Fig. 4: The α4 helix of hsPEX19 variants is a specific binding site for the N17 domain of Httex1-51Q.

a Sequences of the N17 domain of Httex1-51Q and TMDs of peroxisomal and mitochondrial membrane proteins and their Grand Average of Hydropathy (GRAVY) scores¹⁰³. All listed membrane proteins are known to interact with hsPEX19 during their targeting to peroxisome or mitochondria^39,46,58,59. PEX26, PEX11B, and ACBD5 are peroxisomal tail-anchored membrane proteins (TAs), whereas Fis1 is a dually localized TA in mitochondria and peroxisomes^58,59. PEX13 and PMP34 are multi-spanning peroxisomal membrane proteins (PMPs)^39,59. ATAD1 is an N-terminal signal-anchored membrane protein localized in both mitochondria and peroxisome⁴⁶. b Schematic representation of Httex1-51Q, N17-MBP, MBP-WT, and PEX26. The isolated N17 sequence was N-terminally fused to MBP (maltose binding protein). The recombinant PEX26 protein contains the N-terminal 2 × Strep-tagged SUMO domain and the PEX26 targeting sequences (237–305 aa) encompassing the TMD and C-terminal charged tail of PEX26⁶³. c, d Bpa crosslinking assay of hsPEX19^Bpa with MBP-WT, N17-MBP, N17-MBP variants. e, f In vitro aggregation assay to monitor the chaperone activity of hsPEX19-FV and SGTA toward Httex1-51Q. SDS-insoluble Httex1-51Q aggregates in (e) and their replicates were quantified and shown in (f) (n = 3, mean ± SD). g, h Representative images of His₆-PEX19 pulldown assay with PEX26 in (g) and their quantification in (h). I, FT, and E denote total input, flowthrough, and elution, respectively. The amounts of PEX26 bound to hsPEX19 were analyzed by western blot against Strep (PEX26) and PEX19 antibodies. Data in (h) are shown as mean ± SD, with n = 3 (technical replicates). i–k Bpa crosslinking assay of hsPEX19^Bpa with either Httex1-51Q or PEX26. The Bpa crosslinking assays with Httex1-51Q were carried out under the same conditions as Fig. 3h and i. Prior to the Bpa crosslinking assay, 0.75 µM of PEX26 was incubated with 3 µM of hsPEX19^Bpa at room temperature for 5 min. “*” represents the SDS-resistant PEX26 dimers in (k). All Bpa crosslinking assays were performed three times independently (n = 3). Source data are provided as a Source Data file.