Figure 3. Proteolysis of membrane-integrated SecA and various phospholipid-integrated SecA with trypsin.

(A) Liposomes (20 μg) of E. coli lipid mixtures and 20 μg BA13 inverted inner membrane (Mb) were mixed with SecA (10 μg) in 100 μl DTK buffer on ice. Trypsin treatment was undertaken at the concentrations indicated. (B) The same reactions were carried out as in panel (A), except that 1 μg of [³⁵S]SecA was used. (C) The same reactions were carried out as in panel (A) except that the reactions were incubated at 37 °C for 15 min, chilled on ice prior to the addition of trypsin. (D) 2 μg of [³⁵ S]SecA was mixed with different amounts of liposomes (as indicated). Half of the reaction mixtures were incubated at 37 °C for 15 min, while the other half were incubated on ice. All samples were cooled on ice following the reactions. Trypsin (final concentration 1 μg/ml) treatment was used, as described in the Materials and Methods.