Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Modulation of P-glycoproteins by Auxin Transport Inhibitors Is Mediated by Interaction with Immunophilins

2008; Elsevier BV; Volume: 283; Issue: 31 Linguagem: Inglês

10.1074/jbc.m709655200

ISSN

1083-351X

Autores

Aurélien Bailly, Valpuri Sovero, Vincent Vincenzetti, Diana Santelia, Dirk Bartnik, Bernd W. Koenig, Stefano Mancuso, Enrico Martinoia, Markus Geisler,

Tópico(s)

Plant Stress Responses and Tolerance

Resumo

The immunophilin-like FKBP42 TWISTED DWARF1 (TWD1) has been shown to control plant development via the positive modulation of ABCB/P-glycoprotein (PGP)-mediated transport of the plant hormone auxin. TWD1 functionally interacts with two closely related proteins, ABCB1/PGP1 and ABCB19/PGP19/MDR1, both of which exhibit the ability to bind to and be inhibited by the synthetic auxin transport inhibitor N-1-naphylphtalamic acid (NPA). They are also inhibited by flavonoid compounds, which are suspected modulators of auxin transport. The mechanisms by which flavonoids and NPA interfere with auxin efflux components are unclear. We report here the specific disruption of PGP1-TWD1 interaction by NPA and flavonoids using bioluminescence resonance energy transfer with flavonoids functioning as a classical established inhibitor of mammalian and plant PGPs. Accordingly, TWD1 was shown to mediate modulation of PGP1 efflux activity by these auxin transport inhibitors. NPA bound to both PGP1 and TWD1 but was excluded from the PGP1-TWD1 complex expressed in yeast, suggesting a transient mode of action in planta. As a consequence, auxin fluxes and gravitropism in twd1 roots are less affected by NPA treatment, whereas TWD1 gain-of-function promotes root bending. Our data support a novel model for the mode of drug-mediated P-glycoprotein regulation mediated via protein-protein interaction with immunophilin-like TWD1. The immunophilin-like FKBP42 TWISTED DWARF1 (TWD1) has been shown to control plant development via the positive modulation of ABCB/P-glycoprotein (PGP)-mediated transport of the plant hormone auxin. TWD1 functionally interacts with two closely related proteins, ABCB1/PGP1 and ABCB19/PGP19/MDR1, both of which exhibit the ability to bind to and be inhibited by the synthetic auxin transport inhibitor N-1-naphylphtalamic acid (NPA). They are also inhibited by flavonoid compounds, which are suspected modulators of auxin transport. The mechanisms by which flavonoids and NPA interfere with auxin efflux components are unclear. We report here the specific disruption of PGP1-TWD1 interaction by NPA and flavonoids using bioluminescence resonance energy transfer with flavonoids functioning as a classical established inhibitor of mammalian and plant PGPs. Accordingly, TWD1 was shown to mediate modulation of PGP1 efflux activity by these auxin transport inhibitors. NPA bound to both PGP1 and TWD1 but was excluded from the PGP1-TWD1 complex expressed in yeast, suggesting a transient mode of action in planta. As a consequence, auxin fluxes and gravitropism in twd1 roots are less affected by NPA treatment, whereas TWD1 gain-of-function promotes root bending. Our data support a novel model for the mode of drug-mediated P-glycoprotein regulation mediated via protein-protein interaction with immunophilin-like TWD1. Bioactive flavonoids derived from plant secondary metabolism serve as important nutraceuticals (1Taylor