for her excellent technical assistance and Ms.Karen Goodlet for manuscript preparation. ... Mills WR, Joy KW (1980) A rapid method for isolation of purified ...
Plant Physiol. (1991) 97, 913-920 0032-0889/91/97/091 3/08/$01 .00/0
Received for publication March 21, 1991 Accepted June 17, 1991
Partial Purification and Characterization of Ribulose- 1 ,5-bisphosphate Carboxylase/Oxygenase Large Subunit N-Methyltransferasel Robert L. Houtz*, Malcolm Royer, and Michael E. Salvucci Department of Horticulture and Landscape Architecture, Plant Physiology/Biochemistry/Molecular Biology Program (R.L.H., M.R.), and United States Department of Agriculture-Agricultural Research Service (M.E.S.), University of Kentucky, Lexington, Kentucky 40546
Ribulose-P2 carboxylase2 catalyzes the reduction of atmospheric CO2 during photosynthesis. Much is known about the quaternary structure, catalytic mechanism, active site residues, and in vivo regulatory mechanisms for this abundant enzyme (1). The crystal structure of the higher plant enzyme from spinach has been thoroughly analyzed and described in detail (17). The hexadecameric protein is composed of eight nuclear-encoded SS and eight chloroplast-encoded LS which contain the active site. The LS ofribulose-P2 carboxylase from tobacco and muskmelon plants has been shown to be posttranslationally modified by trimethylation of the e-amine group of Lys-14 in the N terminus. However, wheat and spinach ribulose-P2 carboxylase contain an unmodified lysyl residue at position 14 in the LS (14). The significance of this modification and the reason(s) for the species differences are unknown. The N-terminal region of the LS of ribulose-P2 carboxylase is essential for maximum catalytic activity (15), participates in formation of the active site (17, 18), and shows catalytic dependent conformational changes during catalysis as judged by reduced tryptic accessibility of N-terminal lysyl residues (13). Cyt c (lysine) 'N-methyltransferase (7), ribosomal protein (lysine) 'N-methyltransferase (4), histone (lysine) 'N-methyltransferase (33), and calmodulin (lysine) 'N-methyltransferase (10, 30) all show a high degree of protein substrate specificity. Of these 'N-methyltransferases, only calmodulin (lysine) 'Nmethyltransferase (30), and to a lesser extent Cyt c (lysine) 'N-methyltransferase, has been purified and characterized. An
ABSTRACT The large subunit (LS) of tobacco (Nicotiana rustica) ribulose1,5-bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase) contains a trimethyllysyl residue at position 14, whereas this position is unmodified in spinach ribulose-P2 carboxylase. A protein fraction was isolated from tobacco chloroplasts by rate-zonal centrifugation and anion-exchange fast protein liquid chromatography that catalyzed transfer of methyl groups from S-adenosyl[methyl-3H]-L-methionine to spinach ribulose-P2 carboxylase. 3HMethyl groups incorporated into spinach ribulose-P2 carboxylase were alkaline stable but could be removed by limited tryptic proteolysis. Reverse-phase high-performance liquid chromatography of the tryptic peptides released after proteolysis showed that the penultimate N-terminal peptide from the LS of spinach ribulose-P2 carboxylase contained the site of methylation, which was identified as lysine-14. Thus, the methyltransferase activity can be attributed to S-adenosylmethionine:ribulose-P2 carboxylase LS (lysine) 'N-methyltransferase, a previously undescribed chloroplast enzyme. The partially purified enzyme was specific for ribulose-P2 carboxylase and exhibited apparent Km values of 10 micromolar for S-adenosyl-L-methionine and 18 micromolar for ribulose-P2 carboxylase, a V,,,. of 700 picomoles CH3 groups transferred per minute per milligram protein, and a broad pH optimum from 8.5 to 10.0. S-Adenosylmethionine:ribulose-P2 carboxylase LS (lysine)'N-methyltransferase was capable of incorporating 24 3H-methyl groups per spinach ribulose-P2 carboxylase holoenzyme, forming I mole of trimethyllysine per mole of ribulose-P2 carboxylase LS, but was inactive on ribulose-P2 carboxylases that contain a trimethyllysyl residue at position 14 in the LS. The enzyme did not distinguish between activated (Mg2+ and C02) and unactivated forms of ribulose-P2 carboxylase as substrates. However, complexes of activated ribulose-P2 carboxylase with the reaction-intermediate analogue 2'-carboxy-D-arabinitol1,5-bisphosphate, or unactivated spinach ribulose-P2 carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS 'EN-methyltransferase.
