ER import blockade triggers differential survival responses. (A) Model- DPP8/9 and UBRs work in concert to enforce the correct localization of secretory proteins. Secretory proteins contain a cleavable N-degron that is exposed upon their mislocalization. MetAP1/2 cleave first the initiating Met followed by cleavage of N-terminal di-peptide post proline by DPP8/9. Cleavage events expose an N-degron for UBR E3s. UBRs recognize now the mislocalized protein and promote its ubiquitination and its destruction in the proteasome. (B) Cells were treated with 25 nM Apratoxin A (Apra) for 48 h. To assess the effect on ER import blockade, Western blotting was performed on both the culture media (“Media”) and total cell extracts (“TCE”). Proteins were probed with antibodies against GFP to detect the presence of ARSJ-GFP, a secreted reporter protein or COX-IV, a non-secreted control protein. Ponceau staining was used to verify equal protein loading in the media samples. (C and D) Survival assay was performed on DPP8/9 and UBR KO (C) or BAG6, DPP8/9, and BAG6 + DPP8/9 TKO lines (D) following treatment with 25 nM Apra for 72 h. Data are presented as percent of surviving cells by dividing Apra treated to untreated cells (incubated with DMSO [vehicle]). (E) Colony formation assay on the same genetic backgrounds as in D following 10 days of Apra treatment. Colony size was measured and presented as ratio between Apra treated cells and untreated controls. For C–E, n = 3. ns = not significant; * P value< 0.0001, ** P value< 0.007, one-way ANOVA test. Source data are available for this figure: SourceData F8.