Figure 7.
Deficiency in ER exit of PSEN1 affects PEN-2 but not NCT packaging in COPII vesicles. (A) Sequence alignment of the deleted sequence in FAD PSEN1ΔE9. Bold red: COPII binding motif DPE. Green: Identical residues. The motif is highly conserved in mammals and birds but absent in lower vertebrates (fish) and fly. (B) Representative Western blot of COPII vesicles in PSEN-dKO MEFs stably rescued with EGFP-PSEN1WT or EGFP-PSEN1APA using antibodies against NCT, GFP (GFP-FL-PSEN1, white arrowhead; and GFP-PSEN1-NTF), and PEN-2. Filled arrowheads: Immature NCT (NCTimm) packaged in COPII vesicles. ERGIC53/p58: Positive control blocked by mutant Sar1 H79G. (C) Quantification of B. Mutating DPE to APA affected COPII packaging of FL PSEN1 and PEN-2 but not NCT. Mean ± SEM; n = 6. (D) Buckle-up model for γ-secretase assembly. Monomer subunits and dimers exit the ER with NCT/APH1 and PSEN1/PEN-2 preferring Sec24C/D and Sec24A, respectively. In WT cells, NCT/APH1A and PSEN1/PEN-2 dimers are preformed in the ER and buckle up in a full complex after ER exit, likely during ERES-to-ERGIC transition. Prior dimer formation may prevent premature degradation of monomeric subunits, but the exact mechanisms of how they are sorted into COPII vesicles versus retained in the ER remain to be investigated. Assembly to a full complex may hide known/unknown interaction motifs, allowing the complex to exit the early biosynthetic compartments to post-Golgi compartments. In KO backgrounds, and due to a failed assembly, preceding dimers (in NCT, APH1, PSEN1-KO) and trimers (PEN-2-KO) are increasingly recovered in COPII vesicles. Assembled monomers may be routed to degradation (generated by Somersault 18:24; https://www.somersault1824.com/).

Deficiency in ER exit of PSEN1 affects PEN-2 but not NCT packaging in COPII vesicles. (A) Sequence alignment of the deleted sequence in FAD PSEN1ΔE9. Bold red: COPII binding motif DPE. Green: Identical residues. The motif is highly conserved in mammals and birds but absent in lower vertebrates (fish) and fly. (B) Representative Western blot of COPII vesicles in PSEN-dKO MEFs stably rescued with EGFP-PSEN1WT or EGFP-PSEN1APA using antibodies against NCT, GFP (GFP-FL-PSEN1, white arrowhead; and GFP-PSEN1-NTF), and PEN-2. Filled arrowheads: Immature NCT (NCTimm) packaged in COPII vesicles. ERGIC53/p58: Positive control blocked by mutant Sar1 H79G. (C) Quantification of B. Mutating DPE to APA affected COPII packaging of FL PSEN1 and PEN-2 but not NCT. Mean ± SEM; n = 6. (D) Buckle-up model for γ-secretase assembly. Monomer subunits and dimers exit the ER with NCT/APH1 and PSEN1/PEN-2 preferring Sec24C/D and Sec24A, respectively. In WT cells, NCT/APH1A and PSEN1/PEN-2 dimers are preformed in the ER and buckle up in a full complex after ER exit, likely during ERES-to-ERGIC transition. Prior dimer formation may prevent premature degradation of monomeric subunits, but the exact mechanisms of how they are sorted into COPII vesicles versus retained in the ER remain to be investigated. Assembly to a full complex may hide known/unknown interaction motifs, allowing the complex to exit the early biosynthetic compartments to post-Golgi compartments. In KO backgrounds, and due to a failed assembly, preceding dimers (in NCT, APH1, PSEN1-KO) and trimers (PEN-2-KO) are increasingly recovered in COPII vesicles. Assembled monomers may be routed to degradation (generated by Somersault 18:24; https://www.somersault1824.com/).

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