Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3043-3046, April 1991 Biochemistry Methylation and demethylation reactions of guanine nucleotide- binding 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 , Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115 Communicated by William von Eggers Doering, January 2, 1991 (receivedfor review October 26, 1990)

ABSTRACT Retinal transducin was previously shown to ducin (Ty) is farnesylated at (14). As expected, this be farnesylated on its y subunit. This farnesylation reaction on farnesylcysteine residue is also methylated (15). Here we a cysteine residue near the carboxyl terminus is followed by show that bovine retinal rod outer segments (ROS) contain an peptidase cleavage at the cysteine. Thus the modified cysteine S-adenosyl-L-methionine (SAM)-linked methyltransferase becomes the carboxyl terminus. It is shown here that the free activity that can methylate endogenous and added transdu- carboxyl group can be methylated by an S-adenosyl-L- cin, or synthetic substrates. In addition, the methylation of methionine-dependent methyltransferase associated with the the a subunit of phosphodiesterase (16) and of a group of 23- rod outer segment membranes. This process can be inhibited by to 29-kDa membrane proteins (17) is also observed. The 23- S-adenosyl-L-homocysteine and sinefungin. Moreover, syn- to 29-kDa polypeptides have been postulated to belong to a thetic N-acetyl-S-farnesyl-L-cysteine, but not N-acetyl-L- family of small G proteins (17). We previously demonstrated cysteine, is a for the . Rapid demethylation of that these latter proteins could be prenylated in vitro (14). The N-acetyl-S-farnesyl-L-cysteine methyl ester can be observed in terminal cysteine of TY is the site of methylation in this G the membranes. Transducin is also enzymatically demethyl- . The methylating activity is inhibited by S-adenosyl- ated by the rod outer segment membranes. Moreover, the 23- L-homocysteine (SAH) and sinefungin. In the presence of to 29-kDa small G proteins are methylated and demethylated inhibitor, the membranes hydrolyze previously methylated in this system. These data suggest that methylation/demeth- transducin and the 23- to 29-kDa small G proteins as well as ylation may play a regulatory role in visual signal transduction. synthetic substrates. Thus, transducin and the small G pro- teins are shown to be reversibly methylated and demethyl- The reversible methylation ofenzymes at acidic residues can ated by separate enzymatic activities. Since signal transduc- have important regulatory consequences (1). Recently, meth- tion is best understood in the vertebrate visual system, these ylation processes have been shown to be combined with studies pave the way for a quantitative understanding of the in the posttranslational modifications of "small" role of methylation in signal transduction. guanine nucleotide-binding proteins (G proteins), including Ras (2, 3), and the heterotrimeric G proteins (4, 5). The MATERIALS AND METHODS putative sequence of events here involves the initial prenyl- ation of cysteine residues that are part of a CAAX motif Materials. Frozen bovine retinas were obtained from (where C = cysteine, A = an aliphatic , and X = Wanda Lawson Co. (Lincoln, NE). [methyl-3HJSAM (85 any amino acid) located at the carboxyl-terminal end of the Ci/mmol; 1 Ci = 37 GBq) and Amplify were from Amersham. protein (6). The prenylation process can involve either far- Sinefungin, dithiothreitol, soybean , phenyl- nesylation (C15) (7) or geranylgeranylation (C20) (8). These methylsulfonyl fluoride, GTP, and N-acetyl-L-cysteine were modifications involve the enzymatic transfer of the prenyl from Sigma. SAH, endoproteinase Glu-C (Staphylococcus aureus V8 protease), leupeptin, pepstatin, and aprotinin were group from farnesyl or geranylgeranyl pyrophosphate to the obtained from Boehringer Mannheim. trans,trans-Farnesyl cysteine residue ofthe protein, forming a new thioether bond bromide was from Aldrich. in the process. Recently, a - Preparation of ROS, Transducin, and Washed ROS Mem- dependent farnesyltransferase has been purified (9). Follow- branes. The protocol used was based on a published method ing prenylation, the AAX sequence is thought to be cleaved (18). A fraction ofthe ROS collected at the interface ofa step by a specific protease, resulting in the formation of a car- gradient from 25% to 35% (wt/wt) sucrose was resuspended boxyl-terminal prenylated cysteine residue (2). The free in 50 mM Hepes Na, pH 7.4/100 mM NaCl/5 mM MgCl2/0.1 carboxyl terminus can be then methylated (10). It is thought mM phenylmethylsulfonyl fluoride/0.1 mM dithiothreitol that the function ofthese posttranslational modifications is to (buffer A) and stored in small aliquots at -80°C until used. anchor the G protein to the target membrane, allowing the G The protein to express its activity (6). This association may or remaining ROS membranes were washed by a series of may not be receptor-mediated. The overall process appears centrifugation steps and transducin was eluted from the to be critical in cellular function, because interruption of the membranes with 100 ,uM GTP. Washed ROS membranes prenylation reaction via mutagenesis or with inhibitors leads were then resuspended in buffer A and stored at -80°C until to a decrease in cellular growth and function (11-13). used. Since the carboxyl methylation reaction is the only one of Synthesis of N-Acetyl-S-trans,trans-Farnesyl-L-Cysteine the three posttranslational modifications likely to be revers- (AFC) and Its Methyl Ester. AFC was prepared from N-ace- ible, this process is apt to be important in controlling the tyl-L-cysteine and trans,trans-farnesyl bromide by a method activities of prenylated proteins. We previously showed that similar to that described previously (19). AFC was treated the y subunit of the heterotrimeric retinal G protein trans- Abbreviations: SAM, S-adenosyl-L-methionine; SAH, S-adenosyl- L-homocysteine; AFC, N-acetyl-S-trans,trans-farnesyl-L-cysteine; The publication costs of this article were defrayed in part by page charge ROS, rod outer segment(s); G protein, guanine nucleotide-binding payment. This article must therefore be hereby marked "advertisement" protein; Ty, y subunit of transducin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

