Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays

Anke Reinders, Waltraud Schulze, Safia Thaminy, Igor Stagljar, Wolf B. Frommer, John M. Ward

Research output: Contribution to journalArticlepeer-review

50 Scopus citations

Abstract

Interaction of two separately expressed halves of sucrose transporter SUT1 was detected by an optimized split-ubiquitin system. The halves reconstitute sucrose transport activity at the plasma membrane with affinities similar to the intact protein. The halves do not function independently, and an intact central loop is not required for membrane insertion, plasma membrane targeting, and transport. Under native conditions, the halves associate into higher molecular mass complexes. Furthermore, the N-terminal half of the low-affinity SUT2 interacts functionally with the C-terminal half of SUT1. Since the N terminus of SUT2 determines affinity for sucrose, the reconstituted chimera has lower affinity than SUT1. The split-ubiquitin system efficiently detects intramolecular interactions in membrane proteins, and can be used to dissect transporter structure.

Original languageEnglish (US)
Pages (from-to)763-772
Number of pages10
JournalStructure
Volume10
Issue number6
DOIs
StatePublished - 2002

Bibliographical note

Funding Information:
We are grateful to Christine Bürki (Institute of Veterinary Biochemistry, University of Zürich-Irchel) for assistance with vector construction, Dr. Alonso Rodriguez-Navarro (Universidad Politecnica de Madrid) for the 59m15 yeast strain, Dr. Julian I. Schroeder (UC San Diego) for the plasmid containing AtKAT1, Dr. Doris Rentsch for pDR196, and Dr. Stephan te Heesen for strong support with the project in the early stages, and thanks to Dr. Claudia Oecking for helpful advice concerning “blue native” gels. This work was supported by the Deutsche Forschungsgemeinschaft (grants RE 1454/1 to A.R. and SFB446 to W.B.F.). W.S had a stipend from Deutsche Studienstiftung. S.T. and I.S. were supported by the Kanton of Zürich, Zürcher Krebsliga, Gebert-Rüf Stiftung, Walter Honegger Stiftung, Bonizzi-Theler Stiftung, EMDO Stiftung, and Swiss National Science Foundation (31-58798.99).

Keywords

  • Assembly
  • Dimer
  • Heteromeric interaction
  • Membrane protein
  • Structure
  • Sucrose transporter

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