Arabidopsis immunophilin-like TWD1 functionally interacts with vacuolar ABC transporters.
Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-beta-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.
Geisler
Markus
M
Zürich-Basel Plant Science Center, University of Zurich, Institute of Plant Biology, Molecular Plant Physiology, CH-8008 Zurich, Switzerland. markus.geisler@botinst.unizh.ch
Girin
Marjolaine
M
Brandt
Sabine
S
Vincenzetti
Vincent
V
Plaza
Sonia
S
Paris
Nadine
N
Kobae
Yoshihiro
Y
Maeshima
Masayoshi
M
Billion
Karla
K
Kolukisaoglu
Uner H
UH
Schulz
Burkhard
B
Martinoia
Enrico
E
D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000081:Acetamides / Q000032:analysis / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000081:Acetamides; D029681:Arabidopsis Proteins; D002148:Calmodulin-Binding Proteins; C495596:MRP1 protein, Arabidopsis; C495597:MRP2 protein, Arabidopsis; C111981:PAS1 protein, Arabidopsis; C471286:TWD1 protein, Arabidopsis; D022021:Tacrolimus Binding Proteins; C051786:metolachlor