Deficiency of a given γ-secretase component allows ER exit of remaining subunits in subcomplexes. (A) BN-PAGE of γ-secretase in 0.5% DDM-extracted COPII vesicles of WT, (B) NCT-KO and APH1-tKO, and (C) PSEN-dKO and PEN-2-KO SICs. Budding reactions without cytosol or nucleotides or with Sar1-H79G were for background subtraction and specificity, respectively. Antibodies against γ-secretase subunits identified monomers and subcomplexes (Fraering et al., 2004b). Full complexes did not exit the ER Sar1 dependently (compare lanes 2 and 3), whereas KO SICs efficiently packaged preceding monomers/subcomplexes (dimeric NCT/APH1 [asterisks] in PSEN-dKO, FL PSEN1/PEN-2 [filled circles] in NCT-KO and APH1-tKO; NCT/APH1A/FL-PSEN1 [open triangles] in PEN-2-KO; lane 2 in all blots) in an Sar1-dependent manner. Lane 1, without ATP/GTP = negative control.