Steady-state levels of several TA-proteins decrease drastically upon WRB/TRC40

Interestingly, many TA-proteins showed a diverse degree of dependence on the TRC pathway. Several were altered upon the knockdown of WRB and TRC40 (Fig.

26B). The simultaneous loss of the receptor (both WRB and CAML are drastically decreased, Fig. 27B and Fig. 27D) and TRC40 affected the steady-state levels of an overlapping yet distinct set of TA-proteins.

Stx5 was the most affected TA-protein (Fig. 28A) along with USE1 (Fig. 29A), Stx6 (Fig. 28B) and Sec22b (Fig. 30C). Stx5’s strong dependence on the TRC pathway has been previously described (Rivera-Monroy et al. 2016; Norlin et al. 2016; Casson et al.

2017; Norlin, Parekh, and Edlund 2018). Therefore, it is known to be a bona fide substrate of the TRC pathway. Stx5 showed a reduction of 88% at the steady-state level in the membrane fraction (compared to 50% reduction in whole cell lysate, (Rivera-Monroy et al. 2016)) in the combined knockdown of WRB and TRC40. Upon TRC40 knockdown steady-state levels were reduced by 58% with no effects on the steady-state levels when BAG6 was down-regulated (Fig. 28A). The vesicle transport protein USE1 is a SNARE protein believed to be involved in the retrograde transport from the Golgi apparatus to the ER (Dilcher et al. 2003). Steady-state levels of USE1 were severely decreased to 18% (Fig. 29A). Similarly, Stx6, a Golgi-resident protein involved in vesicular traffic, showed a 75% reduction at the steady-state level (Fig.

28B). Sec22b, an ER-resident SNARE protein, reported to be involved in anterograde and retrograde transport (Yiting Liu and Barlowe 2002; Burri et al. 2003), showed a reduction at the steady-state level of around 73% (Fig. 30C). The Ubiquitin-conjugating enzyme E2 J1 (UBE2J1), an ER-resident TA-protein involved in ERAD, showed a decrease of 64% in comparison to the control cells (Fig. 29C). VAPB is an ER-resident protein involved in linking the ER to other organelles (Costello, Castro, Hacker, et al.

2017; Hua et al. 2017; Gomez-Suaga et al. 2017; Dong et al. 2016) and in lipid trafficking, VAPB was found to be decreased to 59% at the steady-state level (Fig.

29D).

104

Figure 28. Quantification of a panel of tail-anchored proteins tested upon knockdown of TRC40, the TRC40 receptor or BAG6. (A) Quantification of Stx5 signal intensities from the blots performed in Fig. 26B. (B) Quantification of Stx6 signal intensities from the blots performed in Fig. 26B. (C) Quantification of Stx1 signal intensities from the blots performed in Fig. 26B. (D) Quantification of Stx8 signal intensities from the blots performed in Fig. 26B. Four biological replicates were analyzed. The graphs show the mean and the error bars represent standard error of the mean. * indicates a p-value <

0.05; ** a p-value < 0.05; *** a p-value < 0.001.

A B

Figure 28. Quantification of a panel of tail-anchored proteins tested upon knockdown of TRC40, the TRC40 receptor or BAG6. (A) Quantification of Stx5 signal intensities from the blots performed in Fig26B. (B) Quantification of Stx6 signal intensities from the blots performed in Fig26B. (C) Quantification of Stx1 signal intensities from the blots performed in Fig26B. (D) Quanti-fication of Stx8 signal intensities from the blots performed in Fig26B. Four biological replicates were analysed. The graphs show the mean and the error bars represent standard error of the mean. * indicates a p-value < 0.05; ** a p-value < 0.05; *** a p-value < 0.001.

105

Figure 29. Quantification of the tail-anchored proteins tested upon knockdown of TRC40, the TRC40 receptor or BAG6. (A) Quantification of USE1 signal intensities from the blots performed in Fig.

26B. (B) Quantification of EMD signal intensities from the blots performed in Fig. 26B. (C) Quantification of UBE2J1 signal intensities from the blots performed in Fig. 26B. (D) Quantification of VAPB signal intensities from the blots performed in Fig. 26B. Four biological replicates were analyzed. The graphs show the mean and the error bars represent standard error of the mean. * indicates a p-value < 0.05; **

a p-value < 0.05; *** a p-value < 0.001.

A B

Figure 29. Quantification of the tail-anchored proteins tested upon knockdown of TRC40, the TRC40 receptor or BAG6. (A) Quantification of USE1 signal intensities from the blots performed in Fig26B. (B) Quantification of EMD signal intensities from the blots performed in Fig26B. (C) Quantification of UBE2J1 signal intensities from the blots performed in Fig26B. (D) Quantification of VAPB signal intensities from the blots performed in Fig26B. Four biological repli-cates were analysed. The graphs show the mean and the error bars represent standard error of the mean. * indicates a p-value < 0.05; ** a p-value < 0.05; *** a p-value < 0.001.

106

Figure 30. Quantification of a panel of tail-anchored proteins tested upon knockdown of TRC40, the TRC40 receptor or BAG6 plus GM130. (A) Quantification of Vti1a signal intensities from the blots performed in Fig. 26B. (B) Quantification of VAPA signal intensities from the blots performed in Fig.

26B. (C) Quantification of Sec22b signal intensities from the blots performed in Fig. 26B. (D) Quantification of GM130 signal intensities from the blots performed in Fig. 26A. Four biological replicates were analyzed. The graphs show the mean and the error bars represent standard error of the mean. * indicates a p-value < 0.05; ** a p-value < 0.05; *** a p-value < 0.001.

A B

Figure 30. Quantification of a panel of tail-anchored proteins tested upon knockdown of TRC40, the TRC40 receptor or BAG6 plus GM130. (A) Quantification of Vti1a signal intensities from the blots performed in Fig26B. (B) Quantification of VAPA signal intensities from the blots performed in Fig26B. (C) Quantification of Sec22b signal intensities from the blots performed in Fig26B. (D) Quantification of GM130 signal intensities from the blots performed in Fig26A. Four biological replicates were analysed. The graphs show the mean and the error bars represent stand-ard error of the mean. * indicates a p-value < 0.05; ** a p-value < 0.05; *** a p-value < 0.001.

C D

107 EMD is a protein of the inner nuclear membrane and is also localized at the ER (Manilal, Nguyen, and Morris 1998; Pfaff et al. 2016). It showed a decrease of 56%

(Fig. 29B). EMD was already reported to be a substrate of the TRC pathway (Pfaff et al. 2016) and is known to be affected by the lack of the TRC receptor (Rivera-Monroy et al. 2016). Stx8 is a SNARE protein preferentially located at early endosomes, but also in late endosomes (Prekeris et al. 1999; Subramaniam et al. 2000; Kazuo Kasai et al. 2008). Stx8 steady-state levels were reduced 62% in the combined