Voltage Sensing in Bacterial Protein Translocation.

Denis G Knyazev,Ana-Nicoleta Bondar,Peter Pohl,Christine Siligan,Roland Kuttner,Mirjam Zimmerman

doi:10.3390/biom10010078

Denis G Knyazev, Ana-Nicoleta Bondar + Show 4 more

Open Access

https://doi.org/10.3390/biom10010078

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Abstract

The bacterial channel SecYEG efficiently translocates both hydrophobic and hydrophilic proteins across the plasma membrane. Translocating polypeptide chains may dislodge the plug, a half helix that blocks the permeation of small molecules, from its position in the middle of the aqueous translocation channel. Instead of the plug, six isoleucines in the middle of the membrane supposedly seal the channel, by forming a gasket around the translocating polypeptide. However, this hypothesis does not explain how the tightness of the gasket may depend on membrane potential. Here, we demonstrate voltage-dependent closings of the purified and reconstituted channel in the presence of ligands, suggesting that voltage sensitivity may be conferred by motor protein SecA, ribosomes, signal peptides, and/or translocating peptides. Yet, the presence of a voltage sensor intrinsic to SecYEG was indicated by voltage driven closure of pores that were forced-open either by crosslinking the plug to SecE or by plug deletion. We tested the involvement of SecY’s half-helix 2b (TM2b) in voltage sensing, since clearly identifiable gating charges are missing. The mutation L80D accelerated voltage driven closings by reversing TM2b’s dipolar orientation. In contrast, the L80K mutation decelerated voltage induced closings by increasing TM2b’s dipole moment. The observations suggest that TM2b is part of a larger voltage sensor. By partly aligning the combined dipole of this sensor with the orientation of the membrane-spanning electric field, voltage may drive channel closure.

Highlights

Subsequent to their synthesis, many of the bacterial membrane and secretory proteins take the translocation pathway across the heterotrimeric SecYEG complex of the evolutionary conserved Sec family [1,2]
The main translocation unit of the translocon, SecY, has a central pore, which is closed for the passage of small molecules in its resting state by a hydrophobic ring (HR) of six amino acids and by a re-entrant loop (TM2a), called the plug domain (PD) [3]
SecYEG was overexpressed for 3 h in E. coli c43 (DE3) cells from a pBad22 vector and induced with 2 g/L of arabinose

Summary

Introduction

Subsequent to their synthesis, many of the bacterial membrane and secretory proteins take the translocation pathway across the heterotrimeric SecYEG complex of the evolutionary conserved Sec family [1,2]. The bacterial SecYEG has a striking homology to its archaeal and eukaryotic protein family members SecYEβ and Sec61αβγ. The channel opens upon binding of the signal sequence of translocating peptides between the gate posts [5,6]. This enables subsequent polypeptide segments to translocate through SecY’s central pore [7]. Hydrophobic helices exit via the lateral gate into the lipid phase, while hydrophilic segments enter the periplasm [8]

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