Regulatory Properties and Interaction of the C- and N-Terminal Domains of BetP, an Osmoregulated Betaine Transporter from Corynebacterium glutamicum

Abstract

The glycine betaine carrier BetP from Corynebacterium glutamicum responds to changes in external osmolality by regulation of its transport activity, and the C-terminal domain was previously identified to be involved in this process. Here we investigate the structural requirements of the C-terminal domain for osmoregulation as well as interacting domains that are relevant for intramolecular signal transduction in response to osmotic stress. For this purpose, we applied a proline scanning approach and amino acid replacements other than proline in selected positions. To analyze the impact of the surrounding membrane, BetP mutants were studied in both C. glutamicum and Escherichia coli, which strongly differ in their phospholipid composition. A region of approximately 25 amino acid residues within the C-terminal domain with a high propensity for alpha-helical structure was found to be essential in terms of its conformational properties for osmodependent regulation. The size of this region was larger in E. coli membranes than in the highly negatively charged C. glutamicum membranes. As a novel aspect of BetP regulation, interaction of the C-terminal domain with one of the cytoplasmic loops as well as with the N-terminal domain was shown to be involved in osmosensing and/or osmoregulation. These results support a functional model of BetP activation that involves the C-terminal domain shifting from interaction with the membrane to interaction with intramolecular domains in response to osmotic stress.

Regulatory Activation Is Accompanied by Movement in the C Terminus of the Na-K-Cl Cotransporter (NKCC1)

“…In a FRET study similar to this one, Bykova et al (36) found a large conformational change in CLC-0 associated with slow gating of the channel. A similar mechanism of regulation has recently been proposed for the Na ϩ /betaine symporter, BetP (37,38). In this transporter, transport activity is thought to be controlled by a "molecular switch" involving electrostatic interactions between the surrounding lipid membrane, the N and C termini, and cytoplasmic loop 8 (37).…”

Ectoine and Hydroxyectoine as Protectants against Osmotic and Cold Stress: Uptake through the SigB-Controlled Betaine-Choline- Carnitine Transporter-Type Carrier EctT from Virgibacillus pantothenticus

“…The transport activity of BetP is activated in less than a second in response to osmotic stress (32). BetP activation is elicited by an osmotic stressderived cellular signal (possibly increases in the intracellular K ϩ pool) that modulates the intersubunit interaction of the extended carboxy terminus of BetP within the BetP trimer assembly (39,42). The EctT transporter from V. pantothenticus lacks such an extended carboxy terminus, and our finding that the transport activity of EctT is not strongly activated by an osmotic stimulus (Fig.…”

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

“…Uptake of [ 14 C]-betaine in E. coli cells was performed as described (64). E. coli MKH13 cells expressing a particular strep-betP mutant were cultivated at 37°C in LB medium containing carbenicillin (50 μg/mL) and induced at an OD 600 of 0.5 by adding anhydrotetracycline (200 μg/L).…”