NCT does not harbor a region/motif required for cargo sorting into COPII vesicles. (A) NCT-KO cells were stably transduced with retroviral particles expressing FL NCT, deletion forms of NCT (Δecto: missing the ectodomain; ΔIC: missing the intracellular domain), NCT with a swapped TMD from TLN (NCT TMD_TLN), or TLN with the TMD of NCT (TLN TMD_NCT). Western blot analysis shows expression of all constructs, but only FL NCT and NCTΔIC rescue γ-secretase function, as monitored by a drop in APP CTF accumulation. (B) All cell lines described in A, except for NCT Δecto, were tested for glycosylation of NCT or TLN by endoH and endoF treatment (arrow: untreated; black arrowhead: endoH resistent; open arrowhead: endoH/F sensitive). For TLN TMD_NCT, FL TLN was expressed as a control for glycosylation. Although FL NCT and NCTΔIC expressed in NCT-KO cells are similarly glycosylated as NCT in WT cells, NCT TMD_TLN glycosylation failed to mature. TLN TMD_NCT glycosylation was similar to WT TLN. (C) SDS-PAGE and Western blot analysis of in vitro generated COPII vesicles from SICs of cell lines described in A: 4.2% of SICs (input) and 100% of COPII-coated vesicles were analyzed with the indicated antibodies. Reactions without nucleotides (ATP + GTP) or cytosol were used for background subtraction. COPII budding is specifically inhibited by the GTP-restricted mutant Sar1b H79G. Representative Western blot analysis of the COPII packaging using antibodies detecting ERGIC53/p58 as a positive control compared with antibodies against the different γ-secretase members. Antibodies to ribophorin were used as a negative control. In all cell lines, the different immature NCT variants or TLN and PSEN1 FL exited the ER in an Sar1-dependent manner.