Hydrophobicity is a major contributor for the TRC-dependence in vivo

TMD hydrophobicity has always been considered as a relevant factor in TA-protein biogenesis. It has been proposed that moderately hydrophobic TMDs were substrates of the TRC pathway (F. Wang et al. 2010; Rao et al. 2016; Costello, Castro, Camões, et al. 2017; Guna et al. 2018; Borgese, Colombo, and Pedrazzini 2003; Borgese, Brambillasca, and Colombo 2007). From the different TMD-hydrophobicity analyses carried out (Fig. 33-36), it is clear that hydrophobicity is indeed a determinant factor in the fate of the endogenous TA-proteins in vivo. The majority of the TA-protein affected by WRB/TRC40 knockdown presented moderate-to-high hydrophobic TMDs whereas the majority of the unaffected TA-proteins had low hydrophobic TMDs. Using the GRAVY scoring (based on the Kyte and Doolittle scale) the group of affected and unaffected TA-proteins could be separated (Fig. 35A). However, for the other hydrophobicity scales, some TA-proteins remained in an intermediary region, similar to the Sec61b-TMD hydrophobicity score, with no clear separation between affected

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and unaffected groups. This result was independent of the scale used: the TMD tendency scale (Fig. 33A), the Kyte and Doolittle scale (Fig. 34A) and for the apparent free-energy (Fig. 36A). The answer to why proteins that have TMDs with very similar hydrophobicity yielded different behaviors may lie in the amino acid composition of the TMDs. Helical wheels projections, generated from the predicted TMD sequences, showed little evidence of which residues could be crucial for the TRC-pathway dependence (Fig. 54, Fig. 55). However, a deeper analysis of the TMD sequences from those TA-proteins sensitive to the WRB/TRC40 knockdown yielded an interesting result. The sequence logo showed the relevance of aromatic amino acids at the beginning of the TMD and the presence of isoleucines, phenylalanines, leucines or valines (Fig. 56A). The algorithm chosen for the logo representation highlights amino acid enrichment and emphasizes the relevant parts (Thomsen and Nielsen 2012). It is not a consensus sequence and cannot be treated as such, since neither the experimental approach nor the sample size is large enough for making that claim.

Other than that, it is an analysis that showed that the presence of an aromatic amino acid was relevant at either the beginning or the end of the TMD. Moreover, the central part of the TMD also appeared to be important since the relative weight of certain hydrophobic amino acids (almost exclusively isoleucines, phenylalanines, leucines) was higher. In contrast, the sequence logo for the TMD of the unaffected TA-proteins showed an enrichment in aromatic amino acids in the last third of the TMD (Fig. 56B).

The enrichment in hydrophobic amino acids is lower than in the case of the affected TA-protein TMDs.

Taken together, hydrophobicity is an important factor in TA-targeting as seen in the TMD-hydrophobicity dot-plots, especially using the GRAVY score. However, hydrophobicity on its own cannot explain the in vivo dependence on the TRC pathway for every TA-protein, especially those with moderately hydrophobic TMD close to the hydrophobicity of Sec61b. The zone of hydrophobicity close to Sec61b could be a region where other pathways overlap in vivo, as pointed out for certain TA-proteins in a previous study (Guna et al. 2018).

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Figure 54. Helical wheel projections of TMDs of the TA-proteins affected by the WRB/TRC40 knockdown. TA-proteins were ordered according to the affectation in steady-state level in the knockdown cells, descendent order. Numbers indicate the TMD hydrophobicity score according to the hydrophobicity scale developed by (Kyte and Doolittle 1982) divided by the length of the TMD (in aa), for obtaining the GRAVY score. Those TA-proteins affected by the presence of TRC40D74E are highlighted in green or in red if they remain unchanged. The helical wheel analysis was done using the application wheel.pl v1.4 (Zidovetzki et al.

2003). Available here: http://rzlab.ucr.edu/scripts/wheel/wheel.cgi

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Figure 55. Helical wheel projections of TMDs of the TA-proteins that remained unaffected upon the WRB/TRC40 knockdown. TA-proteins whose steady-state levels were not altered upon the WRB/TRC40 knockdown were displayed in a random order. Numbers indicate the TMD hydrophobicity score according to the hydrophobicity scale developed by (Kyte and Doolittle 1982) divided by the length of the TMD (in aa), for obtaining the GRAVY score. Those TA-proteins affected by the presence of TRC40D74E are highlighted in green or in red if they remain unchanged. The helical wheel analysis was done using the application wheel.pl v1.4 (Zidovetzki et al. 2003). Available here: http://rzlab.ucr.edu/scripts/wheel/wheel.cgi

GOSR2 2,168 Sec61ß

2,048 Vti1b

2,104

SQS 1,342 PTP1B

1,570 Stx18

1,617

Figure xx. Helical wheel projections of TMDs of the TA-proteins that remained unaffected upon the WRB/TRC40 knockdown. TA-proteins whose steady-state levels were not altered upon the WRB/TRC40 knockdown were displayed in a random order. Numbers indicate the TMD hydrophobicity score according to the hydrophobicity scale developed by (Kyte and Doolittle 1982) divided by the length of the TMD, for obtaining the GRAVY score. The helical wheel analysis was done using the application wheel.pl v1.4 (Zidovetzki et al. 2003). Available here: http://rzlab.ucr.edu/scripts/wheel/wheel.cgi

Sec61ß 2,048

PTP1B 1,570

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Figure 56. Sequence logo of the TMDs of TA-proteins tested upon the WRB/TRC40 knockdown.

(A) Sequence logo of the twelve 19 aa TMDs of the affected TA-proteins. (B) Sequence logo of the six 21 aa TMDs of the unaffected TA-proteins. The probability weighted Kullback-Leibler logo. The height of the amino acids represents the probability times their log-odds score. The code of color used is similar to the one used in the helical wheel projections (Fig. 54, Fig. 55). The sequence logo was generated using the web-based generator Seq2Logo 2.0(Thomsen and Nielsen 2012). Available here:

http://www.cbs.dtu.dk/biotools/Seq2Logo/. Parameters used for the analysis are available in section 7.5.3 within Appendix.

Figure 56. Sequence logo of the TMDs of TA-proteins aftected upon the WRB/TRC40 knock-down. Sequence logo of the twelve TMDs using the probability weighted Kullback-Leibler. The height of the amino acids represents the probability times their log-odds score. The sequence logo was generated using the web-based generator Seq2Logo 2.0 (Thomsen and Nielsen 2012). Available here: http://www.cbs.dtu.dk/biotools/Seq2Logo/

Amino acids

Bits

A

Bits

B

Amino acids

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4.2.3.2. The silent role of the cytoplasmic domain in the subcellular