Figure 1. ER exit of individual... | Rockefeller University Press

Figure 1.

$ER exit of individual γ-secretase subunits does not require expression of all subunits. SDS-PAGE of in vitro COPII vesicles from WT, PSEN-dKO, NCT-KO, and APH1-tKO SICs: 4.2% of SICs (input) and 100% of COPII vesicles were analyzed. Reactions without nucleotides (ATP + GTP) or cytosol were used for background subtraction. COPII budding was inhibited by mutant Sar1b H79G. (A) Scheme of assay and Western blot of COPII packaging using antibodies against ERGIC53/p58 (positive control), ribophorin (negative control), and γ-secretase subunits. Asterisks indicate an unrelated band as detected with anti-PSEN1 antibodies in PSEN-dKO fractions. In the different SICs, except PSEN-dKO, only PSEN1 FL exited the ER in an Sar1-dependent manner (open arrowheads). Except for their respective KO SICs, both APH1A and PEN-2 were recovered in COPII vesicles next to immature NCT (NCTimm; filled arrowheads). (B) Quantification of COPII budding efficiencies as the ratio of signal intensity of the input fraction to the COPII vesicle fraction, corrected for the background obtained in the absence of nucleotides. Graphs show mean budding efficiency ± SEM of at least three or four independent assays.$

ER exit of individual γ-secretase subunits does not require expression of all subunits. SDS-PAGE of in vitro COPII vesicles from WT, PSEN-dKO, NCT-KO, and APH1-tKO SICs: 4.2% of SICs (input) and 100% of COPII vesicles were analyzed. Reactions without nucleotides (ATP + GTP) or cytosol were used for background subtraction. COPII budding was inhibited by mutant Sar1b H79G. (A) Scheme of assay and Western blot of COPII packaging using antibodies against ERGIC53/p58 (positive control), ribophorin (negative control), and γ-secretase subunits. Asterisks indicate an unrelated band as detected with anti-PSEN1 antibodies in PSEN-dKO fractions. In the different SICs, except PSEN-dKO, only PSEN1 FL exited the ER in an Sar1-dependent manner (open arrowheads). Except for their respective KO SICs, both APH1A and PEN-2 were recovered in COPII vesicles next to immature NCT (NCTimm; filled arrowheads). (B) Quantification of COPII budding efficiencies as the ratio of signal intensity of the input fraction to the COPII vesicle fraction, corrected for the background obtained in the absence of nucleotides. Graphs show mean budding efficiency ± SEM of at least three or four independent assays.

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