van der Heide T, Stuart M C, Poolman B
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
EMBO J. 2001 Dec 17;20(24):7022-32. doi: 10.1093/emboj/20.24.7022.
The osmosensing mechanism of the ATP-binding cassette (ABC) transporter OpuA of Lactococcus lactis has been elucidated for the protein reconstituted in liposomes. Activation of OpuA by osmotic upshift was instantaneous and reversible and followed changes in volume and membrane structure of the proteoliposomes. Osmotic activation of OpuA was dependent on the fraction of anionic lipids present in the lipid bilayer. Also, cationic and anionic lipophilic amphiphiles shifted the activation profile in a manner indicative of an osmosensing mechanism, in which electrostatic interactions between lipid headgroups and the OpuA protein play a major role. Further support for this notion came from experiments in which ATP-driven uptake and substrate-dependent ATP hydrolysis were measured with varying concentrations of osmolytes at the cytoplasmic face of the protein. Under iso-osmotic conditions, the transporter could be activated by high concentrations of ionic osmolytes, whereas neutral ones had no effect, demonstrating that intracellular ionic strength, rather than a specific signaling molecule or water activity, signals osmotic stress to the transporter. The data indicate that OpuA is under the control of a mechanism in which the membrane and ionic strength act in concert to signal osmotic changes.
乳酸乳球菌ATP结合盒（ABC）转运蛋白OpuA的渗透感应机制已通过重组到脂质体中的蛋白质得以阐明。渗透上调对OpuA的激活是瞬时且可逆的，并伴随着蛋白脂质体体积和膜结构的变化。OpuA的渗透激活取决于脂质双分子层中阴离子脂质的比例。此外，阳离子和亲脂性两性离子型两亲物以一种表明渗透感应机制的方式改变激活曲线，其中脂质头部基团与OpuA蛋白之间的静电相互作用起主要作用。这一观点进一步得到了实验的支持，在这些实验中，在蛋白质细胞质面用不同浓度的渗透溶质测量了ATP驱动的摄取和底物依赖性ATP水解。在等渗条件下，转运蛋白可被高浓度的离子型渗透溶质激活，而中性渗透溶质则无作用，这表明细胞内离子强度而非特定的信号分子或水活性向转运蛋白发出渗透应激信号。数据表明，OpuA受一种机制的控制，在该机制中，膜和离子强度共同作用以发出渗透变化信号。
