The Twisted Dwarf's ABC

Abstract

There is increasing evidence that immunophilins function as key regulators of plant development. One of the best investigated members, the multi-domain FKBP TWISTED DWARF1 (TWD1)/FKBP42, has been shown to reside on both the vacuolar and plasma membranes where it interacts in mirror image with two pairs of ABC transporters, MRP1/ MRP2 and PGP1/PGP19(MDR1), respectively. Twisted dwarf1 and pgp1/pgp19 mutants display strongly overlapping phenotypes, including reduction and disorientation of growth, suggesting functional interaction.

In a recent work using plant and heterologous expression systems, TWD1 has been demonstrated to modulate PGP-mediated export of the plant hormone auxin, which controls virtually all plant developmental processes. Here we summarize recent molecular models on TWD1 function in plant development and PGP-mediated auxin tranport and discuss open questions.

FK506-binding Proteins (FKBPs), together with unrelated cyclophilins, belong to the immunophilins, an ancient and ubiquitous protein family.^1,4,5 They were first described as receptors for immunosuppressive drugs in animal and human cells, FK506 and cyclosporin A, respectively.¹ All FKBP-type immunophilins share a characteristic peptidyl-prolyl cis-trans isomerase domain (PPIase domain or FKBD, Fig. 2A) making protein folding a key feature among immunophilins.² The best investigated example, the human cytosolic single-domain FKBP12, modulates Ca²⁺ release channels^6,7 and associates with the cell cycle regulator TGF-β.⁸ Furthermore, the human FKBP12/FK506 complex is known to bind and inhibit calcineurin activity,⁹ leading to immune response inhibition. However, not all single- and multiple-domain FKBPs own folding activity and, interestingly, many form distinct protein complexes with diverse functions.^3–5

Figure 2.

Model of TWISTED DWARF 1 interacting proteins. (A) Domain structure of TWD1 and putative interacting proteins. FKBD, FK506-binding domain: TPR, tetratricopeptide repeat; CaM(-BD, calmodulin-binding domain; MA, membrane anchor. For details, see text. (B) Functional TWD1-ABC transporter complexes on both the vacuolar and plasma membrane. While for TWD1/PGP pairs, the positive regulatory role on auxin transport was demonstrated,¹⁸ the modulation of MRP-mediated vacuolar import of glutathion conjugates (GS-X) was established using mammalian test substrates¹⁷ because the in vivo substrates are unknown. Note that C-terminal nucleotide binding folds of MRP- and PGP-like ABC transporters interact with distinct functional domains of TWD1, the TPR and FKBD, respectively. The native auxin, IAAH, gets trapped by deprotonization upon uptake into the cell. Export is catalyzed by secondary active export via PIN-like efflux carriers¹⁵ and/or by primary active, ATP-driven P-glycoproteins (PGPs, right panel); loss-of TWD1 function abolishes PGP-mediated auxin export (left panel).

Multiple-Domain FKBPS Function as Key Players in Plant Development

Twenty-three FKBP-type proteins have been identified in the Arabidopsis thaliana genome, however, the function of individual FKBPs in plant biology is poorly understood. Many single-domain FKBPs, like FKBP20-2, seem to be chloroplast targeted and to be involved in redox regulation of photosynthesis.¹⁰ The multiple-domain FKBP mutant pasticcino1 (pas1), defective in cell elongation and proliferation, shows highly disorganized seedlings. The PAS1 protein (FKBP52) is nuclear, shows little PPIase-activity and controls the targeting and the function of a NAC-like transcription factor.^11–13 The Arabidopsis mutant twisted dwarf1 (twd1, allelic to ultracurvata2 (ucu2)¹⁴) lacks the high-molecular weight FKBP42 and displays a drastic pleiotropic phenotype including reduced development and cell elongation, and disoriented growth of all organs, both on the epidermal and whole plant level (Fig. 1A, E and F).^15,16 Based on a yeast two hybrid screen and confirmed by a whole range of methods TWD1 was shown to interact in mirror image with two pairs of ATP-binding cassette (ABC) transporters (Fig. 2B): the multidrug-resistance (MDR)-like P-glycoproteins (PGP) PGP1 and PGP19 (MDR1) on the plasma membrane, and the multidrug-resistance associated (MRP)-like MRP1 and MRP2 on the tonoplast.^16,17 Interestingly, interacting stretches of individual ABC transporters containing C-terminally the nucleotide-binding folds interact with distinct functional domains of TWD1: the FKBD and tripartite tetratricopeptide repeat (TPR) domain, respectively. Strikingly, the double mutant pgp1/pgp19, but not the corresponding single mutants, shares with twd1 highly similar auxin-related phenotypes: dwarfism (Fig. 1A and B) and agravitropic roots.¹⁸ These results suggest a central position for TWD1 in PGP-mediated auxin transport.

