Figure 3.
MOM protein degradation is inhibited by 26S proteasome dysfunction. (a) Western blots and quantification of Parkin on mitochondria purified from control and Psmc1fl/fl;CaMKIIα-Cre cortices at 3, 4 and 6 weeks old. COXIV was used as a loading control at each age for quantification; a representative COXIV is shown. Error bars represent S.E.M. of n=3 mice. *P<0.05 and **P<0.01 by unpaired Student's t-test. (b) K48 polyubiquitin (polyUb) Western blots of mitochondria purified from control and Psmc1fl/fl;CaMKIIα-Cre cortices at 3, 4 and 6 weeks old. Quantification used COXIV as a loading control at each age; a representative COXIV is shown. COXIV and LDH are shown for both mitochondria and cytosol to demonstrate mitochondrial purity. Threefold less cytosol was loaded to prevent overexposure with α-K48 ubiquitin. Efficiency of isolation procedure was also quantified by measuring mitochondrial and cytosol-specific marker enzymes citrate synthase and LDH respectively in total homogenate and then cytosol and mitochondria following the procedure (materials and methods). Error bars represent S.E.M. of n=3 mice. ***P<0.001 by unpaired Student's t-test. (c) Scatter plot showing log2 ratio of abundance of ubiquitinated proteins in 6-week-old control and Psmc1fl/fl;CaMKIIα-Cre cortices and -log10 P-value of t-test. In all, 1885 proteins were identified; 1504 were significantly different between experimental groups (red markers). Relative intensities of ubiquitin, Mfn2, p62 and Ambra1 are highlighted. UBQLN1 and USP2 shown for comparison. (d) UBQLN1 capture of ubiquitinated Mfn2 in control and Psmc1fl/fl;CaMKIIα-Cre cortices at 3, 4 and 6 weeks old using immunoblotting of independent samples from (c); n=3 mice. Specificity of the UBQLN1 pull-down is demonstrated using control beads without the UBQLN1 UBA domain. Although there is some Mfn2 binding to control beads this is not significant as shown by the pixel density (bottom image) at 6 weeks old and is similar for both control and Psmc1fl/fl;CaMKIIα-Cre cortices