TWISTED DWARF1, a Unique Plasma Membrane-anchored Immunophilin-like Protein, Interacts withArabidopsisMultidrug Resistance-like Transporters AtPGP1 and AtPGP19

Abstract

Null-mutations of the Arabidopsis FKBP-like immunophilin TWISTED DWARF1 (TWD1) gene cause a pleiotropic phenotype characterized by reduction of cell elongation and disorientated growth of all plant organs. Heterologously expressed TWD1 does not exhibit cis-trans-peptidylprolyl isomerase (PPIase) activity and does not complement yeast FKBP12 mutants, suggesting that TWD1 acts indirectly via protein-protein interaction. Yeast two-hybrid protein interaction screens with TWD1 identified cDNA sequences that encode the C-terminal domain of Arabidopsis multidrug-resistance-like ABC transporter AtPGP1. This interaction was verified in vitro. Mapping of protein interaction domains shows that AtPGP1 surprisingly binds to the N-terminus of TWD1 harboring the cis-trans peptidyl-prolyl isomerase-like domain and not to the tetratrico-peptide repeat domain, which has been shown to mediate protein-protein interaction. Unlike all other FKBPs, TWD1 is shown to be an integral membrane protein that colocalizes with its interacting partner AtPGP1 on the plasma membrane. TWD1 also interacts with AtPGP19 (AtMDR1), the closest homologue of AtPGP1. The single gene mutation twd1-1 and double atpgp1-1/atpgp19-1 (atmdr1-1) mutants exhibit similar phenotypes including epinastic growth, reduced inflorescence size, and reduced polar auxin transport, suggesting that a functional TWD1-AtPGP1/AtPGP19 complex is required for proper plant development.

Master and servant: Regulation of auxin transporters by FKBPs and cyclophilins

“…A chaperone-like function for TWD1 during ER to plasma membrane trafficking of ABCBs was suggested based on the finding that ABCB1,4,19 are retained on the ER and degraded in the twd1 mutant [8,9]. ABCB-type auxin transporters and TWD1 have been both identified as targets of NPA [37,41,62,111,112]. Micromolar concentrations of NPA were also shown to disrupt the ABCB1-TWD1 interaction (see +NPA) supporting the hypothesis that NPA binds at the ABCB-TWD1 interface [44,130].…”

“…The twd1/ucu2/fkbp42 mutants is marked by a characteristic twisting of the root epidermal cell files and displays a defect in root gravitropism, an auxin-transport regulated phenotype [8,9,62,63] (Table 1). Over-expression of a mutated version of TWD1 lacking its membrane anchor also results in a hypermorphic ("giant") growth phenotype caused by enhanced cell elongation, which is probably due to altered apoplastic auxin concentrations caused by enhanced auxin transport [64] (see Section 4).…”

“…Over-expression of a mutated version of TWD1 lacking its membrane anchor also results in a hypermorphic ("giant") growth phenotype caused by enhanced cell elongation, which is probably due to altered apoplastic auxin concentrations caused by enhanced auxin transport [64] (see Section 4). All attempts to demonstrate PPIase activity in TWD1 have failed [62,65], probably because only 3 out of the 11 key residues inside the hydrophobic pocket of its FK506-binding domain involved in PPIase function are conserved [55] (see Fig. 1).…”

“…The unusual FK506-binding domain in TWD1 was instead found to participate in protein interaction with ABCB-type auxin transporters and is apparently involved in the functional regulation of these transporters [44] (see Section 4). TWD1 also possesses a tetratricopeptide repeat domain (common [131] Regulation and PM-to-ER trafficking of ABCB-type auxin transporters [8,9] Z. mais [133] Interaction with ABCB transporters on PM (via FKB-domain) [8,62], ABCC transporters on tonoplast (via TPR domain) [66], with HSP90 (TPR domain) [62,65] and calmodulin (calmodulin-binding domain) [66] Over-expression of TWD1 lacking its membrane anchor results in hypermorphic growth [64] FKBP72/PAS1 A. thaliana [69] Unclear (C-terminal membrane anchor), nuclear [68,134] Low, inhibited by FK506 and rapamycin [134] Up-regulated cell division, leaf fusions, short hypocotyls, sterile [69] O. sativa [131] Chaperon during translocation of NAC-like transcription factor (AtFAN) into nucleus [68] Z. mais [133] Regulation of very long fatty acid elongation [70] -DGT/CypA/ROC1/CYP1/CYP2/ P. patens [10] Nuclear and cytoplasmic [10,90], phloem sieve elements [79] Significant [77,78], inhibited by CsA [78] Regulates growth [90], gene expression [84,90,92], patterned cell division [88], phloem function [79], ROS balance in root apical meristem [87] A. [137] in many large immunophilins) that was shown to interact with vacuolar ABC transporters of the C subclass and HSP90 [65,66], as w...…”

“…Whereas FKBPs of plants show reductions in their affinity for immunosuppressive drugs as well as decreases in their PPIase activity, plant cyclophilins display conserved affinity for immunosuppressive drugs and presence of PPIase activity. Recent studies identified plant immunophilins as regulators of polar auxin transport, and suggested that immunophilins function in either auxin-transporter cellular trafficking or activity, or both [8][9][10]43,44,62]. Whereas Arabidopsis TWD1/FKBP42 shows preference for binding and functional interaction with ABCB type of auxin transporters [8], tomato cyclophilin DGT/CypA displays high functional preference for PIN auxin transporters [10].…”