Shimonishi, Y. (1990) Nature (London) 346, 658-660. 16. Swanson, R. J. & Applebury, M. L. (1983) J. Biol. Chem. 258, 10599-10605. 17. Ota, I. M. & Clarke, S. (1989) J. Biol. Chem. 264,12879-12884. 18. Wessling-Resnick, M. & Johnson, G. L. (1987) J. Biol. Chem. 262, 3697-3705. 19. Kamiya, Y., Sakurai, A., Tamura, S., Takahashi, N., Tsuchiya, E., Abe, K. & Fukui, S. (1979) Agric. Biol. Chem. 43, 363-369. 20. Christy, K. G., LaTart, D. B. & Osterhoudt, W. (1989) BioTechniques 7, 692-693. 21. Yamane, H. K. & Fung, B. K.-K. (1989) J. Biol. Chem. 264, 20100-20105. 22. Barber, J. R. & Clarke, S. (1984) J. Biol. Chem. 259, 71157122. 23. Pugh, C. S. G., Borchardt, R. T. & Stone, H. O. (1978) J. Biol. Chem. 253, 4075-4077.
