Purification and Reconstitution of an Osmosensor:  Transporter ProP of <i>Escherichia</i> <i>coli</i> Senses and Responds to Osmotic Shifts

L-Carnitine is essential for ␤-oxidation of fatty acids in mitochondria. Bacterial metabolic pathways are used for the production of this medically important compound. Here, we report the first detailed functional characterization of the caiT gene product, a putative transport protein whose function is required for L-carnitine conversion in Escherichia coli. The caiT gene was overexpressed in E. coli, and the gene product was purified by affinity chromatography and reconstituted into proteoliposomes. Functional analyses with intact cells and proteoliposomes demonstrated that CaiT is able to catalyze the exchange of L-carnitine for ␥-butyrobetaine, the excreted end product of L-carnitine conversion in E. coli, and related betaines. Electrochemical ion gradients did not significantly stimulate L-carnitine uptake. Analysis of L-carnitine counterflow yielded an apparent external K m of 105 M and a turnover number of 5.5 s ؊1 . Contrary to related proteins, CaiT activity was not modulated by osmotic stress. L-Carnitine binding to CaiT increased the protein fluorescence and caused a red shift in the emission maximum, an observation explained by ligand-induced conformational alterations. The fluorescence effect was specific for betaine structures, for which the distance between trimethylammonium and carboxyl groups proved to be crucial for affinity. Taken together, the results suggest that CaiT functions as an exchanger (antiporter) for L-carnitine and ␥-butyrobetaine according to the substrate/product antiport principle.

Section: Discussionmentioning

confidence: 99%

CaiT of Escherichia coli, a New Transporter Catalyzing l-Carnitine/γ-Butyrobetaine Exchange

Jung

Buchholz

Clausen³

et al. 2002

“…Transport activity of purified ProP in proteoliposomes (50) or RSO-MV (42) was significantly increased upon an osmotic upshift imposed by NaCl or sucrose. In addition, the results obtained for EnvZ are different to KdpD, a sensor kinase, which responds to K ϩ limitation or an osmotic upshift imposed by salts.…”

Section: Discussionmentioning

confidence: 99%

K+ Stimulates Specifically the Autokinase Activity of Purified and Reconstituted EnvZ of Escherichia coli

Jung

Hamann

Revermann

2001

The histidine kinase/response regulator system EnvZ/ OmpR of Escherichia coli regulates transcription of the genes ompF and ompC, encoding two porins of the outer membrane. Although the total amount of OmpF and OmpC remains constant, the relative levels of the two proteins fluctuate in a reciprocal manner depending on medium osmolality. The membrane-anchored sensor EnvZ somehow monitors changes in environmental osmolality. To characterize the nature of the stimulus perceived by EnvZ, this protein was overproduced, purified, and reconstituted into proteoliposomes. Autokinase activity of purified and reconstituted EnvZ was stimulated by an increase of the K ؉ concentration. Rb

“…The suitability of a reconstituted system for this purpose has recently been shown for the osmoregulated ProP from E. coli (14). Proteoliposomes are an attractive approach for this goal mainly because of two aspects.…”

mentioning

confidence: 99%

Osmosensor and Osmoregulator Properties of the Betaine Carrier BetP from Corynebacterium glutamicum in Proteoliposomes

Rübenhagen¹,

Rönsch²,

Jung³

et al. 2000

120

189

The secondary glycine betaine uptake system BetP of Corynebacterium glutamicum was purified from Escherichia coli membranes in strep-tagged form after heterologous expression of the betP gene and was reconstituted in E. coli lipids. BetP retained its kinetic properties (V max and K m for betaine and Na ؉ ) as compared with intact cells. The influence of driving forces (Na ؉ gradient and/or electrical potential) on betaine uptake was quantified in proteoliposomes. BetP was effectively regulated by the external osmolality and was stimulated by the local anesthetic tetracaine. A shift of the optimum of osmotic stimulation to higher osmolalities was linearly correlated with an increasing share of phosphatidyl glycerol, the major lipid of the C. glutamicum plasma membrane in the E. coli lipid proteoliposomes. This finding correlates with results demonstrating an identical shift when betP was expressed in E. coli instead of C. glutamicum. These data indicate that (i) BetP comprises all elements of osmosensing and osmoregulatory mechanisms of betaine uptake, (ii) osmoregulation of BetP is directly related to protein/membrane interactions, (iii) the turgor pressure presumably plays no major role in osmoregulation of BetP, and (iv) the regulatory properties of BetP may be related to the physical state of the surrounding membrane.