Get2CD linker mutants attenuated TA insertion into the ER in vivo. (A) Schematic of the model TA substrate used for the in vivo insertion assay. BirA-Bos1 contains, from the N to the C terminus, an N-terminal 3xHA tag, the BirA protein, the TMD of Bos1, and an opsin tag that is efficiently glycosylated (glyc) upon insertion into the ER. “Y” depicts the glycosylation site. (B) Representative autoradiographs are shown for the pulse-chase assays to measure the kinetics of TA translocation to ER in vivo for yeast strains with different Get2 variants as indicated. The complete set of replicates is shown in Fig. S5 B. (C) Quantification of the data from B and additional biological replicates in Fig. S5 B. The steady-state TA translocation levels are the same among yeast strains with different Get2 variants (Fig. S5 A). Hence, all the TA translocation values were normalized to the end point. Error bars denote SD, with n = 5–8 biological replicates. (D) Statistical analysis on the difference between WT and each Get2 variant using an unpaired two-sided t test (assuming that both populations have the same SD and follow Gaussian distribution). The P values for each significant different (P ≤ 0.05) pair are P^0min_WT/H1 = 0.0332; P^1min_WT/ΔH1 = 0.0028; P^1min_WT/ΔH2 = 0.0083; P^1min_WT/ΔH1ΔH2 = 0.0036; and P^2min_WT/ΔH1 = 0.0192. *, P ≤ 0.05; **, P ≤ 0.01.