Figure 3: Amphipathic segments of PEX19 play distinct roles in chaperone and targeting activities.

(a) Identification of amphipathic segments in the PEX19 N-terminal domain. The y axis indicates the predicted probability of disorder. Amphipathic segments are labelled. Disorder probability was also calculated for concatenated amphipathic (middle panel) and intervening sequences (right panel). This figure is complemented by Supplementary Fig. 8. (b) Helical wheel projections of Neurospora amphipathic segment. Hydrophobic residues are coloured green, acidic residues are blue and basic residues are red. Asterisks mark the residues mutated to alanine to make the alpha 4A mutants. This figure is complemented by Supplementary Fig. 8. (c) A sequence alignment shows conservation of the indicated amphipathic segments. Colouring is according to b. This figure is complemented by Supplementary Fig. 8. (d) The schematic shows the overall structure of PEX19. Features are labelled and specified in the legend. CTD, C-terminal domain. (e) Chaperone activity of the indicating PEX19 4A mutants by gel filtration. Fractions along with void volume and size markers are indicated. This figure is complemented by Supplementary Fig. 10. (f) Function of the indicated PEX19 4A mutants in cell-free targeting (upper panels, scale bar, 1 μm) and ability to complement a pex19 deletion strain (lower panels, scale bar, 10 μm) as assessed by matrix protein (mCherry-PTS1) import. This figure is complemented by Supplementary Fig. 15a. (g) The schematic depicts hydrophobic residues of alpha-a and alpha-d and their swapped variants. This figure is complemented by Supplementary Fig. 9. (h) The indicated PEX19 variants were assayed for chaperone activity (upper panels, scale bar, 2 μm), cell-free targeting (middle panels, scale bar, 1 μm) and complementation of the pex19 deletion strain (lower panels, scale bar, 10 μm). This figure is complemented by Supplementary Fig. 15b. (i) Pull-down assay reveals the ability of the PEX19 4A mutants to physically associate with bead-associated PEX3. Input and bound fractions are shown. Proteins are visualized by staining with Coomassie Brilliant Blue. (j) Surface plasmon resonance analysis of the interaction between the indicated alpha segment-mCherry fusion proteins and immobilized PEX3. For simplicity, the binding curves at a concentration of 5 μM are shown. A full data set can be found in Supplementary Fig. 11. (k) TMD-PEX26 pulls down PEX3 in the presence of PEX19 and PEX19 d-4A but not PEX19 a-4A. TMD-PEX26 was translated in the presence of the indicated PEX19 variants and then pulled down after being presented with a biotinylated version of cytoplasmic domain of PEX3. The bound fraction was separated by SDS–PAGE and analysed by western blotting against the indicated proteins.