Figure 2.
Secretion via MAPS does not require substrate unfolding. A, a schematic illustration of constructs used in this study. B, α-Syn–DHFR–FLAG secretion is stimulated by USP19. HEK293T cells were cotransfected with α-Syn–DHFR–FLAG or α-Syn–FLAG together with either an empty vector (E.V.) or a plasmid expressing FLAG-tagged USP19. Medium and lysates were analyzed by immunoblotting using antibodies against the indicated proteins. C, aminopterin changes the conformation of α-Syn–DHFR–FLAG. α-Syn–FLAG or α-Syn–DHFR–FLAG was purified from either control cells or cells treated with aminopterin (10 μm; 5 h). Purified proteins were treated with trypsin as indicated followed by SDS-PAGE and silver staining. D, aminopterin has no effect on secretion of α-Syn–DHFR. Secretion of α-Syn–FLAG or α-Syn–DHFR–FLAG from 293T cells transfected as indicated was analyzed by immunoblotting. E, spontaneous refolding of β-gal is negligible. β-Gal treated with 8 m urea (orange bars) or left untreated was diluted in DMEM (1:1000). β-Gal was measured at the indicated time points. F and G, secretion of LacZ–α-Syn is mediated by USP19 and DNAJC5. F, secretion of LacZ–α-Syn from cells transfected with LacZ–α-Syn together with the indicated plasmids was analyzed by the β-gal assay. The level of LacZ–α-Syn secretion was normalized by the level of α-Syn in cell lysate (error bars represent mean ± S.E.; n = 3 independent experiments; *, p value <0.05). G, secretion of LacZ–α-Syn from control or USP19-null CRISPR cells transfected with LacZ–α-Syn was analyzed as in D (error bars represent mean ± S.E.; n = 3 independent experiments; *, p value <0.05). H, secreted LacZ–α-Syn is mostly folded. Cells were transfected as in D. LacZ–α-Syn in medium was determined by both β-gal assay and ELISA (error bars indicate variations of two independent experiments). Med., medium; Lys., lysate; A.U., arbitrary units.