Abstract
The Na+/proline transporter PutP of Escherichia coli is a member of a large family of Na+/substrate symporters. Previous work on PutP suggests an involvement of the region ranging from Asp-55 to Gly-58 in binding of Na+ and/or proline (Pirch, T., Quick, M., Nietschke, M., Langkamp, M., Jung, H. (2002) J. Biol. Chem. 277, 8790-8796). In this study, a complete Cys scanning mutagenesis of transmembrane domain II (TM II) of PutP was performed to further elucidate the role of the TM in the transport process. Strong defects of PutP function were observed upon substitution of Ala-48, Ala-53, Trp-59, and Gly-63 by Cys in addition to the previously characterized residues Asp-55, Ser-57, and Gly-58. However, except for Asp-55 none of these residues proved essential for function. The activity of eight mutants was sensitive to N-ethylmaleimide inhibition with the sensitive positions clustering predominantly on a hydrophilic face in the cytoplasmic half of TM II. The same face was also highly accessible to the bulky sulfhydryl reagent fluorescein 5-maleimide in randomly oriented membrane vesicles, suggesting an unrestricted accessibility of the corresponding amino acid positions via an aqueous pathway. Na+ stimulated the reactivity of Cys toward fluorescein 5-maleimide at two positions while proline inhibited reaction of the sulfhydryl group at nine positions. Taken together, the results demonstrate that TM II of PutP is of particular functional importance. It is proposed that hydrophilic residues in the cytoplasmic half of TM II participate in the formation of an aqueous cavity in the membrane that allows Na+ and/or proline binding to residues located in the middle of the TM (e.g. Asp-55 and Ser-57). In addition, the data indicate that TM II participates in Na+- and proline-induced conformational alterations.
Highlights
Whereby N- or C-terminal extensions may occur [3, 5,6,7]
It is proposed that hydrophilic residues in the cytoplasmic half of transmembrane domain II (TM II) participate in the formation of an aqueous cavity in the membrane that allows Na؉ and/or proline binding to residues located in the middle of the transmembrane domains (TM) (e.g. Asp-55 and Ser-57)
Previous work on the role of amino acids Ser-54 to Gly-58 has demonstrated that Asp-55 and Ser-57 are of particular importance for PutP function [13,14,15,16]
Summary
Materials—L-[U-14C]Proline (240 Ci/mol) and sheep anti-(mouse IgG)-horseradish peroxidase conjugate were purchased from Amersham Biosciences. Proline Transport Assays—Transport was measured in E. coli WG170 transformed with plasmid pT7-5/putP(⌬Cys) harboring the given mutations. NEM Inhibition—NEM inhibition of active transport was measured in E. coli WG170 transformed with plasmid pT7-5/putP(⌬Cys) carrying the given mutations. An aliquot of the cell suspension prepared as described above was incubated with 2 mM NEM for 20 min on ice, followed by addition of 20 mM -mercaptoethanol to stop the reaction. Cys Accessibility Analyses—To investigate accessibility of individual introduced Cys residues to the sulfhydryl-specific dye fluorescein 5-maleimide, 500 ml of E. coli WG170 transformed with pTrc99a/putP(⌬Cys) harboring given mutations were grown aerobically to an A420 nm of 1.5 before induction with 0.5 mM isopropyl-thio--D-galactoside and further growth for 2 h. 150-l aliquots of the membrane suspension containing 10 mg/ml total protein were incubated with or without Naϩ and/or proline at given concentrations at 25 °C for 10 min. Protein Determination—Determination of protein was performed according to Bradford [23] for purified protein and a modified Lowry method [24] for total membrane protein with bovine serum albumin as standard
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
