Figure 4: Hydrophobicity at the base of PEX3 is required for direct TA protein targeting.

(a) A schematic of PEX3 secondary structure is shown with helical segments numbered according to ref. 32. Position of the TMD, HA-tag, protease site () and residues mutated to make the PEX3-2A mutant are indicated. The lower region indicates the fragments produced during treatment with PreScission protease (1, 1′, 2 and 2′). (b) Ability of the indicated PEX3 mutants to complement a pex3 deletion strain. Scale bar, 10 μm. Matrix is revealed by mCherry-PTS1. TL, transmitted light microscopy. This figure is complemented by Supplementary Fig. 12. (c) Mutants that disrupt the PEX3 hydrophobic surface are defective in TMD-PEX26 integration. Isolated peroxisomes from the indicated strains were used in the cell-free targeting assay and immunostained (α-HA) to assess levels of PEX3. A strain in which PEX3 is not tagged (no tag) serves as a negative control for antibody staining. Scale bar, 1 μm. (d) Pull-down assays show that PEX3-2A binds to PEX19 and PEX19 d-4A as well as wild-type PEX3. The assay was conducted as described in Fig. 3i. (e) Peroxisomes derived from wild-type or PEX3 strain are incubated in the presence () and absence (−) of PreScission proteins and then fractionated by centrifugation to produce total (T), supernatant (S) and pellet (P) fractions. Pellet fractions were resuspended, extracted in the indicated solutions and then centrifuged to regenerate T, S and P fractions. Band identities are defined in a. (f) Peroxisomes in which PEX3 has been cleaved are competent for TMD-PEX26 integration. PreScission protease-treated () and -untreated (−) peroxisomes were used in the cell-free targeting assay. Scale bar, 1 μm. (g) The PEX3-2A mutant is defective in TMD-independent membrane association. Release of the PEX3 cytosolic domain by treatment with PreScission protease was assessed for the indicated versions of PEX3 as in e.