Figure 5.

Lys-561 methylation stabilizes Hspa8. A, Western blot analysis of mutated Hspa8 after cycloheximide-induced inhibition of translation. B, Western blot analysis showing the effect of Hspa8 overexpression (Hspa8^OE) on Mef2A and Mef2D proteins in myotubes. Primary myoblasts were induced to differentiate for 2 days and then were transduced with Hspa8^OE or control adenoviral vectors for 3 more days. C, luciferase assays showing the effects of Hspa8 on the transcriptional activity of Mef2A, Mef2C, and Mef2D using the 3XMEF2-luc reporter. Error bars represent mean + S.D. of five independent biological experiments. * indicates p < 0.05 (Student's t test). D, Western blot analysis of indicated proteins in sarcoplasm and myonuclei proteins of SOL muscles from HET and KO mice. E, qPCR analysis of Mettl21c in differentiated myoblasts transduced with Mettl21c-GFP (Mettl21c^OE) or GFP only (as control) adenoviral vectors. Primary myoblasts were induced to differentiate for 2 days and then were transduced with Mettl21c^OE or control adenoviral vectors for 3 more days. Error bars represent mean + S.D. of five independent biological experiments. * indicates p < 0.05 (Student's t test). F, Western blot analysis of indicated proteins in control and Mettl21c^OE myotubes. G, Western blot analysis of indicated proteins in control and Mettl21c^OE myotubes after Lamp2 knockdown. Primary myoblasts were treated with lentivirus for 2 days and selected in puromycin (1 μg/ml) for 2 additional days. These primary myoblasts were then transduced with Mettl21c^OE or control adenoviral vectors. Two days after adenoviral transduction, the myoblasts were induced to differentiate for 3 days. H, diagram summarizing the role of Mettl21c in type I myofibers. Mettl21c methylates Hspa8 at Lys-561 to interrupt its binding with an E3-ligase CHIP, and to prevent its ubiquitination and proteasome-dependent degradation. The surviving Hspa8 carries Mef2A and Mef2D to lysosome for degradation in chaperone-mediated autophagy pathway.