Effect of precursor ribose binding protein on the SecA penetration into phospholipid vesicles

Abstract

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 ...