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

Abstract

The Na+/proline transporter of Escherichia coli (PutP) is a member of the Na+/solute contransporter family (SCF) and catalyzes the uptake of proline by a Na+ 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.