The P54S V57G Sbh1p tm-domain double mutant is unable to rescue loss of Sbh1p and Sbh2p. A, A wheel presentation of the P54S V57G Sbh1p tm-domain mutant. B, Growth of sbh1Δ sbh2Δ cells (H3232) transformed with plasmids encoding SBH1 tm-domain mutants with or without BIO-tag. C, UTA translocation assay on sbh1Δ sbh2Δ cells (H3543) transformed with plasmids encoding reporter proteins Suc223, Sec2277, Dap2300, or the empty vector pRS314, or with plasmids encoding full length Sbh1p with P54S V57G mutations (Sbh1-FL(DM)), Sbh1p tm-domain with P54S (Sbh1-TM(SM1)), Sbh1p tm-domain with V57G (Sbh1-TM(SM2)) or Sbh1p tm-domain with P54S V57G (Sbh1-TM(DM)) or the empty plasmid. The growth of transformants was tested on SCD-Trp-Leu or on SCD-Trp-Leu-Ura plates to score for translocation of the Ura3p containing reporters. D, Western blot analysis with anti-HA (for Rtn1p-HA), Sec61p antibodies or with HRP conjugated streptavidin (for versions of BIO-Sbh1p) of pull-downs from sbh1Δ cells (H3429) expressing BIO-tagged Sbh1p(P54S V57G), Sbh1p TM(P54S), Sbh1p TM(V57G), Sbh1p TM(P54S V57G) or an empty plasmid p426ADH.