Journal: iScience

Article Title: A dual role of the conserved PEX19 helix in safeguarding peroxisomal membrane proteins

doi: 10.1016/j.isci.2024.109537

Figure Lengend Snippet: The cytosolic domain of PEX3-WT activates PEX26 dissociation from PEX19-WT, whereas PEX3-G138E does not (A‒D) (A and C) Representative western blot images of PEX26 Bpa dissociation from PEX19-WT (A) and PEX19-WT F (C). In the absence and presence of 400 nM PEX3ΔN variants, PEX19-WT·PEX26 Bpa and PEX19-WT F ·PEX26 Bpa complexes at the final concentrations of 400 nM PEX19 proteins were incubated with 20 μM CaM. The zero time point samples were collected before the addition of CaM to the PEX19·PEX26 Bp a . At the indicated time points, aliquots of the reactions were frozen and analyzed by UV crosslinking. PEX26, PEX3ΔN, and CaM were probed in immunoblots with Strep, Flag, and HA antibodies, respectively. (B and D) Quantification of PEX26 dissociation from the Bpa crosslinking data in (A) and (C) and their replicates. (E‒H) (E and G) Representative western blot images of PEX26 Bpa dissociation from PEX19-αd4A (E) and PEX19-αd4A F (G). PEX26 Bpa dissociation assays from PEX19-αd4A (E) and PEX19- αd4A F (G) were conducted in the same manner as in (A) and (C). (F and H) Quantification of PEX26 dissociation from the Bpa crosslinking data in (E) and (G) and their replicates. All values in (B), (D), (F), and (H) are reported as mean ± SD, with n = 3. Error bars are shown but may not be visible in some cases.

Article Snippet: After adding 1.5% SDS and 50 mM DTT, the crosslinked cell lysate was boiled at 95°C for 10 min, loaded onto 10% Tris-Glycine gel, and analyzed by western blotting using PEX19 (1:3000 dilution, Novus Biologicals) and PEX3 (1:3000 dilution, Novus Biologicals) primary antibodies and IRDye800 secondary antibody (1:20,000 dilution, LiCor).

Techniques: Western Blot, Incubation

Journal: iScience

Article Title: A dual role of the conserved PEX19 helix in safeguarding peroxisomal membrane proteins

doi: 10.1016/j.isci.2024.109537

Figure Lengend Snippet: The αd helix of PEX19 interacts with the α1 of PEX3 (A) Crystal structure of PEX19-αa-bound PEX3ΔN (PDB 3MK4 ). The amino acid residues in the PEX19-F125 Bpa -crosslinked PEX3 peptide identified from MS analysis were highlighted in green. The identified PEX19-F125 Bpa -binding residue M72 in PEX3ΔN-α1 was shown in red. The known PEX19-αa-binding residue W104 and the disease-causing residue G138 (G138E) in PEX3ΔN-α2 were shown in purple and magenta, respectively. (B) A schematic representation of PEX19-F125 Bpa crosslinking to PEX3ΔN variants; 400 nM PEX19-F125 Bpa was incubated in the absence and presence of 100 nM PEX26 at room temperature for 1 min; 400 nM PEX3ΔN-WT and its variants were added to the reaction and further incubated at room temperature for 5 min. The frozen reaction samples were subjected to UV crosslinking. (C‒E) Western blot analysis of PEX19-F125 Bpa crosslinking to PEX3ΔN variants. The crosslinked proteins were probed using antibodies against PEX19, Flag (PEX3ΔN), and Strep (PEX26). “∗” represents the SDS-resistant PEX26 dimers in (E). (F) The number of matched peptides for PEX19-F125 Bpa -PEX3 crosslink identified from MS analysis. The M72 residue in the PEX3 peptide ( 64 TCNMTVLS M LPTLR 77 ) was most frequently crosslinked with F125-Bpa in the PEX19 peptide ( 115 VGSDMTSQQE Bpa TSCLK 130 ) (also see Figure S5 H). (G) Western blot analysis of PEX19-F125 Bpa crosslinking to PEX3ΔN-WT, PEX3ΔN-M72K, and PEX3ΔN-M72E. The reactions were carried out in the same way as in (B). (H) Western blot analysis of PEX19-F125 Bpa crosslinking to the endogenous PEX3 in semi-intact HeLa cells; 200-nM PEX19-F125 Bpa was incubated with semi-permeabilized HeLa cells at room temperature for 1 h, after which the cell lysates were subjected to UV crosslinking as described in .

