FIGURE 6.

Recombinant His-YLR137W/Rkm5 produces monomethyllysine. Intact ribosomes isolated from the ΔYLR137W/rkm5 deletion strain were methylated in vitro with His-YLR137W/Rkm5 as described in the legend to Fig. 5. Intact ribosomes (45 μg of protein) isolated from the ΔYLR137W/rkm5 yeast deletion strain were incubated with 1 μm [³H]AdoMet (PerkinElmer; 75–85 Ci/mmol, from a 7 μm stock solution in 10 mm H₂SO₄/ethanol (9:1, v/v) (upper panel) or 20 μm [¹⁴C]AdoMet (Amersham Biosciences; 55.8 mCi/mmol, from a 448 μm stock solution in dilute sulfuric acid, pH 2.5–3.5) (lower panel) with 58 μg of the recombinant His-YLR137W/Rkm5 protein (prepared as described under “Experimental Procedures,” in 100 mm NaCl, 100 mm sodium phosphate at pH 7.0) at 30 °C for 16 h in a final volume of 100 μl. Methylated proteins were precipitated by the addition of an equal volume of 25% (w/v) trichloroacetic acid, incubation for 30 min at room temperature, and centrifugation at 2,700 × g for 30 min. The precipitated protein pellet was washed with 100 μl of cold acetone, acid hydrolyzed, and dried as described above. After mixing the pellet with 5 μl of water and 10 nmol of each of the amino acid standards, ascending thin layer chromatographic separation was performed using a silica-coated plate as described in the Fig. 4 legend. The standards were visualized using ninhydrin, and radioactivity was detected for each 5-mm section (indicated by vertical lines) as described in the Fig. 4 legend. The arrow marks the origin by fraction 1; the solvent front was run near the end of the plate to fraction 38. The slightly slower migration of the ³H-methyl-labeled monomethyllysine derivative (upper panel) as compared with the ¹⁴C methyl-labeled derivative (lower panel) appears to be due to a tritium isotope effect (see text).