Membrane topology of helices VII and XI in the lactose permease of Escherichia coli studied by lacY-phoA fusion analysis and site-directed spectroscopy.
The use of lactose permease-alkaline phosphatase fusions (lacY-phoA) demonstrates that the lactose permease of Escherichia coli contains 12 transmembrane domains and that approximately half of a transmembrane domain is required to translocate alkaline phosphatase to the periplasmic surface of the membrane [Calamia, J., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937-4941]. We have now used fusion analysis in combination with site-directed spectroscopy to examine more precisely the topology of putative helices VII and XI which contain the interacting residues Asp237 and Lys358, respectively. For this purpose, alkaline phosphatase was fused to alternate amino acid residues in transmembrane domains VII and XI. A sharp increase in alkaline phosphatase activity is observed as the fusion junction proceeds from Try228 to Ile230 in helix VII and from Phe354 to Phe356 in helix XI, suggesting that these residues approximate the middle of the corresponding transmembrane helices. Analysis of fluorescence quenching of the pyrene-labeled single-Cys mutants Asp237 --> Cys or Lys358 --> Cys, as well as measurement of collision frequencies between freely diffusing paramagnetic probes and a nitroxide spin-label at these sites, also indicates that Asp237 and also Asp240, which interacts with Lys319 (helix X), are located in transmembrane domains. However, Asp237 and Asp240 are accessible both from the aqueous phase and from within the membrane. The results provide more direct evidence that the three residues are located within transmembrane helices and suggest that Asp237 and Asp240 are either located near the periplasmic surface of the membrane or exposed within a solvent-filled cleft in the permease.
Ujwal
M L
ML
Howard Hughes Medical Institute, Department of Physiology, Los Angeles, California, USA.
Jung
H
H
Bibi
E
E
Manoil
C
C
Altenbach
C
C
Hubbell
W L
WL
Kaback
H R
HR
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005453:Fluorescence; D008565:Membrane Proteins / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013057:Spectrum Analysis; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; C021499:lactose permease; D000469:Alkaline Phosphatase