Figure 1.

The twisted dwarf1 (twd1) mutant displays a pleiotropic developmental phenotype that correlates with reductions in auxin transport. (A) Growth phenotypes of one-month-old soil-grown plants; from left to right: wild-type (ecotype Wassilewskija), pgp1, pgp19, pgp1/pgp19 and twd1. Inset: mature pgp1/pgp19 and twd1 plants. Bars, 5 cm. (B) Growth phenotypes of light-grown seedlings 5 dag; from left to right: wild-type, pgp1, pgp19, pgp1/pgp19 and twd1. Bar, 1 cm. (from ref. 16). (C) Reduced polar IAA transport in hypocotyls of young seedlings reflects growth phenotypes. Means ± S.D. (from ref. 16, modified). (D) Cellular IAA export in mutant protoplasts is decreased compared to wild-type (open square) in the order wt > pgp1 (open triangle) > pgp19 (upside-down open triangle) >> pgp1/pgp19 (open diamond) ≥ twd1 (open circle). Dark-treated (de-energized) plant material (filled square) was used as negative control. Means ± S.D. (data taken from ref. 16 and 31). (E) Electron micrograph of the twd1 hypocotyl. The “twisted syndrome” of all organs is perceptible at the epidermal level. Bar, 100 µm. (picture taken from ref. 31, modified). (F) The twd1 silique displays reduced and disoriented growth. Bar, 500 µm.

TWD1 Functions as Positive Modulator of P-Glycoprotein-Mediated Auxin Transport

Inline with this hypothesis, constitutive upregulation of PGP1 and PGP19 enhanced IAA net efflux, while overexpression of TWD1 did not alter wild type levels, excluding any direct auxin transport activity.¹⁸ Developmental plant phenotypes correlate perfectly with decreased auxin transport rates on the organ (Fig. 1C) and cellular level (Fig. 1D). Moreover, the use of a novel highly selective IAA microelectrode¹⁹ allowed monitoring decreased auxin influxes in twd1 and pgp1/pgp19 mutant root apices compared to the single PGP mutants or the wild type.¹⁸ As a consequence, free IAA levels are greatly elevated in mature pgp1/pgp19 and twd1 roots compared to single mutants or wild type, indicating a reduction of PGP-mediated polar auxin transport in twd1.

The regulatory role of TWD1 on PGP1-mediated auxin transport was confirmed by functional coexpression in S. cerevisiae and mammalian HeLa cells. Interestingly, IAA efflux assays in yeast¹⁸ and functional complementation of the auxin-hypersensitive yeast mutant yap1²⁰ revealed an inhibitory effect of TWD1 on PGP1 function while coexpression in HeLa cells resulted in enhanced auxin export. This difference might result from the lack of higher eukaryotic components in S. cerevisiae.

Together, these findings further support the idea of a positive regulatory role for TWD1 on PGP-driven auxin transport in planta.

Does the Regulatory Function of TWD1 Involve Chaperone Activity?