3043 Downloaded by guest on September 26, 2021 3044 Biochemistry: Pdrez-Sala et al. Proc. Natl. Acad. Sci. USA 88 (1991) with methanolic HCl (0.05 M) to afford AFC methyl ester. When the radioactively labeled 6-kDa protein was ana- NMR spectroscopic and mass spectrometric data for both lyzed by HPLC, the radioactivity was coeluted with T. (Fig. compounds were in complete accordance with the assigned 2A). When the radioactive polypeptide was cleaved with V8 structures. protease and analyzed by HPLC, two main peaks of radio- In Vitro Methylation Reactions. The basic reaction mixture activity were detected, one at 3 min, in the position expected contained 20 GCi of [methyl-3H]SAM (2.34 ,uM) and an for radioactive methanol, and a second peak at 42 min (Fig. aliquot of ROS (120 gg of total protein) or of washed ROS 2B). Analysis ofthis peptide by Edman degradation gave the membranes (80 ,ug of total protein), as the source of meth- sequence Leu-Lys-Gly-Gly-Xaa, which corresponds to the yltransferase, in 100 ,ul of buffer A. Purified transducin was carboxyl-terminal fragment of T.,, confirming that methyl- added to this mixture at 5 uM final concentration. AFC, ation occurs at the cysteine residue, as has been recently N-acetylcysteine, or AFC methyl ester was added in 2 ,ul of reported (15). dimethyl sulfoxide to give a final concentration of 20 p.M. Nature of the Methylation Process. The results described Incubations were carried out at 370C. above show that the in vitro methylation of T., occurs at the SDS/PAGE and Fluorography. For SDS/PAGE, aliquots terminal cysteine residue. The nature of the methylation of the reaction mixture were processed as described (14) and process was investigated next. The enzymatic activity was run in 15% gels. To improve the resolution of low molecular destroyed by heat and inhibited by two well-characterized weight polypeptides, 0.1 M sodium acetate was included in inhibitors of SAM-dependent methyltransferases, SAH (22) the anode buffer (20). Radioactive polypeptides were visu- and sinefungin (23) (Fig. 1B). In addition to SAH and alized by fluorography (14). Exposure was at -70'C for 3-6 sinefungin, AFC also inhibited the incorporation oflabel into days. proteins (Fig. 1B). Importantly, AFC also serves as a sub- Proteolysis of [3H]Methylated T. Purified [3H]methylated strate for the methyltransferase (Fig. 3). The identity of the Ty (30 Lg, 10-5 dpm) was freeze-dried and redissolved in 300 as AFC [3H]methyl ester was demonstrated by ttl of 0.1 M ammonium bicarbonate containing 1.5 pug of S. and HPLC criteria. aureus V8 protease. Proteolysis was carried out at 37°C for coelution with authentic standard by TLC 4 hr.

