Quaternary structure of the lactose transport protein of Streptococcus thermophilus in the detergent-solubilized and membrane-reconstituted state.
The quaternary structure of LacS, the lactose transporter of Streptococcus thermophilus, has been determined for the detergent-solubilized and the membrane-reconstituted state of the protein. The quaternary structure of the n-dodecyl-beta-d-maltoside-solubilized state was studied using a combination of sedimentation velocity and equilibrium centrifugation analysis. From these measurements it followed that the detergent-solubilized LacS undergoes reversible self-association with a monomer to dimer mode of association. The association constants were 5.4 +/- 3.6 and 4.4 +/- 1.0 ml mg(-1) as determined from the velocity and equilibrium sedimentation measurements, respectively. The experiments did not indicate significant changes in the shape of the protein-detergent complex or the amount of detergent bound in going from the monomeric to dimeric state of LacS. Importantly, a single Cys mutant of LacS is labeled by 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid in a substrate-dependent manner, indicating that the detergent-solubilized protein exhibits ligand binding activity. The quaternary structure of membrane-reconstituted LacS was determined by freeze-fracture electron microscopy analysis. Recent developments in the analysis of freeze-fracture images (Eskandari, S. P., Wright, E. M., Freman, M., Starace, D. M., and Zampighi, G. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11235-11240) allowed us to directly correlate the cross-sectional area of the transmembrane segment to a dimeric state of the functionally membrane-reconstituted LacS protein. The cross-sectional area of the LacS protein was calibrated using the membrane-reconstituted transmembrane domain of the mannitol transporter enzyme II, an intramembrane particle for which the cross-sectional area was obtained from maps of two-dimensional crystals. The consequences of the determined quaternary structure for the transport function and regulation of LacS are discussed.
Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
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Friesen
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Knol
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Poolman
D003902:Detergents / Q000494:pharmacology; D029968:Escherichia coli Proteins; D005690:Galactose / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins; D020836:Protein Structure, Quaternary; D027981:Symporters
D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C040358:dodecyl maltoside; C021499:lactose permease; D005690:Galactose