Influence of membrane composition on osmosensing by the betaine carrier BetP from Corynebacterium glutamicum.
The glycine betaine carrier BetP from Corynebacterium glutamicum was recently shown to function as both an osmosensor and osmoregulator in proteoliposomes made from Escherichia coli phospholipids by sensing changes in the internal K+ concentration as a measure of hyperosmotic stress (Rübenhagen, R., Morbach, S., and Krämer, R. (2001) EMBO J. 20, 5412-5420). Furthermore, evidence was provided that a stretch of 25 amino acids of the C-terminal domain of BetP is critically involved in K+ sensing. This K+-sensitive region has been further characterized. Glu572 turned out to be important for osmosensing in E. coli cells and in proteoliposomes made from E. coli phospholipids. BetP mutants E572K, E572P, and E572A/H573A/R574A were unable to detect an increase in the internal K+ concentration in this membrane environment. However, these BetP variants regained their ability to detect osmotic stress in membranes with increased phosphatidylglycerol content, i.e. in intact C. glutamicum cells or in proteoliposomes mimicking the composition of the C. glutamicum membrane. Mutants E572P and Y550P were still insensitive to osmotic stress also in this membrane background. These results led to the following conclusions. (i) The K+ sensor in mutants E572Q, E572D, and E572K is only partially impaired. (ii) Restoration of activity regulation is not possible if the correct conformation or orientation of the C-terminal domain is compromised by a proline residue at position 572 or 550. (iii) Phosphatidylglycerol in the membrane of C. glutamicum seems to stabilize the inactive conformation of BetP C252T and other mutants.
Schiller
Dirk
D
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706-1544, USA.
Ott
Vera
V
Krämer
Reinhard
R
Morbach
Susanne
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001622:Betaine / Q000737:chemistry; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D009995:Osmosis; D010715:Phosphatidylglycerols / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D027981:Symporters
D001426:Bacterial Proteins; C101652:BetP protein, Corynebacterium glutamicum; D002352:Carrier Proteins; D008055:Lipids; D008081:Liposomes; D009841:Oligonucleotides; D010715:Phosphatidylglycerols; D010743:Phospholipids; D027981:Symporters; D001622:Betaine; D011392:Proline; D011188:Potassium