L.P. Grotewold E. Curr. Opin. Plant Biol. 2005; 8: 317-323Crossref PubMed Scopus (460) Google Scholar). They have health-promoting effects, including antioxidant, anticarcinogenic, antiviral, and anti-inflammatory activities; however, the cellular targets of the in vivo protein remain largely unknown (1Taylor L.P. Grotewold E. Curr. Opin. Plant Biol. 2005; 8: 317-323Crossref PubMed Scopus (460) Google Scholar, 2Morris M.E. Zhang S. Life Sci. 2006; 78: 2116-2130Crossref PubMed Scopus (232) Google Scholar). In plants, among other functions, flavonoids such as quercetin, kaempferol, and other aglycone molecules have been shown to inhibit cell-to-cell/polar auxin transport (PAT) 3The abbreviations used are: PAT, polar auxin transport; NPA, N-1-naphylphtalamic acid; ATI, auxin transport inhibitor; NBP, NPA-binding protein; PGP, P-glycoprotein; MDR, multidrug resistance; ABC, ATP-binding cassette; FKBP, FK506-binding protein; PPIase, peptidyl-prolyl cis-trans isomerase; TWD, Twisted Dwarf; BRET, bioluminescence resonance energy transfer; YFP, yellow fluorescent protein; GFP, green fluorescent protein; CFP, cyan fluorescent protein; IAA, indole-3-acetic acid; BA, benzoic acid; rLuc, Renilla luciferase; NBD, nucleotide-binding domain; HA, hemagglutinin. and consequently to enhance localized auxin accumulation (1Taylor L.P. Grotewold E. Curr. Opin. Plant Biol. 2005; 8: 317-323Crossref PubMed Scopus (460) Google Scholar, 3Brown D.E. Rashotte A.M. Murphy A.S. Normanly J. Tague B.W. Peer W.A. Taiz L. Muday G.K. Plant Physiol. 2001; 126: 524-535Crossref PubMed Scopus (572) Google Scholar, 4Jacobs M. Rubery P.H. Science. 1988; 241: 346-349Crossref PubMed Scopus (494) Google Scholar, 5Peer W.A. Bandyopadhyay A. Blakeslee J.J. Makam S.N. Chen R.J. Masson P.H. Murphy A.S. Plant Cell. 2004; 16: 1898-1911Crossref PubMed Scopus (313) Google Scholar, 6Murphy A. Peer W.A. Taiz L. Planta. 2000; 211: 315-324Crossref PubMed Scopus (285) Google Scholar). During PAT, the plant hormone auxin, which determines many aspects of plant physiology and development, is moved directionally in a cell-to-cell mode (7Blakeslee J.J. Peer W.A. Murphy A.S. Curr. Opin. Plant Biol. 2005; 8: 494-500Crossref PubMed Scopus (241) Google Scholar, 8Kerr I.D. Bennett M.J. Biochem. J. 2007; 401: 613-622Crossref PubMed Scopus (63) Google Scholar, 9Vieten A. Sauer M. Brewer P.B. Friml J. Trends Plant Sci. 2007; 12: 160-168Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The regulatory impact of flavonoids on PAT initially was based on their ability to compete with N-1-naphtylphtalamic acid (NPA), a synthetic auxin transport inhibitor (ATI) (4Jacobs M. Rubery P.H. Science. 1988; 241: 346-349Crossref PubMed Scopus (494) Google Scholar, 10Lomax T.L. Muday G.K. Rubery P.H. Davies P.J. Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer, Dordrecht, Netherlands1995: 509-530Crossref Google Scholar, 11Luschnig C. Curr. Biol. 2001; 11: R831-833Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 12Morris D.A. Plant Growth Regul. 2000; 32: 161-172Crossref PubMed Scopus (69) Google Scholar) and herbicide (naptalam, alanap), for transporter binding sites. This concept is further supported by auxin-related phenotypes of Arabidopsis mutants with altered flavonoid levels (1Taylor L.P. Grotewold E. Curr. Opin. Plant Biol. 2005; 8: 317-323Crossref PubMed Scopus (460) Google Scholar, 3Brown D.E. Rashotte A.M. Murphy A.S. Normanly J. Tague B.W. Peer W.A. Taiz L. Muday G.K. Plant Physiol. 