Fig. 5.

KEAP1 controls the degradation of full-length PGAM5. A. KEAP1 interacts only with full-length PGAM5. The left panel shows that full-length PGAM5-FLAG, but not cleaved PGAM5, co-immunoprecipitates with KEAP1-HA in HEK cells. Note also that PGAM5 E79A/S80A co-immunoprecipitates with KEAP1 less efficiently than PGAM5-WT-FLAG. The right panel shows quantification of full length and cleaved PGAM5-WT from total cell lysate and KEAP1 interacting fraction. ****P < 0.0001 compared with respective total cell lysate group, n = 3 independent IP-s, one-way ANOVA followed by Sidak's multiple comparison test. B. KEAP1 overexpression induces degradation of PGAM5. The left panel shows the representative Western blot image of PGAM5 expression in the presence of KEAP1-FLAG or KEAP1 R380A/R415A-FLAG in HEK cells. Note also that KEAP1-FLAG but not KEAP1 R380A/R415A-FLAG is co-immunoprecipitating with PGAM5-HA. The right panel shows quantification of full length and cleaved PGAM5 from total cell lysate. **P < 0.01, ns: not significant, n = 4 independent experiments, one-way ANOVA followed by Sidak's multiple comparison test. C. KEAP1 silencing increases the level of endogenous PGAM5. Representative Western blot image (left) and analysis of PGAM5 expression (right) in PC6 cells expressing scrambled or KEAP1 shRNA. ***P < 0.001 and ****P < 0.0001, n = 4, t-test. D. PGAM5 is accumulating in the cytosol after proteasome inhibition. Representative superresolution Airyscan images showing PC6 cells expressing PGAM5-YPet and mitochondrially targeted Kate2 treated with DMSO or 25 μM MG132 for 5 h. E. Proteasome inhibition leads to cytosolic accumulation of full-length PGAM5. Representative Western blot image of PGAM5-Flag levels in cytosolic and mitochondrial fractions of PC6 cells treated DMSO or 25 μM MG132 for 5 h. ATP5A and GAPDH as markers of mitochondria and cytosol, respectively, demonstrate the purity of fractions.