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 mem brane-reconstituted state of the protein. The quaternary structure of the n -dodecyl-beta -D-maltoside-solubilized state was studied using a combinatio n of sedimentation velocity and equilibrium centrifugation analysis. From t hese measurements it followed that the detergent-solubilized LacS undergoes reversible self-association with a monomer to dimer mode of association. T he association constants were 5.4 +/- 3.6 and 4.4 +/- 1.0 ml mg(-1) as dete rmined from the velocity and equilibrium sedimentation measurements, respec tively. The experiments did not indicate significant changes in the shape o f the protein-detergent complex or the amount of detergent bound in going f rom the monomeric to dimeric state of LacS. Importantly, a single Cys mutan t 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 memb rane-reconstituted LacS was determined by freeze-fracture electron microsco py analysis. Recent developments in the analysis of freeze-fracture images (Eskandari, S, P,, Wright, E, Ri,, 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 mem brane-reconstituted transmembrane domain of the mannitol transporter enzyme II, an intramembrane particle for which the cross-sectional area was obtai ned from maps of two-dimensional crystals. The consequences of the determin ed quaternary structure for the transport function and regulation of LacS a re discussed.