Article Snippet: After adding 1.5% SDS and 50 mM DTT, the crosslinked cell lysate was boiled at 95°C for 10 min, loaded onto 10% Tris-Glycine gel, and analyzed by western blotting using PEX19 (1:3000 dilution, Novus Biologicals) and PEX3 (1:3000 dilution, Novus Biologicals) primary antibodies and IRDye800 secondary antibody (1:20,000 dilution, LiCor).

Techniques: Binding Assay, Residue, Incubation, Western Blot

Journal: iScience

Article Title: A dual role of the conserved PEX19 helix in safeguarding peroxisomal membrane proteins

doi: 10.1016/j.isci.2024.109537

Figure Lengend Snippet: PEX19-αd4A significantly reduced the peroxisomal targeting of PEX26 (A) 2×Strep-PEX19 pull-down experiments. 2×Strep-PEX19 variants (WT, Δ αa, and αd4A) and GFP-PEX26 were co-expressed in HEK293T cells, and the cytosolic fractions were subjected to 2×Strep-PEX19 pull-down experiments. I and E denote input and elution, respectively. The I and E fractions were subjected to SDS-PAGE and western blotting with antibodies against Strep and GFP. (B) The 2×Strep-PEX19·GFP-PEX26 complexes from the E fractions (A) were used for PEX26 targeting experiments in semi-permeabilized HeLa cells, as shown in Figure S4 A. After incubating the 2×Strep-PEX19·GFP-PEX26 complexes for 1 h, the cells were fixed and further analyzed by immunofluorescence (scale bar: 10 μm); 5.3-fold enlarged images of the boxed areas were shown separately in the right panel (scale bar: 2 μm). (C) Colocalization rates of PEX26 with the peroxisomal membrane protein (PMP70). A total of 130 cells from three biological replicates were analyzed using LAS X software. The lines indicate the mean values of the colocalization rate of PEX26 for each condition (n = 130). ∗p < 0.0001 (Student’s t test). (D) Mean intensity of colocalized PEX26 with PMP70. The mean intensities of peroxisome-localized PEX26 in (C) were analyzed using LAS X software. Values are reported as mean ± SD, with n = 3 (three biological replicates). Error bars are shown but may not be visible in some cases. (E) A proposed model of PEX26 targeting to the peroxisome. PEX19 rapidly captures free PEX26 in the cytosol (step 1). As shown in (A), a majority of PEX19 proteins in the cytosol are farnesylated (asterisk). Farnesylated PEX19 displays an increased binding affinity to PEX26. The αd helix of PEX19 protects against PEX26 loss to off-pathway chaperones (step 2). The αa helix of PEX19 primarily interacts with the cytosolic domain of PEX3. The secondary interaction of PEX19-αd helix with PEX3 destabilizes the PEX19·PEX26 complex, thereby inducing the release of PEX29 to the membrane and further leading to its membrane insertion (steps 3–4).

Article Snippet: After adding 1.5% SDS and 50 mM DTT, the crosslinked cell lysate was boiled at 95°C for 10 min, loaded onto 10% Tris-Glycine gel, and analyzed by western blotting using PEX19 (1:3000 dilution, Novus Biologicals) and PEX3 (1:3000 dilution, Novus Biologicals) primary antibodies and IRDye800 secondary antibody (1:20,000 dilution, LiCor).

Techniques: SDS Page, Western Blot, Immunofluorescence, Membrane, Software, Binding Assay

Journal: iScience

Article Title: A dual role of the conserved PEX19 helix in safeguarding peroxisomal membrane proteins

doi: 10.1016/j.isci.2024.109537

Figure Lengend Snippet:

Article Snippet: After adding 1.5% SDS and 50 mM DTT, the crosslinked cell lysate was boiled at 95°C for 10 min, loaded onto 10% Tris-Glycine gel, and analyzed by western blotting using PEX19 (1:3000 dilution, Novus Biologicals) and PEX3 (1:3000 dilution, Novus Biologicals) primary antibodies and IRDye800 secondary antibody (1:20,000 dilution, LiCor).

Techniques: Virus, Recombinant, Protease Inhibitor, Lysis, Sequencing, Transfection, Modification, Mass Spectrometry, Mutagenesis, Software, Imaging, Microscopy