Figure S3. Subcomplexes identified by... | Rockefeller University Press

Figure S3.

$Subcomplexes identified by BN-PAGE are confirmed in rat glia and by coimmunoprecipitation; the DXE motif in exon 9 is crucial for COPII-dependent export of PSEN1 from ER.(A) BN-PAGE and Western blot analysis of γ-secretase in COPII vesicles generated from rat glial SICs. Monomeric NCT and dimeric NCT/APH1A show COPII-dependent budding. (B) A representative Western blot is shown comparing total cell extracts of WT, APH1-tKO, PSEN-dKO, and PEN-2-KO MEFs with the respective unbound and coimmunoprecipitated (using anti-NCT mAb 9C3, bound) fractions. For each cell line, omission of either 9C3 or extract was used as a negative control. Whereas 9C3 coimmunoprecipitates all subunits in WT extracts, only immature NCT was pulled down in APH1A-tKO extracts, and both immature NCT and APH1A were pulled down in PSEN-dKO extracts. In PEN-2-KO extracts, immature NCT, APH1A, and FL PSEN1 coimmunoprecipitated. These interactions are reminiscent of the subcomplexes identified by BN-PAGE. Note that APH1A is increasingly more present in the immunoprecipitates of PSEN-dKO and PEN-2-KO, respectively, and compared with WT extracts, confirming the accumulation of corresponding subcomplexes. Asterisk indicates an unrelated band also present in control lanes. (C) WT human PSEN1, the DPE mutant (D302A/D304A), and PSEN1ΔE9 were in vitro translated in SICs followed by the in vitro budding assay in the absence or presence of ATP. Radiolabeled PSEN1 variants from the pellet (SIC input) and vesicle fractions were compared on 10% SDS-PAGE gel. (D) WT human PSEN1 and PSEN1 mutants (DPE and ΔE9) were stably transfected, and next, SICs were made to determine the ER export efficiency of these PSEN1 variants at steady state. To better understand the DPE consensus site molecularly, we made subtler mutations, including the reversal of the two acidic amino acids followed by the analysis in C. Any manipulation of the DPE motifs dramatically impaired the ER export. WB, Western blot. (E) Quantification of the radiolabeled PSEN1 retrieved from the vesicle fractions that budded from the SICs (mean ± SD, n = 3). (F) PSEN-dKO were stably transduced with lentiviral vectors expressing either eGFP-tagged hPSEN1 or a mutant variant wherein the DPE motif in the cytosolic loop domain is altered to APA (eGFP-hPSEN1 APA) using site-directed mutagenesis. Western blot analysis shows that eGFP-hPSEN1 and –PSEN1 APA are equally expressed and accessible to endoproteolysis. In addition, mature glycosylation of NCT and PEN-2 stability are restored, whereas APP-CTF levels, the direct substrate for PSEN1, are not different, indicating that the APA mutation does not affect γ-secretase complex formation and activity. (G) Cell surface biotinylation of eGFP-hPSEN1 and –hPSEN1 APA compared with WT and NCT-KO MEFs shows no defect in the trafficking of the APA mutant to the cell surface. Input lanes represent 10 µg of protein; bound fractions are recovered from 500 µg total cell lysate using streptavidin pull-down assays. Equal amounts of PSEN1-NTF (using an anti-hPSEN1–specific antibody) as well as mature NCT are recovered in the bound fraction. A murine specific antibody was used to detect endogenous mPSEN1-CTF in WT and NCT-KO cells. Note that in NCT-KO MEFs, endoproteolysis of mPSEN1 is inhibited and some FL mPSEN1 (asterisk) reaches the cell surface. TfR and actin are used as positive and negative controls, respectively.$

Subcomplexes identified by BN-PAGE are confirmed in rat glia and by coimmunoprecipitation; the DXE motif in exon 9 is crucial for COPII-dependent export of PSEN1 from ER. (A) BN-PAGE and Western blot analysis of γ-secretase in COPII vesicles generated from rat glial SICs. Monomeric NCT and dimeric NCT/APH1A show COPII-dependent budding. (B) A representative Western blot is shown comparing total cell extracts of WT, APH1-tKO, PSEN-dKO, and PEN-2-KO MEFs with the respective unbound and coimmunoprecipitated (using anti-NCT mAb 9C3, bound) fractions. For each cell line, omission of either 9C3 or extract was used as a negative control. Whereas 9C3 coimmunoprecipitates all subunits in WT extracts, only immature NCT was pulled down in APH1A-tKO extracts, and both immature NCT and APH1A were pulled down in PSEN-dKO extracts. In PEN-2-KO extracts, immature NCT, APH1A, and FL PSEN1 coimmunoprecipitated. These interactions are reminiscent of the subcomplexes identified by BN-PAGE. Note that APH1A is increasingly more present in the immunoprecipitates of PSEN-dKO and PEN-2-KO, respectively, and compared with WT extracts, confirming the accumulation of corresponding subcomplexes. Asterisk indicates an unrelated band also present in control lanes. (C) WT human PSEN1, the DPE mutant (D302A/D304A), and PSEN1ΔE9 were in vitro translated in SICs followed by the in vitro budding assay in the absence or presence of ATP. Radiolabeled PSEN1 variants from the pellet (SIC input) and vesicle fractions were compared on 10% SDS-PAGE gel. (D) WT human PSEN1 and PSEN1 mutants (DPE and ΔE9) were stably transfected, and next, SICs were made to determine the ER export efficiency of these PSEN1 variants at steady state. To better understand the DPE consensus site molecularly, we made subtler mutations, including the reversal of the two acidic amino acids followed by the analysis in C. Any manipulation of the DPE motifs dramatically impaired the ER export. WB, Western blot. (E) Quantification of the radiolabeled PSEN1 retrieved from the vesicle fractions that budded from the SICs (mean ± SD, n = 3). (F) PSEN-dKO were stably transduced with lentiviral vectors expressing either eGFP-tagged hPSEN1 or a mutant variant wherein the DPE motif in the cytosolic loop domain is altered to APA (eGFP-hPSEN1 APA) using site-directed mutagenesis. Western blot analysis shows that eGFP-hPSEN1 and –PSEN1 APA are equally expressed and accessible to endoproteolysis. In addition, mature glycosylation of NCT and PEN-2 stability are restored, whereas APP-CTF levels, the direct substrate for PSEN1, are not different, indicating that the APA mutation does not affect γ-secretase complex formation and activity. (G) Cell surface biotinylation of eGFP-hPSEN1 and –hPSEN1 APA compared with WT and NCT-KO MEFs shows no defect in the trafficking of the APA mutant to the cell surface. Input lanes represent 10 µg of protein; bound fractions are recovered from 500 µg total cell lysate using streptavidin pull-down assays. Equal amounts of PSEN1-NTF (using an anti-hPSEN1–specific antibody) as well as mature NCT are recovered in the bound fraction. A murine specific antibody was used to detect endogenous mPSEN1-CTF in WT and NCT-KO cells. Note that in NCT-KO MEFs, endoproteolysis of mPSEN1 is inhibited and some FL mPSEN1 (asterisk) reaches the cell surface. TfR and actin are used as positive and negative controls, respectively.