2001; 126: 524-535Crossref PubMed Scopus (572) Google Scholar, 13Peer W.A. Brown D.E. Tague B.W. Muday G.K. Taiz L. Murphy A.S. Plant Physiol. 2001; 126: 536-548Crossref PubMed Scopus (271) Google Scholar), although fundamental physiological processes occur in the absence of flavonoids. Currently the flavonoids are seen as transport regulators or modulators (14Peer W.A. Murphy A.S. Trends Plant Sci. 2007; 12: 556-563Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar); nevertheless, the mechanisms by which flavonoids interfere with auxin efflux components are not yet clear (1Taylor L.P. Grotewold E. Curr. Opin. Plant Biol. 2005; 8: 317-323Crossref PubMed Scopus (460) Google Scholar). The auxin efflux complex is thought to regulate PAT on the molecular level and consists of at least two proteins: a membrane integral transporter and an NPA-binding protein (NBP) regulatory subunit (11Luschnig C. Curr. Biol. 2001; 11: R831-833Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 15Bernasconi P. Physiol. Plant. 1996; 96: 205-210Crossref Scopus (53) Google Scholar, 16Cox D.N. Muday G.K. Plant Cell. 1994; 6: 1941-1953PubMed Google Scholar, 17Michalke W. Katekar G.F. Geissler A.E. Planta. 1992; 187: 254-260Crossref PubMed Scopus (33) Google Scholar). Recently, ABCB/P-glycoprotein (PGP)/multidrug resistance (MDR) proteins, members of the expanded Arabidopsis ABC (ATP-binding cassette) transporter family (18Martinoia E. Klein M. Geisler M. Bovet L. Forestier C. Kolukisaoglu U. Muller-Rober B. Schulz B. Planta. 2002; 214: 345-355Crossref PubMed Scopus (311) Google Scholar, 19Verrier P.J. Bird D. Burla B. Dassa E. Forestier C. Geisler M. Klein M. Kolukisaoglu U. Lee Y. Martinoia E. Murphy A. Rea P.A. Samuels L. Schulz B. Spalding E.J. Yazaki K. Theodoulou F.L. Trends Plant Sci. 2008; PubMed Google Scholar), have been identified as both auxin transporters (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 21Geisler M. Blakeslee J.J. Bouchard R. Lee O.R. Vincenzetti V. Bandyopadhyay A. Titapiwatanakun B. Peer W.A. Bailly A. Richards E.L. Ejendal K.F. Smith A.P. Baroux C. Grossniklaus U. Muller A. Hrycyna C.A. Dudler R. Murphy A.S. Martinoia E. Plant J. 2005; 44: 179-194Crossref PubMed Scopus (430) Google Scholar, 22Geisler M. Murphy A.S. FEBS Lett. 2006; 580: 1094-1102Crossref PubMed Scopus (296) Google Scholar, 23Santelia D. Vincenzetti V. Azzarello E. Bovet L. Fukao Y. Duchtig P. Mancuso S. Martinoia E. Geisler M. FEBS Lett. 2005; 579: 5399-5406Crossref PubMed Scopus (173) Google Scholar, 24Terasaka K. Blakeslee J.J. Titapiwatanakun B. Peer W.A. Bandyopadhyay A. Makam S.N. Lee O.R. Richards E.L. Murphy A.S. Sato F. Yazaki K. Plant Cell. 2005; 17: 2922-2939Crossref PubMed Scopus (290) Google Scholar) and high-affinity NBPs (25Murphy A.S. Hoogner K.R. Peer W.A. Taiz L. Plant Physiol. 2002; 128: 935-950Crossref PubMed Scopus (170) Google Scholar, 26Noh B. Murphy A.S. Spalding E.P. Plant Cell. 2001; 13: 2441-2454Crossref PubMed Scopus (445) Google Scholar). High NPA concentrations cause inhibition of auxin efflux catalyzed by ABCB1/PGP1 and ABCB19/PGP19/MDR1 (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 21Geisler M. Blakeslee J.J. Bouchard R. Lee O.R. Vincenzetti V. Bandyopadhyay A. Titapiwatanakun B. Peer W.A. Bailly A. Richards E.L. Ejendal K.F. Smith A.P. Baroux C. Grossniklaus U. Muller A. Hrycyna