Fig. 1. The covalently linked SecYEG dimer is functional for pre protein translocation. (A) Overproduction of SecYYEG suppresses the SecDFyajC^– cold-sensitive and SecY thermo-sensitive phenotypes. Strain BL325 (BL21 tgt::kan-araC⁺ -P_BAD::yajCsecDF) or CJ107 (Wolfe et al., 1985) were transformed with the isopropyl-β-d-thio galactopyranoside (IPTG)-inducible plasmid pTrc99 or pTrc99 encoding SecYE_HAG (pTrcEYG) or SecYYE_HAG (pTrcEYYG). Transformants were plated on LB/ampicillin (50 µg/ml) contain ing 1 mM IPTG and incubated at the restrictive temperature. (B) Immunostaining of IMV preparations. About 1 µg of IMV proteins prepared from E.coli BL21 overexpressing SecYE_HAG (IMVs-Y) or SecYYE_HAG (IMVs-YY) were analyzed by SDS–PAGE and immunostained with the indicated antibodies. In the presence of SDS, SecY (48.5 kDa) and SecYY (97 kDa) migrate aberrantly. (C and D) Translocase activity of the IMV preparations. Translocation reactions were performed and analyzed as described in Materials and methods.