Figure 3. PEX14 acts as a proximity... | Rockefeller University Press

Figure 3.

$PEX14 acts as a proximity partner and membrane anchor for OPTN. (A) Schematic representation of the domains and key interactions of the miniTurbo-OPTN-GFP fusion protein. CYLD, cylindromatosis; HLH, helix-loop-helix; HTT, huntingtin; LZ, leucine zipper domain; LC3/GABARAP, microtubule-associated protein 1A/1B-light chain 3/γ-aminobutyric acid receptor-associated protein; Rab, Ras-related protein; TBC1D17, TBC1 domain family member 17; TBK1, TANK (TRAF-associated NF-kB activator)-binding kinase 1. (B) Volcano plot showing the log2-fold changes (FC) in protein abundance and corresponding log10-fold adjusted P values (PAdj) for 2,856 proteins identified via streptavidin-affinity purification and identification/quantification by LC-MS/MS. The analysis was conducted in HEK-293 cells expressing miniTurbo-OPTN-GFP (n = 4 biological replicates), with or without 50 µM biotin. Green, blue, and red dots indicate miniTurbo-OPTN-GFP, known OPTN interactors, and bona fide peroxisomal proteins detected in the affinity-purified fractions, respectively. Vertical and horizontal dotted lines represent the twofold change (FC) and PAdj (0.05) cutoffs, respectively. (C) PEX14 is required for OPTN recruitment to peroxisomal membranes. HEK-293 cells deficient in PEX3 (Δ3), PEX5 (Δ5), or PEX14 (Δ) were transfected or not with a plasmid encoding the peroxisomal membrane marker PMP34-GFP or OPTN-GFP. 2 days later, the cells were treated with 200 nM Baf A1 for 2 h, fixed with methanol, and immunostained using the indicated antibodies. Nuclei were counterstained with DAPI. Scale bars: 10 µm.$

PEX14 acts as a proximity partner and membrane anchor for OPTN. (A) Schematic representation of the domains and key interactions of the miniTurbo-OPTN-GFP fusion protein. CYLD, cylindromatosis; HLH, helix-loop-helix; HTT, huntingtin; LZ, leucine zipper domain; LC3/GABARAP, microtubule-associated protein 1A/1B-light chain 3/γ-aminobutyric acid receptor-associated protein; Rab, Ras-related protein; TBC1D17, TBC1 domain family member 17; TBK1, TANK (TRAF-associated NF-kB activator)-binding kinase 1. (B) Volcano plot showing the log₂-fold changes (FC) in protein abundance and corresponding log₁₀-fold adjusted P values (PAdj) for 2,856 proteins identified via streptavidin-affinity purification and identification/quantification by LC-MS/MS. The analysis was conducted in HEK-293 cells expressing miniTurbo-OPTN-GFP (n = 4 biological replicates), with or without 50 µM biotin. Green, blue, and red dots indicate miniTurbo-OPTN-GFP, known OPTN interactors, and bona fide peroxisomal proteins detected in the affinity-purified fractions, respectively. Vertical and horizontal dotted lines represent the twofold change (FC) and PAdj (0.05) cutoffs, respectively. (C) PEX14 is required for OPTN recruitment to peroxisomal membranes. HEK-293 cells deficient in PEX3 (Δ3), PEX5 (Δ5), or PEX14 (Δ) were transfected or not with a plasmid encoding the peroxisomal membrane marker PMP34-GFP or OPTN-GFP. 2 days later, the cells were treated with 200 nM Baf A1 for 2 h, fixed with methanol, and immunostained using the indicated antibodies. Nuclei were counterstained with DAPI. Scale bars: 10 µm.