SecA protein of Escherichia coli, when added externally to the vesicles composed of phosphatidylethanolamine, dioleoylphosphatidylglycerol and cardiolipin, was found to be fragmented by trypsin encapsulated within the vesicles. In the presence of ATP or its non-hydrolyzing analogue, ATP-γS, the number of fragments and extent of hydrolysis were much less than in the absence of these compounds. When ADP was added, however, the hydrolysis products were similar to those when no nucleotide was present. Quenching of SecA fluorescence by vesicle-entrapped iodide corroborated the digestion results. These experiments demonstrated that the SecA protein traverses the lipid bilayer and its membrane topology depends on the kind of nucleotide present.
The refolding precursor ribose binding protein (RBP) added externally was found to inhibit the digestion of SecA by the trypsin inside the vesicles but a signal peptide, also added externally, promoted the digestion. The presence of refolding pRBP induced dequenching of SecA fluorescence by $I^-$ entrapped within the interior space of vesicles. These observations suggest that the refolding precursor RBP retards the lipid bilayer penetration by SecA while the signal peptide enhances it. This discrepancy was found to be due to reduced SecA binding to the vesicles in the presence of the precursor RBP while the signal peptide had no effect. The extraction of vesicle-bound SecA either by KCl or urea was reduced appreciably by signal peptides. In membrane-free solution, the precursor RBP decreased the intensity of intrinsic tryptophan fluorescence of SecA and 1-anilino-8-naphthalene sulfonate (ANS) binding to SecA, but the signal peptide gave exactly the opposite results. We also observed that, upon interaction with precursor RBP, SecA became resistant to heat and guanidine hydrochloride-induced unfolding but signal peptide had no effect. These results suggest that SecA assumes more closed conformation upon interaction with pRBP and signal peptide induces more open structure of SecA. Kinetic studies of ANS binding to SecA after this were mixed with unfolded precursor RBP showed a two-step reaction. The initial increase in ANS binding caused by the fast interaction between the signal peptide of the precursor RBP and SecA was followed by the release of ANS which presumably occur upon subsequent interaction between the mature domain of the precursor RBP and SecA.
The precursor RBP also enhanced the digestion of SecA added to the E. coli inverted vesicles while the signal peptide had an opposite effect. These results are consistent with the results obtained with the model membrane above. When the pRBP and ATP were present together, however, the penetration of SecA increased dramatically underlining the importance of the SecY/E complex for the membrane insertion of SecA.
