Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays.
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.
Reinders
Anke
A
Plant Biology, University of Minnesota, 220 Biological Sciences Center, 1445 Gortner Avenue, St. Paul, MN 55108, USA.
Schulze
Waltraud
W
Thaminy
Safia
S
Stagljar
Igor
I
Frommer
Wolf B
WB
Ward
John M
JM
D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism*; D025801:Ubiquitin; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D026901:Membrane Transport Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C078729:sucrose transport protein, plant; D013395:Sucrose