Figure 4. PEX26 targeting to the ER is dependent on the GET machinery.

(a–c) GAL1 promoter-driven pulse expression of PEX26-ops in yeast. Opsin glycosylation reports entry into the ER. (a) GET-dependent but SEC62/SEC72-independent entry of PEX26-ops into the ER in yeast. Strains defective in post-translational ER targeting (DAmP-sec62, Δsec72)⁶³ or the GET pathway were transformed with PEX26-ops and expression was induced for 120 min. Western blot with anti-opsin antibodies. Both halves of each panel were cropped from the same blot. Quantifications of band intensities can be found in Supplementary Table S1 (n ≥ 3). (b) Extrachromosomal GET5-expression restores ER entry of PEX26-ops in Δget5-yeasts. Induction for 120 min. Western blot with anti-opsin antibodies. (c) EGFP-PEX26 does not integrate into the ER if the GET-pathway is impaired, but co-localizes with the peroxisomal marker HcRed-SKL. Co-expression of EGFP-PEX26 with Sec63-RFP ER marker or peroxisomal marker HcRed-SKL in a Δget3 strain. Bar = 5 μm. (d–f) Integration of PEX26-ops into the ER is TRC40-independent. Co-transfection of PEX26-ops with TRC40^WT, TRC40^SW1, TRC40^GR or mock plasmid. (d) TRC40 and TRC40 mutants do not influence ER targeting of PEX26-ops. Western blot. HeLa cells were co-transfected for 15 hours with PEX26-ops and mock, TRC40 or TRC40 mutant. All parts of each panel were cropped from the same blot. (e,f) TRC40-independent ER targeting of PEX26. Overexpression of the dominant TRC40 mutant SW1 impairs ER entry of Ramp4-ops but not of PEX26. HeLa cells co-expressing (e) Ramp4-ops or (f) PEX26 and TRC40 variants were labelled for 15 min with [³⁵S]-methionine and [³⁵S]-cysteine. (e) Ramp4-ops and (f) PEX26 were immunoprecipitated with anti-opsin antibodies and analyzed by autoradiography. Band intensities were quantified. Full-size blots and autoradiograph are presented in Supplementary Figure S1.