It was recently suggested that brassinosteroid-mediated signal transduction in twd1 may require the activity of the HSP90 chaperone complex.²⁴ Consistent with these findings, citrate synthase aggregation assays²⁵ revealed a chaperone-like activity requiring the TWD1 HSP90-binding TPR domain (Fig. 2A) while the FKBD alone did not prevent aggregation.²⁵ Previous studies suggested yeast single-domain FKBP12 to function as a regulator of murine PGP-like proteins, but independently of its PPIase activity.^21,22 Interestingly, TWD1 function on PGP1-mediated IAA efflux was only slightly mimicked by Arabidopsis FKBP12.¹⁸ Moreover, the FKBD of TWD1 displays no PPIase activity²³ and TWD1 does not complement yeast FKBP12.¹⁷ Furthermore, TWD1 does not bind FK506²³ which, based on structural data, is due to sterical reasons.²⁶ In agreement it was established that impaired auxin transport in twd1 was neither caused by changes in PGP1 or PIN1/PIN2 auxin efflux carrier¹⁵ expression nor by localization, confirming that the regulatory effect is due to physical interaction. Finally, the biochemically and immunologically demonstrated membrane location of TWD1,¹⁶ recently suggested to adopt an orientation perpendicular to the membrane using solid-state NMR,²⁷ are in disagreement with current models of chaperone function. Although the in vivo lack of TWD1 chaperone function in the auxin-signaling pathway still needs to be demonstrated, it seems likely that the multi-domain immunophilins have gained a distinct function in protein-protein interaction through evolution.

Outlook

The positive regulatory role of TWD1 is well documented but the question remains why nature invented such a complicated mechanism of auxin transport regulation. One plausible explanation might be that regulation via protein-protein interaction is a very economic way of regulation that on the other hand allows very precise fine-modulation. This might be conferred by plant drugs disrupting protein-protein interaction. In this respect it is worth mentioning that excess of NPA removes TWD1 from NPA chromatographies.¹⁶

Although reductions of PGP-mediated auxin transport in twd1 are in agreement with dwarfism and other auxin-related phenotypes, loss of TWD1 action does not directly explain the “twisted syndrome”. However, many helical orientations caused by mutations are due to misassembly of microtubuli. Interestingly, mammalian multi-domain FKBPs have been shown to bind dynein via the N-terminal PPIase domain.²⁸

Finally, it remains to be solved why twd1 mutant plant show more pronounced phenotypes at later stages compared to pgp1/pgp19 (Fig. 1A, inset). One possibility is that other TWD1-interactors contribute to the full twisted phenotype. These might be MRP1/MRP2, but unfortunately their in vivo substrate is hard to guess and quadruplet knock-out lines are difficult to get due to genetical distances. Other obvious candidates include interacting partner HSP90, a well known developmental regulator²⁹ and calmodulin (CaM). TWD1 shares some unique structural properties with the human homologue FKBP38.³⁰ Beside their common putative membrane anchoring, TWD1 binds CaM which is essential for HsFKBP38 function. An active FKBP38/CaM/Ca²⁺ complex is required for Bcl-2 interaction, a key protein in apoptosis control. Strong indications are now pointing to the upcoming role of TWD1-homolog heterocomplexes functioning in regulation of MDR-type transporters and reveal the potential benefits of such investigations for agronomy and human health.

Note

A recent paper by Grinzin et al. (J Mol Biol 2006; 364:799–809) describes the crystal structure of TWD1 representing the first three-dimensional structure of a multi-domain immunophilin. While the overall architecture of the two domains matches the known characteristics of the FKBP and TPR folds, respectively, their arrangement is unique and sheds light on the probable binding modes of key interaction partners, including HSP90 and two classes of ABC transporters.

Addendum to: Granzin J, Eckhoff A, Weiergräber OH. Crystal structure of a multi-domain immunophilin from Arabidopsis thaliana: a paradigm for regulation of plant ABC transporters. J Mol Biol. 2006;364:799–809. doi: 10.1016/j.jmb.2006.09.052.