RESULTS 1.0 In Vitro Methylation of T.,. Incubation of bovine ROS with [methyl-3H]SAM results in the radioactive labeling of several 0.5- polypeptides with apparent molecular masses of 88, 60, 23-29, and 6 kDa (Fig. 1A). The methylation of the 88-kDa 0.01, t15 protein (a subunit ofretinal phosphodiesterase) and ofthe 23- to 29-kDa polypeptides has already been reported (16, 17). 10 ° The 6-kDa polypeptide coincides with TY in SDS/poly- x acrylamide gels (Fig. 1C). Moreover, incubation of purified 5 CLE transducin with extensively washed ROS and [3H]SAM re- sults in the labeling of the 6-kDa polypeptide and the mem- brane-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 ob- B served in cultured cells (3) and the 23-kDa G protein purified from brain (21). This conclusion is based on our previous observation that they are prenylated (14), the fact that they 0.8- are methylated, their molecular masses, and their ability to on membranes (data not shown). II bind GTP nitrocellulose LKGGC* LO I A B C 0.4 V8 3 4 5 K - 106 _ 80

50 0.0 2'- x k* 33 I - 28 0 5 20 25 30 35 40 45 50 _ 19 Retention time, min 14 FIG. 2. (A) HPLC purification of [3H]methylated Ty. [3H]Meth- - - ylated transducin was extracted from the membranes by washing 2.5 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 FIG. 1. Methylation of ROS proteins and puirified transducin. In A, Rainin, Woburn, MA) with eluants A (10 mM trifluoroacetic acid vitro methylation reactions were carried out uinder the conditions in water) and B (10 mM trifluoroacetic acid in acetonitrile), using specified in Materials and Methods. After 3 hr. aliquots of the eluant Afor 5 min and then a0-95% gradient ofB during 45 min. Flow reaction mixtures were processed for SDS/PAG;E and fluorography. rate was 0.75 ml/min. Fractions (0.5 ml) were collected and mixed (A) Methylation of ROS. (B) Methylation of piurified transducin (5 with 7 ml of Hydrofluor (National Diagnostics, Manville, NJ) for ,uM) under standard conditions (lane 1), when RIOS membranes were scintillation counting. The identity of the T. peak was confirmed by heated at 100NC for 5 min before the incubatio)n (lane 2), or in the amino acid analysis. (B) Analysis of digested [3H~methylated TY with presence of 200 .M sinefungin (lane 3), 2 mM 'SAH (lane 4), or 250 the HPLC system described above. The labeled peptide was purified ,uM AFC (lane 5). (C) Coomassie blue staining offpurified transducin. and its sequence was obtained by Edman degradation. Downloaded by guest on September 26, 2021 Biochemistry: Pe'rez-Sala et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3045 1 2 3 KDa

