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. 2015 Apr 27;209(2):261–273. doi: 10.1083/jcb.201408088

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© 2015 Habeck et al.

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Figure 5. — TM-residue Ser68 of the Sbh2 tail anchor is a part of the degron. (A) Sbh2 TA sequence in complex with Ssh1 TM helices 1 and 4. Top view of the interface of Sbh2 and Ssh1. Sbh2 residues 58–79 and Ssh1 residues 26–53 (TM1) and 148–179 (TM4) are shown. Sbh2 Ser61 and Ser68 are depicted in red in ball and stick mode. Picture was generated with PyMOL using the atomic coordinates from Protein Data Bank accession no. 2WWA (Becker et al., 2009). (B) Degradation of HA-tagged Sbh2 WT and S61P and S68A point mutants in ssh1Δ cells. A representative blot is shown. chx chase was performed as in Fig. 1 B. The graph at right shows the mean degradation rates observed from at least three independent experiments. Error bars represent ± SD (C and D) HA-Sbh2(S61P) and HA-Sbh2(S68A) are stable in doa10Δ ssh1Δ cells. chx chase analysis of HA-Sbh2(S61P) and HA-Sbh2(S68A) stability. (E) HA-Sbh2(S61P,S68A) is stable even in ssh1Δ cells. chx chase analysis of HA-Sbh2(S61P,S68A) stability. (F) Suppression of growth defect of sbh1Δ sbh2Δ cells at high temperature by mutant Sbh2 variants (S61P), (S68A), and (S61P,S68A), respectively. Growth assay as in Fig. 3 E. Empty vector and HA-Sbh2 lanes are from one plate. (G) Co-IP analysis of digitonin-solubilized microsomes to investigate the interaction between Sec61 β subunits and Sec61. WT or mutant HA-Sbh2 was ectopically expressed in doa10Δ ssh1Δ cells and was precipitated with anti-HA agarose beads. Precipitates were analyzed by immunoblotting with the indicated antibodies. The TA Ubc6 protein served as a negative control. (H) Co-IP analysis to investigate the interaction between Sec61 β subunits and Sec61. WT HA-Sbh1 or HA-Sbh2 or mutant HA-Sbh2 was ectopically expressed in SEC61-9MYC ssh1Δ cells (W303-1B strain background), and Sec61-9MYC was precipitated with an anti-MYC antibody (rabbit polyclonal). Precipitates were analyzed by immunoblotting with indicated antibodies. IB, immunoblot.