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

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 (Asp 468 -Arg 497 ) forms an intermolecular ␣-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.

Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

“…Recently we showed that the osmolality yielding half-maximal ProP activity (and hence the osmotic activation "threshold") increased in parallel with the proportion of CL when E. coli was cultivated in media of increasing osmolality. This suggested that CL modulates ProP function, adapting the osmolality range over which ProP activity is regulated to match ambient (growth) conditions (7). The C termini of E. coli ProP and its orthologues are longer than those of its paralogues (like LacY) (3,7).…”

“…ProP orthologues with and without C-terminal coiled-coils are osmosensory transporters but orthologues and variants with the coiled-coil activate at lower osmolalities than those without (7). Mutations that disrupt or eliminate the coiled-coil raise the activation threshold (7,25,29). Thus the coiled-coils present on some ProP orthologues tune them to osmoregulate at low osmolalities (7).…”

“…This suggested that CL modulates ProP function, adapting the osmolality range over which ProP activity is regulated to match ambient (growth) conditions (7). The C termini of E. coli ProP and its orthologues are longer than those of its paralogues (like LacY) (3,7). The C terminus of E. coli ProP and some orthologues includes heptad repeats characteristic of proteins that form ␣-helical coiled-coils (Fig.…”

“…The proportions of these lipids vary with growth medium, osmolality, and growth phase. The proportion of CL increases ϳ2-fold at the expense of PE, and PG remains constant, as E. coli is cultivated to exponential phase in minimal media of increasing osmolality (⌸/RT) (7). The CL synthase encoded by the osmotically inducible cls gene is responsible for most CL synthesis but an alternate pathway for CL synthesis is evident in CL synthase-deficient (cls Ϫ ) bacteria (6,8).…”

“…Indeed, antiparallel, homodimeric ␣-helical coiled-coils link the C termini of E. coli ProP dimers (25)(26)(27)(28)(29). ProP orthologues with and without C-terminal coiled-coils are osmosensory transporters but orthologues and variants with the coiled-coil activate at lower osmolalities than those without (7). Mutations that disrupt or eliminate the coiled-coil raise the activation threshold (7,25,29).…”