-106 -80 o75- -50 x -33

E - 28

- 19

- 14 25- - 6 -2.5

~0 60 120 180 FIG. 5. Demethylation of the 23- to 29-kDa G proteins and Incubation time, min transducin. Transducin was incubated with ROS membranes and [3H]SAM. After 2 hr of incubation, methylation was inhibited by FIG. 3. Time-dependent formation of AFC [3H]methyl ester. addition of 200 uM sinefungin. Aliquots of the incubation mixture AFC (o) or AFC methyl ester (s), at a concentration of 20 AM, was were taken 10 min (lane 1), 30 min (lane 2), and 2 hr (lane 3) after the incubated with [3H]SAM and ROS membranes. At the indicated time addition of the inhibitor and processed for SDS/PAGE and fluorog- points, 50-Al aliquots were withdrawn from the incubation mixture raphy. Fluorographic exposure was for 3 days. Quantitation of the and the reaction was quenched with 500 ul ofchloroform/methanol, fluorographic spots showed a decrease of 50% in the radioactivity 1:1 (vol/vol). AFC [3H]methyl ester was extracted by "vortexing" associated with the 23- to 29-kDa proteins and of 20%o in the for 1 min in this mixture. Phase separation was achieved by adding radioactivity associated with TY after 2 hr. 250 tl of water. The chloroform layer, containing 95% of the AFC [3H]methyl ester, was evaporated under argon, resuspended in 15% ester was rapidly hydrolyzed by the demethylase activity in 2-propanol in hexane, and spiked with authentic AFC methyl ester the membranes. Heating of the membranes strongly dimin- standard for UV detection (210 nm). Samples were injected on a ished the demethylase activity, as is expected of an enzy- normal-phase HPLC column (Dynamax 60 A, Rainin) and elution was performed with the same solvent. Radioactivity was counted matic activity. Under similar conditions, the catalyzed de- with an on-line Berthold (Nashua, NH) LB 506-C HPLC radioac- methylation of T. (Fig. 4B) and the putative small G proteins tivity monitor. (Fig. 5), was also demonstrated. It appears that the small G proteins are more rapidly demethylated than T., 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 DISCUSSION substrate activity was demonstrated by showing that N-ace- The results show that T, can be enzymatically methylated by tylcysteine is not a substrate for the methyltransferase (data an SAM-dependent methyltransferase activity bound to the not shown). ROS membranes. It is interesting that as isolated here, TY The Methylation Is a Reversible Process. Incubation of appears to be largely (>80%) methylated, as deduced from nonradioactive AFC methyl ester with [3H]SAM and washed HPLC analysis of control and base-treated transducin (re- ROS membranes resulted in a linear, time-dependent incor- sults not shown). Other proteins are also methylated, includ- poration of radioactivity into AFC methyl ester for at least 2 ing the small G proteins (17) and the a subunit of the retinal hr (Fig. 3). To study the demethylation process directly, AFC phosphodiesterase (16). The methylation site of TY was [3H]methyl ester was incubated with ROS membranes in the shown to be on the terminal cysteine residue, a result in presence of methylation inhibitors (Fig. 4A). The methyl accord with observations made by others using different

