Cramer, P.; Yonemura, Y.; Behrendt, L.; Marszalek, A.; Sannai, M.; Durso, W.; Guenes, C.; Szafranski, K.; Nakamura, N.; Nasrashvili, T.; Mayer, J.; von Eyss, B.; Kaether, C.

Exit from the endoplasmic reticulum is mediated by the Sar1/COPII machinery and a number of accessory factors. How the initial steps of cargo recruitment upstream of Sar1/COPII are mediated remains unclear, but the dihydropyridine FLI-06 inhibits cargo recruitment into ER exit sites. Here, we used chemical genetics screening approaches in conjunction with FLI-06 treatment and identified the ER membrane proteins YIPF5 and GOT1A/B as putative components of early export processes. Surprisingly, the two homologous proteins GOT1A and GOT1B, coded by GOLT1A and GOLT1B, respectively, exhibited opposite functions after treatment with FLI-06: increasing the expression of GOT1A or reducing the expression of GOT1B or YIPF5 prevented inhibition of ER-export by FLI-06. Inhibiting ER export with FLI-06 elicited a specific ER stress-related gene expression signature distinct from the ER-stress signature induced by Thapsigargin. The interactomes of GOT1A and GOT1B suggested a connection to ER-stress mediators. Moreover, RNA-Seq data showed that FLI-06-induced genes are strongly enriched for ATF6 target genes which are suppressed by GOLT1A overexpression or GOLT1B knock-down. This suggests that ATF6 signaling is involved in FLI-06-mediated toxicity, and we could demonstrate that siRNA-mediated knock-down or specific inhibitor of ATF6 rescued cells from FLI-06-mediated cell death. Knock-down or inhibition of ATF6 is sufficient to resume transport from the ER under FLI-06-treatment, suggesting that ATF6 is directly involved in the FLI-06-mediated ER-export block. Surprisingly, our data show that this ATF6 function is independent of de novo transcription, implying a novel, transcription-independent function of ATF6.