Figure 1: Reconstitution of peroxisome TA protein biogenesis.

(a) mRNAs encoding model peroxisome (TMD-PEX26) or ER (TMD-SEC61β) TA proteins were microinjected along with markers of peroxisome matrix (P=mCherry-PTS1) or ER lumen (ER=mCherry-KDEL). After several hours, localization of mRNA translation products was assessed by confocal microscopy. The left panels show an overview of the cell. Scale bar, 8 μm. The dotted circle shows the position of the nucleus and the dotted box indicates the region that is magnified in the three right panels. Scale bar, 2 μm. (b) The indicated GFP-TMD mRNAs were translated in the absence and presence of recombinant PEX19 or GET3. Aggregates were visualized by fluorescence microscopy. Scale bar, 2 μm. (c) The indicated translation reactions were analysed by gel filtration. Fractions along with void volume and size markers are indicated. Translation reactions utilizing a bacterial translation system are labelled bacterial translation. The PEX19 C-terminal domain is referred to as CTD. (d) TMD-PEX26 was translated in the presence of varying amounts of PEX19 and the reaction was centrifuged at 100k g to produce pellet (P) and supernatant (S) fractions. Both TMD-PEX26 and PEX19 are HA tagged. This figure is complemented by Supplementary Figs 2 and 5. (e) Detergent treatment releases TMD-PEX26 from PEX19. TMD-PEX26/PEX19 complexes were pulled down with a His-tag on PEX19, and then washed with the indicated conditions followed by anti-HA western blotting. (f) PEX19 delivers TMD-PEX26 to the peroxisome membrane. Flow cytometry shows that the majority of mCherry-labelled peroxisomes receive TMD-PEX26. The inset panels show typical rings of peroxisome-associated TMD-PEX26. Scale bar, 1 μm. This figure is complemented by Supplementary Figs 3 and 5. (g) Peroxisome-associated TMD-PEX26 is resistant to extraction by sodium carbonate. Membranes from the cell-free reaction were extracted with the indicated treatments. Total (T), supernatant (S) and pellet (P) fractions are analysed. The lower panel shows behaviour of the matrix marker mCherry-PTS1. (h) The effect of apyrase and the indicated nonhydrolyzable nucleotide analogues on TMD-PEX26 translation (upper panels) and targeting (lower panels). Scale bar, 1 μm. This figure is complemented by Supplementary Fig. 4. (i) ER membranes are not required for cell-free targeting. Peroxisomes were immunoisolated, and total (T), bead (B) and flow-through (FT) fractions were analysed by western blotting for PMP22-HA and the ER marker, KAR2 (inset panel). Isolated peroxisomes (on sepharose beads) were incubated with PEX19/TMD-PEX26 complexes and then examined by confocal microscopy for the localization of TMD-PEX26 and peroxisomes (P). Scale bar, 2 μm. Sepharose beads are seen in the TL panel. The dotted box indicates the region that is magnified in the inset panels. Scale bar, 1 μm. TL, transmitted light microscopy.