Osmosensor ProP of Escherichia coli responds to the concentration, chemistry, and molecular size of osmolytes in the proteoliposome lumen

被引:77
作者
Culham, DE
Henderson, J
Crane, RA
Wood, JM [1 ]
机构
[1] Univ Guelph, Dept Microbiol, Guelph, ON N1G 2W1, Canada
[2] Univ Guelph, Guelph & Waterloo Ctr Chem & Biochem, Guelph, ON N1G 2W1, Canada
关键词
D O I
10.1021/bi0264364
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Transporter ProP of Escherichia coli mediates the cellular accumulation of organic zwitterions in response to increased extracellular osmolality. We compared and characterized the osmoregulation of ProP activity in cells and proteoliposomes to define the osmotic shift-induced cellular change(s) to which ProP responds. ProP-(HiS)(6) activity in cells and proteoliposomes was correlated with medium osmolality, not osmotic shift, turgor pressure, or membrane strain. Both K-M and V-max for proline uptake via Prop-(HiS)6 increased with increasing medium osmolality, as would be expected if osmolality controls the proportions of transporter with inactive and active conformations. The osmolality yielding, half-maximal ProP-(HiS)6 activity was higher in proteoliposomes than in cells. The osmolality response of ProP is also attenuated in bacteria lacking soluble protein ProQ. Indeed, the catalytic constant (k(cat)) for ProP-(HiS)(6) in proteoliposomes approximated that of ProP in intact bacteria lacking ProQ. Thus. the proteoliposome system may replicate a primary osmosensory response that can be further amplified by ProQ. ProP(HiS)(6) is designated as an osmosensor because its activity is dependent on the osmolality, but not the composition, of the assay medium to which the cell surface is exposed. In contrast, ProP-(HiS)(6) activity was dependent on both the osmolality and the composition of the lumen in osmolyte-loaded proteoliposomes. For proteoliposomes containing inorganic salts, glucose, or poly(ethylene glycol) 503, transporter activity correlated with total lumenal cation concentration. In contrast, for proteoliposomes loaded with larger poly(ethylene glycol)s, the osmolality, the lumenal cation concentration, and the lumenal ionic strength at half-maximal transporter activity decreased systematically with poly(ethylene glycol) radius of gyration (range 0.8-1.8 nm). These data suggest that ProP-(HiS)(6) responds to osmotically induced changes in both cytoplasmic K+ levels and the concentration of cytoplasmic macromolecules.
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页码:410 / 420
页数:11
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