S-adenosylmethionine:protein (lysine) 'N-methyltransferase specific for the methylation of Lys-14 in the LS of ribuloseP2 carboxylase should be localized in chloroplasts from plants in which the LS of ribulose-P2 carboxylase contains trimethyllysine-14. By analogy with studies characterizing calmodulin 'N-methyltransferase (30), ribulose-P2 carboxylase with an unmodified lysyl residue at position 14 in the LS should serve as a substrate for the assay, isolation, and characterization of ribulose-P2 carboxylase LS 'N-methyltransferase. In this report a method is described for the assay, partial purifi-
' This work was supported by U.S. Department of Agriculture/ Competitive Research Grants Office Grant 89-37262-4482 and Hatch Project KY 00586 to R.L.H. The investigation reported in this paper (No. 90-10-221) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director.
2Abbreviations: ribulose-P2 carboxylase, ribulose- 1 ,5-bisphosphate carboxylase-oxygenase; ribulose-P2, ribulose-1 ,5-bisphosphate; LS (SS), the large (small) subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase; AdoMet, S-adenosyl-L-methionine. 913
HOUTZ ET AL.
cation, and characterization of tobacco ribulose-P2 carboxylase LS 'N-methyltransferase using spinach ribulose-P2 carboxylase as substrate. We also show that the site of methylation in spinach ribulose-P2 carboxylase by ribulose-P2 carboxylase LS EN-methyltransferase is lysyl residue 14 in the LS. MATERIALS AND METHODS Chemicals and Proteins
S-Adenosyl-[methyl-3H]-L-methionine ([methyl-3H]AdoMet, 70-80 Ci/mmol) was purchased from New England Nuclear. Unlabeled AdoMet from Sigma was purified before use by chromatography on Whatman CM-52 as described previously (6). Ribulose-P2 was synthesized from ribose 5phosphate and ATP and purified by chromatography on Dowex-l-chloride with a linear gradient of 0 to 0.5 M LiCl prepared in 5 mm HCI (12). 2'-Carboxy-D-arabinitol-I-,5bisphosphate was prepared as described before (26). TFA (sequanal grade) was obtained from Pierce Chemical Co., and HPLC grade acetonitrile was obtained from Fisher. All aqueous solutions were prepared with Milli-Q deionized water (18 MQ * cm-'). Horse heart Cyt c, bovine muscle actin, calf thymus histone III, and bovine serum albumin were from Sigma. Ribulose-P2 carboxylases (21) and ribulose-P2 carboxylase activase protein (29) were purified from spinach leaves. Calmodulin (VU-1), derived from a cloned synthetic gene (27), was provided by Daniel M. Roberts. Protein was determined by a modified Lowry procedure (2).
Chloroplast Isolation and Purification of Ribulose-P2 Carboxylase LS fN-Methyltransferase Chloroplasts were mechanically isolated from the leaves of 3- to 4-week-old tobacco (Nicotiana rustica cv pulmila) plants (22) with slight modifications. N,N-bis(2-hydroxyethyl)glycine-KOH buffer (50 mM) was used instead of Tricine in the chloroplast extraction medium, and 0.1 % bovine serum albumin was omitted. Usually, two chloroplast preparations each from 50 g ofdeveined and washed leaves were combined. Isolated chloroplasts were lysed by vigorous resuspension in lysis buffer (50 mm Hepes-KOH [pH 8.0], 5 mM MgCl2, 1 mM EDTA, 2 mm DTT) and incubated on ice for 5 min. After centrifugation at 12,500g for 4 min, the supernatant was made 0.12 M sucrose and layered on linear 0.2 to 0.8 M sucrose gradients (31). The gradients were centrifuged at 65,000 rpm for 2 h at 4°C in a Beckman Ti-70 rotor. The sucrose gradients were fractionated from top to bottom into two fractions corresponding to two separate peaks of A280 absorbing material: a 17-S fraction that contained primarily ribulose-P2 carboxylase. The stromal protein fraction from the top of the gradient contained the majority of the "N-methyltransferase activity. The stromal protein fraction was further fractionated by anion-exchange fast protein liquid chromatography on a Mono-Q column (32). Ribulose-P2 carboxylase LS "N-Methyltransferase Assay The radiometric assay of ribulose-P2 carboxylase LS ENmethyltransferase activity was similar to procedures described
Plant Physiol. Vol. 97, 1991
for calmodulin 'N-methyltransferase (24) with slight modifications. Standard assays contained 60 AM [methyl-3H]AdoMet (0.5-1.0 ,uCi/nmol), 1 mg spinach ribulose-P2 carboxylase (decarbamylated), 100 mM Hepes-KOH (pH 8.0), 2 mm DTT, 50 mM KCl, 2 mM MgCl2, and 10 ,uL of enzyme source (5100 ,ug protein) in a final volume of 100 IuL. Reactions were initiated by addition of enzyme, incubated at 30°C for 10-60 min, and terminated by addition of 500,uL 10% TCA. When assays contained