Multimer recognition and secretion by the non-classical secretion pathway in Bacillus subtilis

Liuqun Zhao,Jingqi Chen,Jibin Sun,Dawei Zhang

doi:10.1038/srep44023

Abstract

Non-classical protein secretion in bacteria is a common phenomenon. However, the selection principle for non-classical secretion pathways remains unclear. Here, our experimental data, to our knowledge, are the first to show that folded multimeric proteins can be recognized and excreted by a non-classical secretion pathway in Bacillus subtilis. We explored the secretion pattern of a typical cytoplasmic protein D-psicose 3-epimerase from Ruminococcus sp. 5_1_39BFAA (RDPE), and showed that its non-classical secretion is not simply due to cell lysis. Analysis of truncation variants revealed that the C- and N-terminus, and two hydrophobic domains, are required for structural stability and non-classical secretion of RDPE. Alanine scanning mutagenesis of the hydrophobic segments of RDPE revealed that hydrophobic residues mediated the equilibrium between its folded and unfolded forms. Reporter mCherry and GFP fusions with RDPE regions show that its secretion requires an intact tetrameric protein complex. Using cross-linked tetramers, we show that folded tetrameric RDPE can be secreted as a single unit. Finally, we provide evidence that the non-classical secretion pathway has a strong preference for multimeric substrates, which accumulate at the poles and septum region. Altogether, these data show that a multimer recognition mechanism is likely applicable across the non-classical secretion pathway.

Highlights

Milieu in B. subtilis[19]
We previously reported that RDPE, which converts D-fructose into D-psicose from Ruminococcus sp. 5_1_39BFAA, can be secreted into the medium from B. subtilis without a typical signal peptide
These results indicate that the secretion of RDPE occurs in the early-exponential phase and that the secretion efficiency was nearly constant during the growth phase, even though extensive cell lysis occurred in the late-stationary growth phase

Summary

Introduction

Milieu in B. subtilis[19]. Wang et al listed 45 common non-classically secreted proteins identified in various bacteria[9], hinting that non-classical secretion is a universal phenomenon in bacteria. Results Secretion of cytoplasmic protein RDPE is not due to cell lysis. It is not known how folded RDPE is exported via a non-classical secretion pathway in B. subtilis.

Results

Conclusion