SecB-dependent protein transport in Escherichia coli

SecB is a cytosolic chaperone which facilitates transport of a subset of proteins, including membrane proteins such as PhoE and LamB and some periplasmic proteins such as maltose-binding protein, in Escherichia coli. However, not all proteins require SecB for transport and proteins such as ribose-binding protein are exported efficiently even in SecB null strains. The characteristics which confer SecB dependence on some proteins, but not others, have not been defined. To determine the sequence characteristics that are responsible for the SecB requirement, we have inserted a systematic series of short, polymeric sequences into the SecB-independent protein, alkaline phosphatase (PhoA). The extent to which these simple sequences convert alkaline phosphatase into a SecB-requiring protein was evaluated in vivo. We find that an insert with as few as 10 residues of which 3 are basic, confers SecB dependence and is efficiently exported in the presence of SecB. The alkaline phosphatase mutants most sensitive to loss of SecB are those most sensitive to inhibition of SecA via azide treatment, consistent with the necessity for formation of a preprotein-SecB-SecA complex. Furthermore, the impact of the basic motif depends on the hydrophobicity of the signal sequence, location within the mature protein and parallels the accessibility of the location to the secretion apparatus. ^ We have studied the interaction of SecB with SecA and with SecA-signal peptide complexes in vitro using purified components and synthetic signal peptides. Through a chemical cross-linking approach, we find that the formation of SecA-SecB complexes is accompanied by a decrease in cross-linking of SecA dimers, suggesting that SecB induces a significant conformational change in SecA. Furthermore, functional signal peptides, but not dysfunctional ones, promote the formation of SecA-SecB complexes. Interestingly, the presence of SecB can sufficiently boost the response due to signal peptides of moderate hydrophobicity such that it is comparable to the activity generated by a more hydrophobic peptide in the absence of SecB. The results suggest that SecB directly modulates the activity of SecA and provide a biochemical basis for the enhanced transport efficiency of preproteins in the presence of SecB in vivo.