Osmosensing properties of BetP

During a hyperosmotic stress response, an osmosensor element transduces a signal to the transport protein, which results in an osmotic activation or activity adaptation of the transporter. BetP possesses all functions for signal transduction.

Therefore, it should harbour at least one sensor domain, which reliably transduces an appropriate signal to the catalytic transport domain, thus regulating its activity.

Possible stimuli sensed by bacterial cells are divided into four different categories (Wood, 1999). Those are stimuli directly from the external osmolality, ionic strength, or concentration of particular solutes. Similar parameters are proposed to be relevant at the cytoplasmic site too, because a change in internal water activity is the consequence of a change in external osmolality. This leads to molecular crowding of cytoplasmic macromolecules and might be one relevant signal. Furthermore, all membrane related parameters such as cell turgor and membrane strain may well be important for osmoregulated transporters. Finally, changes in the external osmolality might also directly influence soluble and membrane embedded proteins by changing their surface hydration and thus might result in conformational changes.

When reconstituted into proteoliposomes, BetP showed to be more effectively activated by an increase of the luminal monovalent cations K⁺, Rb⁺ and Cs⁺. Several possible triggers were excluded from the experimental set up, such as changes of external solutes, internal solutes (except K⁺, choline⁺, NH4 ⁺, Na⁺) and membrane strain and cell turgor, because proteoliposomes are lacking turgor pressure. Na^+, NH4+ and choline⁺ were shown to be less efficient in BetP activation and BetP was therefore concluded to be a K⁺-specific chemosensor (Rubenhagen et al., 2001).

The N- and the C-terminal domains of BetP have been shown to influence activation strongly in intact C. glutamicum cells (Peter et al., 1998) (Figure 2).

Truncation of the N-terminal domain did not significantly change the catalytic activity of BetP, however, the activation profile was shifted to higher osmolalities.

Truncations of the C-terminal domain have a more drastic effect. Truncation of 25 to 45 amino acids from the C-terminus result in a deregulation of the transporter.

Truncation of just 12 C-terminal amino acids led only to a partial deregulation. These mutant forms were found to be constitutively active in intact C. glutamicum cells even in the absence of osmotic stress, although at one quarter reduced Vmax (Kramer and Morbach, 2004).

Figure 2| Consequence of N- and C-terminal truncations of BetP on transport activity. Glycine-betaine (betaine) uptake activity in wild type BetP C. glutamicum cells (yellow curve) increases in response to increasing hyperosmotic stress. Truncation of 60 amino acids at the N-terminal domain of BetP resulted in an activation profile that is shifted to higher osmolalities (blue curve). Truncation of 25 amino acids at the C-terminal domain leads to deregulation of BetP (red curve). Its increase at low Na⁺ concentration is caused by the dependence on Na⁺ as a co-substrate. (Adapted from http://www.kraemerlab.uni-koeln.de/osmosensing.php).

From these results it was concluded that the C-terminal domain is involved in osmosensing. Interestingly, they also indicate that the C-terminal domain contains an inhibitory element as well. Hence, this domain seems to be required to keep BetP in an inactive state in the absence of osmotic stress. Furthermore, this property was observed as being influenced by the identity of the lipids in which BetP was embedded (Schiller et al., 2006). By surface plasmon resonance spectroscopy (Ott et al., 2008) it was determined that the C-terminal domain interacts with the membrane surface. Lipid effects on osmodependent regulation have been observed with other

transporters as well, for example ProP of E. coli and OpuA of L. lactis. They respond with substrate transport at higher levels of hyperosmotic stress when the fraction of negatively charged phospholipids is increased in proteoliposomes or cells (Tsatskis et al., 2005; van der Heide et al., 2001).

Recent studies (Ott et al., 2008) have given new insights on the N-terminal domain of BetP, which in previous findings was already deemed to have a contribution to the regulation of BetP (Peter et al., 1998). Truncations of the N-terminal domain not only lead to a shift in sensitivity towards osmotic stress, but the surrounding lipids also affect the impact of N-terminal truncations on the regulatory properties of BetP (Ott et al., 2008). Since this was also observed in mutants that lacked the N-terminal domain completely, this was thought to have an indirect effect on BetP regulation. The regulatory influence of the N-terminal domain is therefore not based on direct peptide-membrane interaction but rather on protein-protein interaction with the opposing C-terminal domain, which in turn interacts directly with the membrane.

Based on these observations, a model for a molecular mechanism of the activation process of BetP was formulated (Figure 3) (Ott et al., 2008):

The membrane bound state of the C-terminal domain can be regarded as the inactive state, since an increased binding of the C-terminal domain to the membrane surface in the presence of negatively charged lipids made BetP activation increasingly difficult. This protein-lipid interaction of the C-terminal domain is an important feature of the inactivation of BetP and critically depends on the phospholipid charge composition of the membrane. The change from the inactive to the active state of the transporter is modulated by interaction changes of the C-terminal domain with part of loops at the cytoplasmic side (protein-protein interaction) as well as with the membrane (protein-lipid interaction). This is described as a functional switch model of BetP between the inactive and the active state depending on the location of the C-terminal domain.

Figure 3| Model of the regulation mechanism of BetP. A, inactive BetP. The green arrows indicate the interaction between the N-terminal and C-terminal domain (protein-protein interaction) and the interaction of the C-terminal domain with the lipids of the membrane (protein-lipid interaction). B, osmotically stimulated BetP. An unknown stimulus, indicated with the pink arrow in the plane of the membrane, together with the increase in K⁺ concentration in the cytoplasm lead to the activation of BetP. The direct or indirect sense of this K⁺ stimulus by the C-terminal domain induces a conformational change that leads to other protein-protein interactions between the C-terminal domain and cytoplasmic loops, in particular loop 8 (light grey). The modulated interactions are indicated with dashed lines. (Adapted after (Ott, 2008)

Obviously this model cannot describe all aspects of BetP activation, since it has been shown that also the composition of the hydrophobic part of the surrounding membrane and/or its physical state seems to influence the activity state of BetP (Ozcan et al., 2007; Ozcan et al., 2005).

In contrast to the BetP activation mechanism after an osmotic upshift, the downregulation after the point of osmotic compensation is unclear, concerning the

stimuli and the responsible signal transduction. As the adapted state is not equivalent to the inactive/resting state before osmotic activation (Figure 1), stimuli such as K⁺ and a change in external parameters were excluded. The regulatory mechanism might therefore be related to stimuli pertaining to the membrane properties, turgor or membrane strain.