Signal peptide determinants of SecA binding and stimulation of ATPase activity

A signal peptide is required for entry of a preprotein into the secretory p athway, but how it functions in concert with the other transport components is unknown. In Escherichia coil, SecA is a key component of the translocat ion machinery found in the cytoplasm and at membrane translocation sites. S ynthetic signal peptides corresponding to the wild type alkaline phosphatas e signal sequence and three sets of model signal sequences varying in hydro phobicity and amino-terminal charge were generated. These were used to esta blish the requirements for interaction with SecA, Binding to SecA, modulati on of SecA conformations sensitive to protease, and stimulation of SecA-Lip id ATPase activity occur with functional signal sequences but not with tran sport-incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region. For signal peptide s of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequi vocally that the signal peptide, in the absence of the mature protein, inte racts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of signal peptide characteristics that prom ote interaction with SecA in vitro and the hierarchy of those observed for function in vivo.