Deubiquitinating enzyme USP30 maintains basal peroxisome abundance by regulating pexophagy

Abstract

The regulation of organelle abundance is critical for cell function and survival; however, the mechanisms responsible are not fully understood. In this study, we characterize a role of the deubiquitinating enzyme USP30 in peroxisome maintenance. Peroxisomes are highly dynamic, changing in abundance in response to metabolic stress. In our recent study identifying the role of USP30 in mitophagy, we observed USP30 to be localized to punctate structures resembling peroxisomes. We report here that USP30, best known as a mitophagy regulator, is also necessary for regulating pexophagy, the selective autophagic degradation of peroxisomes. We find that overexpressing USP30 prevents pexophagy during amino acid starvation, and its depletion results in pexophagy induction under basal conditions. We demonstrate that USP30 prevents pexophagy by counteracting the action of the peroxisomal E3 ubiquitin ligase PEX2. Finally, we show that USP30 can rescue the peroxisome loss observed in some disease-causing peroxisome mutations, pointing to a potential therapeutic target.

Figure 1.

Overexpressed USP30 localizes to peroxisomes. (A and B) COS7 cells expressing USP30-Flag (red) and mitochondrial marker OMP-25-GFP (A) or stained for peroxisome marker PMP70 (green; B). (C) Schematic of USP30-Flag, PMP34-USP30-Flag, and TOM20-USP30-Flag constructs show the site of the catalytic mutation (*C77S). (D) COS7 cells expressing TOM20-USP30-Flag or PMP34-USP30-Flag (red) and stained for PMP70 (green). (E) Thresholded MCC of PMP70-stained peroxisomes (M_PO) colocalized with USP30-Flag, PMP34-USP30, TOM20-USP30, or ATP5A (mock). (F) COS7 cells expressing SA-PEX16-GFP (green) with mitochondrial proteins FIS1-Myc, TOM20-Cer, USP35-HA, or USP30 constructs (red) as indicated. Mitochondrial localization shown in Fig. S1 G. (G) COS7 coexpressing USP30-Flag (red), sa-PEX16 (green), and OMP25-Cer (blue). Statistical significance (Student’s t test): ****, P ≤ 0.0001; ns, P ≥ 0.05 (n = 3; 30 cells/trial). White boxes indicate zoomed region. Bars: 10 µm; 5 µm in zoom.

Figure 2.

Overexpressed USP30 reduces peroxisome loss induced by starvation. (A) Immunofluorescence of HeLa cells transfected with USP30-Flag, PMP34-USP30-Flag, or TOM20-USP30-Flag or mock transfected. Cells grown in DMEM or HBSS for 24 h, stained for PMP70 and Flag. (B) Quantification of the peroxisome density in A and E, relative to mock DMEM (n = 3; 30 cells/trial). (C) HeLa lysates expressing USP30-Flag, analyzed by immunoblotting for PEX14 and GAPDH. (D) Quantification of PEX14/GAPDH density ratio in C compared with mock DMEM (n = 6). (E) HeLa cells expressing USP30-Flag C77S or PMP34-USP30-Flag C77S, costained for PMP70. (F and G) HeLa cells expressing PEX26-RG alone (F) or coexpressed with USP30-Flag (G). (H) Quantification of average percent lysosomal peroxisomes (n = 3; 50 cells/trial). Statistical significance (Student’s t test): *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Bars: 20 µm (A and E); 10 µm (F and G).

Figure 3.

Silencing USP30 induces pexophagy and is opposed by PEX2. (A) Confocal immunofluorescent images of PMP70 in HeLa cells treated with siRNA against USP30 (siUSP30) or a nontargeting siRNA (siCTRL) grown in DMEM or HBSS for 24 h. (B) Quantification of the relative peroxisome density in A, relative to siCTRL DMEM. (C) Immunoblots of PEX14 and GAPDH in HeLa cells treated with siRNA as in A. (D) Average PEX14 levels in C, compared with GAPDH levels and normalized to siCTRL DMEM. (E) ATG5 WT and ATG5^−/− MEFs, treated with siCTRL or siUSP30, under DMEM or HBSS, stained for PMP70. (F) Quantification of E, with all treatment values relative to siCTRL ATG5 WT. (G) HeLa cells treated with siCTRL or siUSP30 were incubated with 5 µM of chloroquine (CQ) and stained for PMP70. (H) Quantification of G relative to siCTRL −CQ. (I) HeLa cells treated with siATG12 or siNBR1 cotreated with either siCTRL or siUSP30, stained for PMP70. (J) Quantification of I relative to siCTRL treatment. (K) Cells treated with siRNA against PEX2 (siPEX2) and codepleted of siUSP30 or siCTRL. Cells were grown in either DMEM or HBSS for the final 24 h and stained for PMP70. (L) Quantification of K, with all treatment groups relative to DMEM siCTRL. Statistical significance (Student’s t test): **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, P ≥ 0.05; n = 3; 50 cells/trial. Bars, 20 µm.

Figure 4.

Knockdown of USP30 results in increased ubiquitined PEX5 and PMP70. (A) Knockdown of PEX5 (siPEX5) in HeLa cells, with overexpression of USP30-GFP grown in DMEM or HBSS for 24 h. Cells stained for PMP70. (B) Quantification of A, all treatment values relative to siCTRL DMEM. (C) Immunoprecipitation of HA-Ub, performed in HEK293 cells stably expressing HA-Ub. Cells were mock-treated or treated with siCTRL or siUSP30 under DMEM and HBSS conditions, probing for PMP70, PEX5, and catalase. (D) Quantification of PEX5 and PMP70 levels immunoprecipitated in C, relative to mock-treated DMEM. Each condition was normalized to the input. (E) HeLa cells stained for endogenous NBR1 (green) and PMP70 (red), either mock transfected or overexpressing USP30-GFP (white). (F) MCC of PMP70 (or M_po) colocalized with NBR1 representative images in E. Statistical significance (Student’s t test): *, P ≤ 0.05; **, P ≤ 0.01, ***, P ≤ 0.001; ns, P ≥ 0.05; n = 3; 30 cells/trial. Bars: 20 µm and 5 µm in zoom.

Figure 5.

USP30 overexpression can rescue peroxisome levels induced by mutation of the AAA ATPase. (A) HeLa cells treated with siRNA against PEX1 (siPEX1) or siCTRL, then mock-transfected or transfected with USP30-Flag, costained for PMP70. (B) Quantification of A with all treatment values calculated relative to mock siCTRL. Representative images for PMP34-USP30 are found in Fig. S2 A. (C) Human fibroblast cells from healthy (WT) or PEX1 G843D mutation overexpressing USP30-Flag and imaged as in A. (D) Quantification of peroxisome density with respect to mock-treated WT cells in C. (E) Model of PEX2 and USP30 in pexophagy. Peroxisomes are maintained by a balance between the action of USP30 and PEX2. Excess of PEX2 results in pexophagy. (F) PEX2 is responsible for ubiquitinating PMP70 and PEX5. PMP70 is deubiquitinated by USP30, while PEX5 can be removed by the AAA ATPase or deubiquitinated by USP30. Statistical significance (Student’s t test): **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, P ≥ 0.05; n = 3; 30 cells/trial. Bars, 20 µm.