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Methylation and Demethylation Reactions of Guanine Nucleotide

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Methylation and Demethylation Reactions of Guanine Nucleotide Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3043-3046, April 1991 Biochemistry Methylation and demethylation reactions of guanine nucleotide- binding 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 (receivedfor review October 26, 1990) ABSTRACT Retinal transducin was previously shown to ducin (Ty) is farnesylated at cysteine (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 substrate for the enzyme. 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- protein. 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 prenylation 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 amino acid, 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 trypsin inhibitor, 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 farnesyl pyrophosphate- 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 product 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.
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