Figure 2. Retro-2 diverts newly-synthesized TA proteins from ER targeting to degradation.

(A) Schematic of the dual-colour reporter consisting of a self-cleaving P2A peptide between a GFP and a RFP with a C-terminal TMD. Genetic and chemical perturbations to targeting pathways will promote destabilization of RFP-TA_TMD, as TA proteins are diverted for degradation by accessory TRC pathway components (not shown) if they are not efficiently captured by ASNA1. (B) Wildtype and ASNA1^KO HEK293T cell lines with indicated reporters were pre-treated with 3 µM DHQZ36.1 for 1 hr prior to induction with dox for approximately 18 hr and FACS analysis. Shown are bar graphs of reporter RFP to GFP ratios with standard deviations derived from three experiments as relative means to their corresponding mock-treated wildtypes. (C) Schematic of the post-translational and co-translational ER targeting of RFP-SEC61B_TMD. and RFP- SEC61B_TMD-BFP, respectively. (D) Cells with indicated genotypes and reporters were treated and analyzed as in part b).

Figure 2—figure supplement 1. ASNA1 knockout and Retro-2/DHQZ36.1 treatment destabilize the transmembrane domain of STX5.

(A) Left: Whole-cell lysates were prepared from wildtype and ASNA1^KO HEK293T cells and subjected to SDS-PAGE and visualized by immunoblotting (IB). SQSTM1 was used as the loading control. ASNA1^KO clone #2 (marked in red) was carried forward for the rest of the experiments. Right: Sequencing for ASNA1^KO clone #2 at the ASNA1 locus. The line represents the sgRNA used to make the deletion. (B) Top: Chemical structures for Retro-2, DHQZ36.1 and DHQZ5. Bottom: Wildtype and ASNA1^KO HEK293T cell lines expressing the GFP-2A-RFP-SEC61B_TMD reporter were pre-treated with the indicated compound for 1 hr prior to induction with dox for approximately 18 hr and FACS analysis. Cells were treated with 10 µM Retro-2, 3 µM DHQZ36.1 or 10 µM DHQZ5. Shown are bar graphs of reporter RFP to GFP ratios as relative means ± standard deviation (three experiments) to mock-treated wildtype cells. (C) Wildtype HEK293T cells expressing the GFP-2A-RFP-SEC61B_TMD reporter were pre-treated with Retro-2 (black circles) or DHQZ36.1 (red squares) for 1 hr prior to induction with dox for approximately 18 hr and FACS analysis. Shown are the dose-response curves for the reporter RFP to GFP ratios as relative means (three experiments) to the mock-treated cells. The dose response was modeled using the four-parameter logistic regression to determine the half maximal effective concentration (EC₅₀ ± standard error). Error bars for the means represent the standard error calculated from the four-parameter logistic regression.