Signal Peptide Determinants of SecA Binding and Stimulation of ATPase Activity

Ligong Wang,Alexander Miller,Debra A Kendall

doi:10.1074/jbc.275.14.10154

Ligong Wang, Alexander Miller + Show 1 more

Open Access

https://doi.org/10.1074/jbc.275.14.10154

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Abstract

A signal peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown. In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites. Synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and three sets of model signal sequences varying in hydrophobicity and amino-terminal charge were generated. These were used to establish the requirements for interaction with SecA. Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-lipid ATPase activity occur with functional signal sequences but not with transport-incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region. For signal peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequivocally that the signal peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of signal peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo.

Highlights

Many proteins that are synthesized in the cytoplasm of cells are found in noncytoplasmic locations
SecA has been shown to interact with preproteins [10, 12, 13], and recently, our laboratory established that the signal peptide region alone can bind SecA in liposomes and stimulate SecA-lipid ATPase activity [14]
If SecA is a key component of the sorting mechanism that discriminates secreted proteins from nonsecreted ones, it must be able to distinguish between functional signal peptides and all other sequences, including some that may be marginally hydrophobic

Summary

Signal Peptide Determinants of SecA Binding and Stimulation of ATPase Activity*

(Received for publication, November 3, 1999, and in revised form, December 21, 1999). Using signal sequence mutants and in vivo analysis, no functional differences are observed for signal peptides with one or three positively charged residues and with core regions rich in leucine residues [21] For those signal peptides that are less hydrophobic, a clear correlation between amino-terminal charge and transport activity is found [22, 23]. The signal peptide core region could be an important recognition element for interaction with the transport machinery and its affinity may be attenuated by flanking charged residues That one such interaction involves SecA has been inferred by the graded response of signal sequence mutants involving titration of amino-terminal charge and core region hydrophobicity to SecA inhibition in vivo [22]. Binding of synthetic peptides corresponding to functional signal sequences was shown to occur for SecA in both aqueous solution and a lipid bilayer

EXPERIMENTAL PROCEDURES

TABLE I Sequences of synthetic signal peptides

RESULTS AND DISCUSSION

Wild type

TABLE III Relative competition of peptides in aqueous solution