FIGURE 2.

METTL21A interacts with and methylates members of the human Hsp70 protein family. A, members of the Hsp70 family co-purify with METTL21A in TAP. Shown is a Coomassie Blue-stained SDS-polyacrylamide gel of lysates and eluates from TAP from a Flp-In T-REx HEK-293-derived cell line treated, where indicated, with doxycycline (Dox) to induce expression of SBP- and CBP-tagged METTL21A. The identity of bait and co-purifying proteins was determined by peptide mass fingerprinting and is indicted to the right. Molecular mass standards (in kilodaltons) are indicated to the left. B, METTL21A methylates HSPA8. 200 pmol of untagged or GST-tagged HSPA8 was incubated with [³H]SAM in the presence or absence of 1 mm ATP and 100 pmol of METTL21A. Upper panel, quantification of incorporated TCA-precipitable radioactivity by liquid scintillation counting. Error bars indicate S.D. (n = 3). *, significant effects of ATP addition (Student's two-tailed t test; alpha level of 0.05, assuming unequal variance). Middle and lower panels, Ponceau S staining and fluorography, respectively, of membranes containing SDS-PAGE-separated methyltransferase reactions corresponding to those in the upper panel. C, activity of METTL21A on various human Hsp70 proteins. The indicated Hsp70 proteins (200 pmol) were incubated with 1 mm ATP and varying amounts of METTL21A in the presence of [³H]SAM. Error bars represent the range of duplicate experiments. No SUB, no substrate.