75 A

x 50 - x 0.vE

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. Downloaded by guest on September 26, 2021 3046 Biochemistry: PNrez-Sala et al. Proc. Natl. Acad. Sci. USA 88 (1991) analytical methods (15). Standard inhibitors of SAM meth- to invent specific inhibitors of these . The finding yltransferase enzymes, such as SAH and sinefungin, potently that AFC and AFC methyl ester are substrates for the inhibited enzymatic activity when T7 was the substrate. respective enzymes augurs well for the potential design of Interestingly, AFC also inhibited TY methylation. This inhi- mechanism-based inhibitors of the methyltransferase and bition occurred because AFC is a substrate for the methyl- esterase enzymes. ating enzyme. The farnesyl moiety is important for activity, since N-acetyl-L-cysteine is without activity as a substrate for This work was supported by U.S. Public Health Service Research the enzyme. Although we have not directly studied the issue, Grant EY03624 from the National Institutes of Health. D.P.-S. and it appears likely that the local peptide structure is relatively F.J.C. are recipients of fellowships from Consejo Superior de unimportant as a substrate determinant. Investigaciones Cientificas (Spain). When the methylated T.,, small G proteins, and AFC 1. Clarke, S. (1985) Annu. Rev. Biochem. 54, 479-506. methyl ester were incubated with ROS membranes in the 2. Gutierrez, L., Magee, A. I., Marshall, C. J. & Hancock, J. F. presence of methyltransferase inhibitors, the methyl esters (1989) EMBO J. 8, 1093-1098. were hydrolyzed by an enzymatic activity in the membranes. 3. Maltese, W. A., Sheridan, K. M., Repko, E. M. & Erdman, It appears, however, that the turnover rate for the 23- to R. A. (1990) J. Biol. Chem. 265, 2148-2155. 29-kDa proteins is faster than that for transducin. The lower 4. Yamane, H. K., Farnsworth, C. C., Xie, H., Howald, W., rate of TY methyl ester hydrolysis might suggest that this Fung, B. K.-K., Clarke, S., Gelb, M. H. & Glomset, J. A. reaction is of little regulatory significance. However, it is (1990) Proc. Nadl. Acad. Sci. USA 87, 5868-5872. the rate is substantially greater under physio- 5. Mumby, S. M., Casey, P. J., Gilman, A. G., Gutowski, S. & possible that Sternweis, P. C. (1990) Proc. Nadl. Acad. Sci. USA 87, 5873- logical conditions. By contrast, the rate of demethylation of 5877. the 23- to 29-kDa proteins seemed to be quite substantial, 6. Hancock, J. F., Magee, A. I., Childs, J. E. & Marshall, C. J. suggesting a possible regulatory role for methylation/ (1989) Cell 57, 1167-1177. demethylation of these proteins in visual transduction. 7. Casey, P. J., Solski, P. A., Der, C. J. & Buss, J. E. (1989) The notion that reversible methylation is important in Proc. Nadl. Acad. Sci. USA 86, 8323-8327. regulating the G-protein-mediated signal-transduction cas- 8. Farnsworth, C. C., Gelb, M. H. & Glomset, J. A. (1990) Sci- cade is attractive. As previously mentioned, the other post- ence 247, 320-322. translational modifications linked to methylation (prenyla- 9. Reiss, Y., Goldstein, J. L., Seabra, M. C., Casey, P. J. & cleavage) are almost certainly irreversible. Brown, M. S. (1990) Cell 62, 81-88. tion and peptidase 10. Clarke, S., Vogel, J. P., Deschenes, R. J. & Stock, J. (1988) This leaves the methylation step as the only one that is Proc. Natd. Acad. Sci. USA 85, 4643-4647. reversible, as is demonstrated here. The effect of methyl- 11. Barbacid, M. (1987) Annu. Rev. Biochem. 56, 779-827. ation/demethylation is chemically significant because the 12. Schafer, W. R., Kim, R., Sterne, R., Thorner, J., Kim, S.-H. neutral ester moiety is converted into a carboxylate anion. & Rine, J. (1989) Science 245, 379-385. This modification would be expected to be important irre- 13. Maltese, W. A. & Sheridan, K. M. (1987) J. Cell. Physiol. 133, spective of whether prenylated proteins associate with mem- 471-481. branes by partitioning or by a receptor-mediated event. 14. Lai, R. K., Pdrez-Sala, D., Cafiada, F. J. & Rando, R. R. Since transducin is the best understood of the signal- (1990) Proc. Nail. Acad. Sci. USA 87, 7673-7677. G it is also the system 15. Fukada, Y., Takao, T., Ohguro, H., Yoshizawa-, T., Akino, T. transducing heterotrimeric proteins, & Shimonishi, Y. (1990) Nature (London) 346, 658-660. that should be most fruitfully investigated with respect to the 16. Swanson, R. J. & Applebury, M. L. (1983) J. Biol. Chem. 258, posttranslational modifications discussed here. It will be 10599-10605. especially interesting to determine what the role of methyl- 17. Ota, I. M. & Clarke, S. (1989) J. Biol. Chem. 264,12879-12884. ation is in the control of the visual transduction process. 18. Wessling-Resnick, M. & Johnson, G. L. (1987) J. Biol. Chem. There are several candidates possible. First, the interaction 262, 3697-3705. of methylated and demethylated transducin with photoacti- 19. Kamiya, Y., Sakurai, A., Tamura, S., Takahashi, N., Tsu- vated rhodopsin will need to be investigated. Other points of chiya, E., Abe, K. & Fukui, S. (1979) Agric. Biol. Chem. 43, possible control include the rates at which Ta hydrolyzes 363-369. with or all, of these 20. Christy, K. G., LaTart, D. B. & Osterhoudt, W. (1989) Bio- GTP and then reassociates Tp3y. Some, Techniques 7, 692-693. mechanisms could be involved in visual adaptation mecha- 21. Yamane, H. K. & Fung, B. K.-K. (1989) J. Biol. Chem. 264, nisms. The role(s) of the 23- to 29-kDa G proteins in visual 20100-20105. transduction processes is currently obscure and will need to 22. Barber, J. R. & Clarke, S. (1984) J. Biol. Chem. 259, 7115- be investigated. 7122. Given that methylation/demethylation steps can be impor- 23. Pugh, C. S. G., Borchardt, R. T. & Stone, H. O. (1978) J. Biol. tant elements in signal transduction, it will be ofsome interest Chem. 253, 4075-4077. Downloaded by guest on September 26, 2021