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. 2011 May 16;108(22):9113–9118. doi: 10.1073/pnas.1018749108

Fig. 4.

Fig. 4.

Pex19p is required for the budding of peroxisomal vesicles from the ER. (A) ER-budding assay was performed with Δpex19 cells coexpressing Pex11p–2HA, Sec61p–3HA, and Pex3p–GFP with either Δpex19 or WT cytosol. The expression of Pex11p–2HA was markedly low in Δpex19 and Δpex3 cells, so RS fractions of five budding reactions were pooled and analyzed together to obtain comparable levels of the protein. Neither Pex11p–2HA nor Pex3p–GFP was detected in the RS 200 KgP when Δpex19 cytosol was used. The budding of Pex11p–2HA was restored with WT cytosol (WT S1) in an ATP-dependent manner. Likewise, Pex3p–GFP budding too was restored with the WT cytosol (WT S1). PYCs in lane 1 represent nearly 3% load of the starting PYCs. (B) A similar ER-budding assay was performed with Δpex3 cells coexpressing Pex11p–2HA and Sec61p–3HA with Δpex3 or WT cytosol. Pex11p–2HA was detected in the RS 200-KgP fraction, indicating that Pex3p is not required for budding of preperoxisomal vesicles. Further addition of WT cytosol did not increase the budding of Pex11p–2HA vesicles. (C) The ER-budding assay was performed with Δpex1, Δpex5, Δpex6, Δpex7, and Δpex14 cells expressing Pex11p–2HA with their respective cytosols. Pex11p–2HA was detected in the RS 200 KgP in all of the mutants.