The Osmotic Activation of Transporter ProP Is Tuned by Both Its C-terminal Coiled-coil and Osmotically Induced Changes in Phospholipid Composition

Yonit Tsatskis,Jumana Khambati,Martina Dobson,Mikhail Bogdanov,William Dowhan,Janet M Wood

doi:10.1074/jbc.m508362200

Abstract

Transporter ProP of Escherichia coli (ProPEc) senses extracellular osmolality and mediates osmoprotectant uptake when it is rising or high. A replica of the ProPEc C terminus (Asp468-Arg497) forms an intermolecular alpha-helical coiled-coil. This structure is implicated in the osmoregulation of intact ProPEc, in vivo. Like that from Corynebacterium glutamicum (ProPCg), the ProP orthologue from Agrobacterium tumefaciens (ProPAt) sensed and responded to extracellular osmolality after expression in E. coli. The osmotic activation profiles of all three orthologues depended on the osmolality of the bacterial growth medium, the osmolality required for activation rising as the growth osmolality approached 0.7 mol/kg. Thus, each could undergo osmotic adaptation. The proportion of cardiolipin in a polar lipid extract from E. coli increased with extracellular osmolality so that the osmolality activating ProPEc was a direct function of membrane cardiolipin content. Group A ProP orthologues (ProPEc, ProPAt) share the C-terminal coiled-coil domain and were activated at low osmolalities. Like variant ProPEc-R488I, in which the C-terminal coiled-coil is disrupted, ProPEc derivatives that lack the coiled-coil and Group B orthologue ProPCg required a higher osmolality to activate. The amplitude of ProPEc activation was reduced 10-fold in its deletion derivatives. The coiled-coil structure is not essential for osmotic activation of ProP per se. However, it tunes Group A orthologues to osmoregulate over a low osmolality range. Coiled-coil lesions may impair both coiled-coil formation and interaction of ProPEc with amplifier protein ProQ. Cardiolipin may contribute to ProP adaptation by altering bulk membrane properties or by acting as a ProP ligand.

Highlights

Somes: Hϩ symporter ProP of Escherichia coli [4], Naϩ symporter BetP of Corynebacterium glutamicum [5], and ATP-binding cassette transporter OpuA of Lactococcus lactis [6]
Osmotic Adaptation of ProPEc—We previously reported that the transport assay medium osmolality required to activate ProPEc-His6 was independent of the osmolality of the medium in which E. coli was grown (NaCl-supplemented MOPS media with osmolalities in the range 0.12– 0.32 mol/kg; Fig. 1B of Ref. 10)
For cells grown in the higher osmolality range, the osmolality required to activate ProPEc depended on the osmolalities of both the growth medium and the assay medium (Fig. 2)

Summary

Introduction

Somes: Hϩ symporter ProP of Escherichia coli [4], Naϩ symporter BetP of Corynebacterium glutamicum [5], and ATP-binding cassette transporter OpuA of Lactococcus lactis [6]. Tuning and Osmotic Activation of Transporter ProP (TMI–TMXII) or determined by NMR spectroscopy of the corresponding synthetic peptide [20] (the C-terminal coiled-coil domain, represented by the white box). B, the NMR solution structure of a peptide replica of residues Asp468–Arg497 of ProPEc. The antiparallel ␣-helical coiled-coil structure appears to be stabilized by two salt bridges, each formed by the interaction of Arg488 on one strand with Asp475 and Asp478 on the other. The coiledcoil domain unique to Group A orthologues is not essential for osmosensing, since ProPCg was found to act as an osmosensor and osmoregulator after expression in E. coli [22]

Objectives

Results

Conclusion