Topology and function of the Na+/proline transporter of Escherichia coli, a member of the Na+/solute cotransporter family

The Na+/proline transporter of Escherichia coli (PutP) is a member of the Na+/solute cotransporter family (SCF) and catalyzes the uptake of proline by a Naf dependent transport mechanism. Hydropathy profile analysis suggests that the protein consists of 12 transmembrane domains (TMs) that traverse the membrane in zigzag fashion connected by hydrophilic loops. However, analysis of a series of putP-phoA (PutP-alkaline phosphatase) and putP-lacZ (PutP-beta-galactosidase) fusions and site-directed labeling of the transporter indicate a 13-helix motif with the N-terminus on the outside and the C-terminus facing the cytoplasm. The findings are discussed with respect to a common topological motif for all members of the SCF. Furthermore, amino acid substitution analysis indicates that the N-terminal part of PutP is important for ion binding. Thus, Asp55 (putative TM II) is essential for transport and proposed to interact directly with Na+. The functional importance of TM II is further confirmed by the observation that replacement of Arg40, Ser50, Ala53, or Ser57 alters transport kinetics dramatically. (C) 1998 Elsevier Science B.V.