SQSTM1/p62 promotes mitochondrial ubiquitination independently of PINK1 and PRKN/parkin in mitophagy

Abstract

The ubiquitination of mitochondrial proteins labels damaged mitochondria for degradation through mitophagy. We recently developed an in vivo system in which mitophagy is slowed by inhibiting mitochondrial division through knockout of Dnm1l/Drp1, a dynamin related GTPase that mediates mitochondrial division. Using this system, we revealed that the ubiquitination of mitochondrial proteins required SQSTM1/p62, but not the ubiquitin E3 ligase PRKN/parkin, during mitophagy. Here, we tested the role of PINK1, a mitochondrial protein kinase that activates mitophagy by phosphorylating ubiquitin, in mitochondrial ubiquitination by knocking out Pink1 in dnm1l-knockout liver. We found mitochondrial ubiquitination did not decrease in the absence of PINK1; instead, PINK1 was required for the degradation of MFN1 (mitofusin 1) and MFN2, two homologous outer membrane proteins that mediate mitochondrial fusion in dnm1l-knockout hepatocytes. These data suggest that mitochondrial ubiquitination is promoted by SQSTM1 independently of PINK1 and PRKN during mitophagy. PINK1 and PRKN appear to control the balance between mitochondrial division and fusion in vivo. Abbreviations: DNM1L/DRP1: dynamin 1-like; KEAP1: kelch-like ECH-associated protein 1; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN1/2: mitofusin 1/2; OPA1: OPA1, mitochondrial dynamin like GTPase; PDH: pyruvate dehydrogenase E1; PINK1: PTEN induced putative kinase 1; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase.

Keywords: Dnm1l/Drp1; PINK1; PRKN/parkin; mitochondria; mitochondrial division; mitophagy.

Figure 1.

Mitochondrial ubiquitination requires SQSTM1, but not PRKN. (A) Frozen liver sections in the indicated mice were subjected to immunofluorescence microscopy with ubiquitin, SQSTM1, and a mitochondrial protein, pyruvate dehydrogenase (PDH). Lower panels are magnified images of boxed regions. Bars in higher panels: 10 µm. Bars in lower panels: 2 µm. (B) Quantification of cells showing the accumulation of ubiquitin on mitochondria. Values are average ± SD (n = 3–5 mice). (C) Mitochondria were isolated from control and Alb-dnm1l KO livers and analyzed by western blotting with antibodies to SQSTM1, GAPDH, VDAC and TOMM20. 20 µg of protein was loaded in each lane. (D-F) Band intensity was quantified, and values are presented as average ± SD (n = 3 mice). Statistical analysis was performed using one-way ANOVA followed by the post-hoc Tukey test in (B) and Student’s t-test in (D-F): *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Mitochondria undergo ubiquitination in the absence of PINK1. (A and B) Frozen liver sections were examined by immunofluorescence microscopy with antibodies against ubiquitin, SQSTM1, and PDH (A) and LC3, SQSTM1, and PDH (B). Lower panels are magnified images of boxed regions. Bars in higher panels: 10 µm. Bars in lower panels: 2 µm. (C and D) Quantification of cells showing the accumulation of ubiquitin (C) and SQSTM1 (D) on mitochondria. Values are average ± SD (n = 3 mice). Statistical analysis was performed using one-way ANOVA followed by the post-hoc Tukey test: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Additional loss of PINK1 restores the amounts of MFN1 and 2 that are decreased in Alb-dnm1l KO hepatocytes. (A) Immunoblotting of livers isolated from the indicated mice at 3 months using antibodies against MFN1 and 2, OPA1, GAPDH, and TOMM20. Lower image represents a model for OPA1 processing. (B–G) Quantification of band intensity of MFN1 (B), MFN 2 (C), OPA1-S3 (D), OPA1-S5 (E), GAPDH (F), and TOMM20 (G). Values are average ± SD (n = 3–4). (H) Model for mitochondrial ubiquitination mediated by the SQSTM1-KEAP1-RBX1 complex in mitophagy and by the PINK1-PRKN pathway in morphological regulation. Statistical analysis was performed using ANOVA followed by the post-hoc Tukey test: *p < 0.05, **p < 0.01.