Fig. 6

Indispensable roles of the AAA+ ATPase and ubiquitin ligase activities of mysterin.

(a) Schematic representation of mysterin enzymatic mutants. D1D2 contains four mutations at conserved amino acids in the two AAA+ ATPase modules, resulting in inactivation of ATPase activity and blockade of oligomer formation. ΔRING lacks the entire RING finger domain, which eliminates ubiquitin ligase activity. (b) Examination of the knockdown efficiency in morphants by RT-PCR. The lower bands represent the impaired splicing caused by the MO (arrow). (c) Ectopic expression of human wild-type and D1D2 mutant mysterin examined by Western blotting using an anti-FLAG antibody. Intact mysterin is indicated by arrow (Also see Supplementary Fig S4b). GAPDH is the loading control. (d) Restoration of fast muscle morphology in mysterin morphants by ectopic expression of wild-type human mysterin, but not of the ATPase mutant (D1D2-3×FLAG). Fast actin fibers are labeled with phalloidin (green). RFP expression is driven by fast muscle-specific GAL4FF and is thus an indicator of GAL4 activity. (e) Ectopic expression of human wild-type and ΔRING mutant mysterin examined by Western blotting using an anti-FLAG antibody. Mysterin band (591 kDa) is indicated by an arrow (Also see Supplementary Fig S4b). GAPDH is the loading control. (f) Restoration of the fast muscle anomaly by expression of wild-type human mysterin, but not of the ubiquitin ligase mutant (ΔRING-3×FLAG). Fast actin fibers are labeled with phalloidin (green). RFP expression is driven by fast muscle-specific GAL4FF and is thus an indicator of GAL4 activity.