C.A. Dudler R. Murphy A.S. Martinoia E. Plant J. 2005; 44: 179-194Crossref PubMed Scopus (430) Google Scholar) (hereafter referred to as PGPs), most probably by binding to the transporter itself (26Noh B. Murphy A.S. Spalding E.P. Plant Cell. 2001; 13: 2441-2454Crossref PubMed Scopus (445) Google Scholar). This is in analogy to flavonoids functioning as inhibitors of plant (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 21Geisler M. Blakeslee J.J. Bouchard R. Lee O.R. Vincenzetti V. Bandyopadhyay A. Titapiwatanakun B. Peer W.A. Bailly A. Richards E.L. Ejendal K.F. Smith A.P. Baroux C. Grossniklaus U. Muller A. Hrycyna C.A. Dudler R. Murphy A.S. Martinoia E. Plant J. 2005; 44: 179-194Crossref PubMed Scopus (430) Google Scholar, 24Terasaka K. Blakeslee J.J. Titapiwatanakun B. Peer W.A. Bandyopadhyay A. Makam S.N. Lee O.R. Richards E.L. Murphy A.S. Sato F. Yazaki K. Plant Cell. 2005; 17: 2922-2939Crossref PubMed Scopus (290) Google Scholar) and mammalian PGPs (2Morris M.E. Zhang S. Life Sci. 2006; 78: 2116-2130Crossref PubMed Scopus (232) Google Scholar) probably by mimicking ATP and competing for PGP nucleotide-binding domains (27Conseil G. Baubichon-Cortay H. Dayan G. Jault J.M. Barron D. Di Pietro A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9831-9836Crossref PubMed Scopus (373) Google Scholar). Overexpression of certain PGPs is associated with increased MDR, whereas loss of function often results in diverse diseases (28Dean M. Methods Enzymol. 2005; 400: 409-429Crossref PubMed Scopus (130) Google Scholar) in mammalians. The immunophilin-like FKBP42, TWISTED DWARF1 (TWD1) (29Geisler M. Bailly A. Trends Plant Sci. 2007; 12: 465-473Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 30Geisler M. Kolukisaoglu H.U. Bouchard R. Billion K. Berger J. Saal B. Frangne N. Koncz-Kalman Z. Koncz C. Dudler R. Blakeslee J.J. Murphy A.S. Martinoia E. Schulz B. Mol. Biol. Cell. 2003; 14: 4238-4249Crossref PubMed Scopus (200) Google Scholar, 31Romano P. Gray J. Horton P. Luan S. New Phytol. 2005; 166: 753-769Crossref PubMed Scopus (89) Google Scholar), belongs to the FKBP (FK506-binding protein)-type family of PPIases (peptidyl-prolyl cis-trans isomerases, EC 5.1.2.8) (29Geisler M. Bailly A. Trends Plant Sci. 2007; 12: 465-473Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 31Romano P. Gray J. Horton P. Luan S. New Phytol. 2005; 166: 753-769Crossref PubMed Scopus (89) Google Scholar, 32Fanghanel J. Fischer G. Front. Biosci. 2004; 9: 3453-3478Crossref PubMed Scopus (198) Google Scholar). Many but not all PPIases catalyze the cis-trans isomerization of cis-prolyl bonds and have been identified as targets of immunosuppressant drugs such as FK506 (tacrolimus) (29Geisler M. Bailly A. Trends Plant Sci. 2007; 12: 465-473Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 31Romano P. Gray J. Horton P. Luan S. New Phytol. 2005; 166: 753-769Crossref PubMed Scopus (89) Google Scholar, 32Fanghanel J. Fischer G. Front. Biosci. 