OXPHOS deficiencies affect peroxisome proliferation by downregulating genes controlled by the SNF1 signaling pathway
- Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, United States
Figures
Figure 1 with 2 supplements
Peroxisome metabolites influence peroxisome size.
(A) Fluorescence microscopy of WT and Δpot1 mutant cells expressing Pex3-GFP driven by the PEX3 promoter and BFP-SKL driven by the GAPDH promoter, grown in oleate for 8 hr. (B) Brief description of …
Figure 1—figure supplement 1
Peroxisome metabolites influence peroxisome size.
(A) Fluorescence microscopy of WT, Δpot1, and Δaox1 Δaox2 cells expressing Pex3-GFP and BFP-SKL driven by the PEX3 the GAPDH promoters, respectively. Cells were grown in oleate and methanol for 8 …
Figure 1—figure supplement 2
NADH-shuttling mutants influence peroxisome size.
Fluorescence microscopy of WT and NADH-shuttling mutant cells expressing Pex3-GFP and BFP-SKL as in Figure 1—figure supplement 1. Cells were grown in oleate and methanol for 8 hr, respectively. Bar: …
Figure 2 with 1 supplement
Dysfunctional mitochondria affect peroxisome proliferation.
Fluorescence microscopy of WT, Δndufa9, ΔnugM, and Δcyt1 mutant cells expressing Pex3-GFP and BFP-SKL, grown for 16 hr in (A) oleate and 8 hr in (B) methanol medium. Bar: 5 μm.
Figure 2—figure supplement 1
Altered mitochondrial respiration in mitochondrial CI mutants.
Equal amounts of WT, Δndufa9, and ΔnugM cells were grown as described in Materials and methods, and mitochondrial metabolic activity was measured in digitonin-permeabilized cells using the PM1 …
Figure 3 with 1 supplement
WT cells treated with the oxidative phosphorylation (OXPHOS) uncoupler, 2,4-dinitrophenol (DNP), share peroxisome proliferation defects with mitochondrial CI and CIII mutant cells.
Fluorescence microscopy of WT cells expressing Pex3-GFP, grown in methanol medium, with or without 0.25 mM DNP. Bar: 5 μm.
Figure 3—figure supplement 1
Cells treated with the oxidative phosphorylation (OXPHOS) uncoupler, 2,4-dinitrophenol (DNP), share same peroxisomal protein expression defects with OXPHOS mutants, without affecting Snf1 phosphorylation.
(A) Western blot for several peroxisomal proteins, as well as the total (T) the phosphorylated forms (P) of Snf1 in cells treated with and without DNP. Specific bands are indicated with an arrow. (B)…
Figure 4 with 1 supplement
Oxidative phosphorylation (OXPHOS) mutants, like SNF1 mutants impair Pot1, Aox1, and Pex11 expression.
(A) Western blot of Pex11-2HA visualized with anti-HA antibodies in WT and ΔnugM mutant cells. (B) Model of transcriptional regulation of peroxisome genes regulated by SNF1 signaling. ? denotes …
Figure 4—figure supplement 1
Peroxisomal protein expression under different environmental conditions and validation of the phospho-AMPKα (Thr172) antibody in P. pastoris.
(A) Western blots for several peroxisomal proteins at different time points from wild-type P. pastoris cells grown in glucose and shifted to no glucose (-glucose), oleate, or methanol medium. …
Figure 5 with 1 supplement
NAD+/NADH ratios and oxygen consumption rates (OCRs).
(A) NAD+/NADH ratios obtained using SoNar sensors from oleate-grown cells (Zhao et al., 2015), as described in Materials and methods in wild-type and indicated mutant strains. (B) Cell number …
Figure 5—figure supplement 1
Extracellular acidification rates (ECARs).
ECAR data from methanol and after adding glucose (2% final concentration) for wild-type and mutant cells from two different parental strains (PPY12 or closely related to PPY12, and GS115). Mutants …
Figure 6 with 1 supplement
Analysis of transcriptional activators, Mxr1 and Mit1, of peroxisomal proteins.
(A) Western blots of Pex3, phospho-Snf1, and Pot1 or Aox1 in WT, Δmxr1, and Δmit1 mutant cells. (B) Western blots of Pot1 and Aox1 in WT, Δgal83, and ΔnugM mutant cells, either expressing or not …
Figure 6—figure supplement 1
PKA inhibition or HOG1 deletion did not rescue peroxisome proliferation defect of ΔnugM mutant cells.
Western blot of Aox1 and Pot1 in WT and ΔnugM mutant cells, with and without PKA inhibition or HOG1 deletion. In P. pastoris, PKA consists of a regulatory subunit dimer (Bcy1) and two catalytic …
Figure 7
Deletion of transcriptional repressors regulated by SNF1 complex signaling rescues ΔnugM and Δgal83 mutant cells.
(A, B) Western blot of Aox1 and Pot1 in WT, ΔnugM and Δgal83 mutant cells, with and without deletions of genes (MIG1, MIG2, and NRG1) encoding the transcriptional repressors regulated by SNF1 …
Figure 8 with 1 supplement
Gal83 nuclear localization during methanol adaptation is inhibited in ΔnugM and Δsak1 mutant cells.
Fluorescence microscopy of WT, Δsak1, and Δnugm mutant cells expressing Gal83-GFP driven by the GAL83 promoter in the presence of 200 ng/ml leptomycin B (LMB) and Sec71-mCherry as perinuclear ER …
Figure 8—figure supplement 1
Gal83 nuclear localization during oleate adaptation is inhibited in ΔnugM mutant cells.
Fluorescence microscopy of WT, Δsak1, and ΔnugM mutant cells expressing Gal83-GFP driven by the GAL83 promoter. Bar: 5 μm.
Figure 9
Cultivation of Δpex14 mutant cells in the respiratory medium, lactate, induces peroxisome proliferation.
Fluorescence microscopy of WT, Δpex14 mutant cells expressing Pex3-GFP in different media for 8 hr. Bar: 5 μm.
Figure 10
Interorganellar communication and signaling pathways in peroxisome proliferation, division, and matrix protein biogenesis.
Feedback loop between peroxisome and mitochondria is shown in red. Fatty acids (FA) uptake and its β-oxidation produce NADH equivalents and acetyl-CoA. However, the peroxisome membrane is …
Figure 11
The Δpex11 cells of P pastoris do not phenocopy the oxidative phosphorylation (OXPHOS) mutant, and can proliferate peroxisomes in oleate and methanol.
Peroxisomes are labeled with Pex3-GFP. Bar: 5 μm.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/75143/elife-75143-mdarchecklist1-v2.docx
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Supplementary file 1
Strains and plasmids table.
- https://cdn.elifesciences.org/articles/75143/elife-75143-supp1-v2.docx
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Supplementary file 2
RT-qPCR primers.
- https://cdn.elifesciences.org/articles/75143/elife-75143-supp2-v2.docx
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Source data 1
Western blots and Ponceau S stained membranes (raw and annotated data).
- https://cdn.elifesciences.org/articles/75143/elife-75143-data1-v2.zip
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Source data 2
Western blots and Ponceau S stained membranes (raw and annotated data).
- https://cdn.elifesciences.org/articles/75143/elife-75143-data2-v2.zip
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Source data 3
Excel files containing the data for Figure 2—figure supplement 1, Figure 4E, Figure 5, and Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/75143/elife-75143-data3-v2.zip
