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.
Friesen
R H
RH
Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Knol
J
J
Poolman
B
B
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