Recognition of secretory proteins in Escherichia coli requires signals in addition to the signal sequence and slow folding

Ipsita Mallik,Ann M Flower,Margaret A Smith

doi:10.1186/1471-2180-2-32

Abstract

BackgroundThe Sec-dependent protein export apparatus of Escherichia coli is very efficient at correctly identifying proteins to be exported from the cytoplasm. Even bacterial strains that carry prl mutations, which allow export of signal sequence-defective precursors, accurately differentiate between cytoplasmic and mutant secretory proteins. It was proposed previously that the basis for this precise discrimination is the slow folding rate of secretory proteins, resulting in binding by the secretory chaperone, SecB, and subsequent targeting to translocase. Based on this proposal, we hypothesized that a cytoplasmic protein containing a mutation that slows its rate of folding would be recognized by SecB and therefore targeted to the Sec pathway. In a Prl suppressor strain the mutant protein would be exported to the periplasm due to loss of ability to reject non-secretory proteins from the pathway.ResultsIn the current work, we tested this hypothesis using a mutant form of λ repressor that folds slowly. No export of the mutant protein was observed, even in a prl strain. We then examined binding of the mutant λ repressor to SecB. We did not observe interaction by either of two assays, indicating that slow folding is not sufficient for SecB binding and targeting to translocase.ConclusionsThese results strongly suggest that to be targeted to the export pathway, secretory proteins contain signals in addition to the canonical signal sequence and the rate of folding.

Highlights

The Sec-dependent protein export apparatus of Escherichia coli is very efficient at correctly identifying proteins to be exported from the cytoplasm
The results indicate that slow folding is not sufficient for SecB binding and subsequent targeting to translocase and further, that secretory proteins contain targeting information in addition to the signal sequence and the folding kinetics
No LA57-N102LT was detected in the periplasm, even in the prlA4 strain, indicating that export did not occur to any significant extent

Summary

Introduction

The Sec-dependent protein export apparatus of Escherichia coli is very efficient at correctly identifying proteins to be exported from the cytoplasm. It was proposed previously that the basis for this precise discrimination is the slow folding rate of secretory proteins, resulting in binding by the secretory chaperone, SecB, and subsequent targeting to translocase. The Sec-dependent protein export pathway of Escherichia coli is responsible for translocation of secretory proteins across the inner membrane to final destinations in the periplasm or outer membrane. But not all, secretory proteins is dependent on interaction with SecB, a cytoplasmic chaperone that maintains the preprotein in a loosely folded conformation competent for translocation. Both SecB and the preprotein provide binding sites for SecA, a peripheral membrane ATPase. Formation of the complete translocase complex promotes an ATP binding and hydrolysis cycle by SecA that results in (page number not for citation purposes)

Methods

Results

Conclusion