![The EMC uses a hydrophilic vestibule for TA protein insertion. (A) Views of the two intramembrane surfaces of the EMC. Residues in EMC3 (purple) lining either the hydrophilic vestibule or hydrophobic crevice were mutated to cysteines for disulfide crosslinking and are highlighted in blue or tan, respectively. EMC4, 7, and 10 are omitted in the inset for clarity. (B) Purified WT or EMC3 cysteine (Cys) mutant EMC was incubated with CaM-SQS containing a cysteine in the TMD at either position T408 (CaM-SQS[T408C]) or L401 (CaM-SQS[L401C]). After substrate release from CaM with EGTA, cysteines in close proximity were crosslinked with the zero-length disulfide crosslinker DPS. Quenched reactions were analyzed by SDS-PAGE and autoradiography. (C) hRMs prepared from EMC3 WT or Cys mutant cell lines were mixed with CaM-SQS(T408C) for crosslinking as described in B. Substrate crosslinks were enriched by denaturing purification of EMC3-GFP. Samples were analyzed by SDS-PAGE followed by autoradiography or Western blotting. Source data are available for this figure: SourceData F2.](https://cdn.rupress.org/rup/content_public/journal/jcb/222/8/10.1083_jcb.202212007/1/jcb_202212007_fig2.png?Expires=1767719270&Signature=OTed3X52-Npb81MU0AlRcpii1m8V2rvHF3PWNOLPbYOYUNf-GVb0Y0WboDa4fH~MSf1PVWegfjUbkTsbyLh3YTaTG97wK7zz4av5uu5krF9NOpBc9n2HiwDGvFUeRWdD-1ei6to8EulT5JWCN90H0e54by18lFykGeG6Pm9W468ERrkPOANzl9iLI~0uVVg--a8w-c8gM1LE9kJKOysRGkRGlIOlcIUNBRJXMqSuUQn-0rsGjZDIUeAJxlVgxwTuUNMRXYb-gIiknFpTC~oouAJxz0~xUmbk3UyjTLW63~2YtmKCIPb504oCvoPdPoqjK9VRY4dmMm6O0Mnqhex6MA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The EMC uses a hydrophilic vestibule for TA protein insertion. (A) Views of the two intramembrane surfaces of the EMC. Residues in EMC3 (purple) lining either the hydrophilic vestibule or hydrophobic crevice were mutated to cysteines for disulfide crosslinking and are highlighted in blue or tan, respectively. EMC4, 7, and 10 are omitted in the inset for clarity. (B) Purified WT or EMC3 cysteine (Cys) mutant EMC was incubated with CaM-SQS containing a cysteine in the TMD at either position T408 (CaM-SQS[T408C]) or L401 (CaM-SQS[L401C]). After substrate release from CaM with EGTA, cysteines in close proximity were crosslinked with the zero-length disulfide crosslinker DPS. Quenched reactions were analyzed by SDS-PAGE and autoradiography. (C) hRMs prepared from EMC3 WT or Cys mutant cell lines were mixed with CaM-SQS(T408C) for crosslinking as described in B. Substrate crosslinks were enriched by denaturing purification of EMC3-GFP. Samples were analyzed by SDS-PAGE followed by autoradiography or Western blotting. Source data are available for this figure: SourceData F2.