Fig 1: GSK3 phosphorlaytes mitochondrial outermembrane proteins and controls their turnover.A Experimental strategy to identify mitochondrial GSK3 interacting partners during quiescence. Created with Biorender. GO term analysis of proteins involved in mitochondrial functions (B) and Ubiquitin/proteasome system (C) identified by GSK3-Apex2 proximity labeling. D In vitro kinase assay tests GSK3 candidate targets identified in (B) (Tom complex components and VDAC) and (C) (UPS components) (n = 3 experimental replicates). Validation of GSK3 Kinase targets TOM22 (E) and VDAC (F) in 293 T cells (n = 4 experimental replicates). G A diagram of the of phosphorylation sites (Red letters) essential for GSK3 regulation of VDAC protein levels and a western examining how the mutation of these sites impact GSK3 mediated turnover of VDAC. Lanes 1 and 2 show the impact of wt GSK3 expression on VDAC stability. Lanes 3 and 4 shows phosphorylation site mutations in block VDAC GSK3 induced VDAC turnover(n = 3 experimental replicates). Significance values are calculated by a two -tailed student’s t-test assuming unequal variance. Error bars represent one standard deviation.
Fig 2: GSK3-mediated phosphorylation of VDAC drives proteasome recruiment.A TMRE mitochondrial membrane potential staining of st10 eggs from a control-RNAi and VDAC-RNAi ovariole. Quantification in Fig. S6A. B Quantification of the number of stage 10 egg chambers that display high levels of the TMRE staining (N = 20 ovaries). C Mitochondria-associated proteasome activity in mitochondrial fractions from st14 eggs from control-RNAi and VDAC-RNAi ovariole (n = 4 experiments on independent biological replicates). D K48 ubiquitin levels and zymography in mitochondrial fractions from st14 eggs from control-RNAi and VDAC-RNAi ovariole (n = 4 experiments on independent biological replicates). E K48 ubiquitination assay for VDAC in active and quiescent oocytes (n = 3 independent experiments). F Mitochondrial associated proteasome activity from control oocytes and oocytes that overexpress a Phospho-resistant form of VDAC (VDAC-PR) (n = 3 independent experiments). G Epistasis analysis of VDAC1 and FAO pathway in regulating mitochondria-associated proteasome activity (n = 5 biological replicates). P values were calculated by one-way analysis of variance (ANOVA). Error bars represent one standard deviation. **p < 0.001, *p < 0.05. Error bars represent 1 X standard deviation.
Fig 3: The proteasome is recruited to the mitochondria during cellular quiescence.A Model of Insulin-AKT-GSK3 regulation of mitochondrial respiration quiescence (MRQ). Created with Biorender. B Proteasome activity from st1–8 oocytes(active), st14 oocytes(quiescent), and embryos (collected 0–2h and 16–20 h posted egg laying) (n = 4 completely independent experiments on independent samples). C In-gel hydrolysis assay to examine total proteasome activity in st1–8 and st14 eggs. This experiment was replicated 3 times with biological replicates. D Western blotting examines K48 ubiquitin levels in total cell lysis in st1–8 and st14 eggs. E Mitochondria-associated proteasome activity in st1–8 and st14 oocytes (n = 6 independent biological replicates). F In-gel hydrolysis assay examine mitochondria-associated proteasome activity in st1–8 and st14 eggs. This experiment was replicated 3 times with biological replicates. G Western blotting to examine K48 ubiquitin levels in mitochondrial fractions in st1–8 and st14 eggs. This experiment was replicated 3 times with biological replicates. H Native Western blotting gels to examine 20 S core and 19 S regulatory components of the assembled proteasome in total cell lysis from st1–8 and st14 eggs. This experiment was replicated 3 times with biological replicates. I TMRE mitochondrial membrane potential staining of wild-type ovarioles treated with DMSO or proteasome inhibitor MG132 (50 µM, 2 h). All error bars represent 1 X standard deviation. J Summary of the effects of proteasome inhibitor MG132 (50 µM, 2 h) on TMRE mitochondrial membrane potential staining in the indicated stages. These data are normalized to the TMRE staining in the adjacent follicle cell (n = >20 biological replicates). In the box-and-whisker plots, the box represents the upper and lower quartiles and the line within the box is the mean. The whiskers represent the maximum and minimum values. P values were calculated by one-way analysis of variance (ANOVA). Error bars represent one standard deviation. **p < 0.001, *p < 0.05. Error bars represent 1X standard deviation.
Fig 4: GSK3 mediate suppression of fatty acid oxidation triggers proteasome recruitment.A Mitochondria-associated proteasome activity, proteasome zymography, and K48 ubiquitin levels in mitochondrial fractions in st14 eggs from control-RNAi and GSK3-RNAi ovarioles. (4 replicate experiments on independent biological replicates). B The oxygen consumption rate (OCR) measurements in st14 eggs from control-RNAi and GSK3-RNAi ovarioles (n = 6 biological replicates). C Acyl-carnitine levels from LC/MS analysis of st14 eggs from control-RNAi and GSK3-RNAi ovarioles(n = 8 biological replicates). In the box-and-whisker plots, the box represents the upper and lower quartiles and the line within the box is the mean. The whiskers represent the maximum and minimum values. D Levels of enzymes involved in mitochondrial fatty acid oxidation pathway (FAO) from proteomic analysis of eggs from st1–8 (active growth), st14 (control-RNAi), and st14 (GSK3-RNAi) ovarioles (n = 2 biological replicates). E, F TMRE mitochondrial membrane potential staining of control-RNAi, MTPa-RNAi(E), and ETFa-RNAi (F) ovarioles. Quantification in Figure S2. G OCR measurements in st10 eggs from a control-RNAi, MTPa-RNAi, and ETFa-RNAi ovariole(n = 9 biological replicates). Mitochondria-associated proteasome activity (n = 3 independent experiments on independent biological samples) and K48 ubiquitin levels in mitochondrial fractions from st14 eggs from control-RNAi, MTPa-RNAi (H), and ETFa-RNAi (I) ovarioles. Significance values are calculated by a two -tailed student’s t-test assuming unequal variance. Error bars represent one standard deviation. **p < .001, *p < .05. Error bars represent 1 X standard deviation.
Supplier Page from Sino Biological, Inc. for Human GSK3B Protein (His Tag)