2004; 9: 3453-3478Crossref PubMed Scopus (198) Google Scholar). The TWD1 C terminus forms a so-called amphipathic in-plane membrane anchor, which probably confers a perpendicular orientation (33Scheidt H.A. Vogel A. Eckhoff A. Koenig B.W. Huster D. Eur. Biophys. J. 2007; 36: 393-404Crossref PubMed Scopus (12) Google Scholar) on both the vacuolar (34Geisler M. Girin M. Brandt S. Vincenzetti V. Plaza S. Paris N. Kobae Y. Maeshima M. Billion K. Kolukisaoglu U.H. Schulz B. Martinoia E. Mol. Biol. Cell. 2004; 15: 3393-3405Crossref PubMed Scopus (81) Google Scholar) and the plasma membrane (30Geisler M. Kolukisaoglu H.U. Bouchard R. Billion K. Berger J. Saal B. Frangne N. Koncz-Kalman Z. Koncz C. Dudler R. Blakeslee J.J. Murphy A.S. Martinoia E. Schulz B. Mol. Biol. Cell. 2003; 14: 4238-4249Crossref PubMed Scopus (200) Google Scholar, 35Kamphausen T. Fanghanel J. Neumann D. Schulz B. Rahfeld J.U. Plant J. 2002; 32: 263-276Crossref PubMed Scopus (92) Google Scholar). TWD1 docks with its N-terminal FK506-binding domain (FKBD), shown to lack PPIase activity and FK506 binding (30Geisler M. Kolukisaoglu H.U. Bouchard R. Billion K. Berger J. Saal B. Frangne N. Koncz-Kalman Z. Koncz C. Dudler R. Blakeslee J.J. Murphy A.S. Martinoia E. Schulz B. Mol. Biol. Cell. 2003; 14: 4238-4249Crossref PubMed Scopus (200) Google Scholar, 35Kamphausen T. Fanghanel J. Neumann D. Schulz B. Rahfeld J.U. Plant J. 2002; 32: 263-276Crossref PubMed Scopus (92) Google Scholar), to C-terminal nucleotide-binding domains (18Martinoia E. Klein M. Geisler M. Bovet L. Forestier C. Kolukisaoglu U. Muller-Rober B. Schulz B. Planta. 2002; 214: 345-355Crossref PubMed Scopus (311) Google Scholar) of PGP1 and PGP19 (see Fig. 1A). In this way, TWD1 acts in planta as a positive regulator of PGP1- and PGP19-mediated auxin efflux by means of protein-protein interaction (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 30Geisler M. Kolukisaoglu H.U. Bouchard R. Billion K. Berger J. Saal B. Frangne N. Koncz-Kalman Z. Koncz C. Dudler R. Blakeslee J.J. Murphy A.S. Martinoia E. Schulz B. Mol. Biol. Cell. 2003; 14: 4238-4249Crossref PubMed Scopus (200) Google Scholar), modulating the movement of auxin out of apical regions and long range auxin transport on the cellular level (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 36Lewis D.R. Miller N.D. Splitt B.L. Wu G. Spalding E.P. Plant Cell. 2007; 19Crossref Scopus (156) Google Scholar). Here, we employed a yeast-based bioluminescence resonance energy transfer (BRET) system to investigate PGP1-TWD1 interaction on a molecular level. Auxin transport inhibitors and flavonoids, inhibitors of mammalian and plant PGPs, disrupt PGP1-TWD1 interaction. Auxin transport inhibitors modulate the regulatory effect of TWD1 on PGP1 activity, supporting a novel mode of PGP regulation via immunophilin-like TWD1 (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). This represents a new concept of drug-mediated ABC transporter modulation via membrane-anchored immunophilins and may have important agronomic and clinical implications. Yeast Constructs, Growth, and Expression Analysis—cDNA covering the N-terminal FKBD of Arabidopsis TWD1 (TWD1-(1-187); At3g21640) were cloned into BamHI and SalI sites of the copper-inducible yeast shuttle vector pRS314CUP (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) resulting in pRS314CUP-FKBD. Point mutations in TWD1 and a stop codon in PGP1 (bp 3.240) were introduced using the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) resulting in pRS314CUP-TWD1C70D,L72E and pNEV-PGP1ΔNBD2-YFP (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Empty vector controls pNEV and pRS314CUP as well as pNEV-PGP1, pNEV-PGP1-YFP, pNEV-PGP1ΔNBD2-YFP, pRS314CUP-FKBD, pRS314CUP-FKBD-rLuc, pRS314CUP-TWD1, and pRS314CUP-TWD1-rLuc were transformed into Saccharomyces cerevisiae strain JK93dα (37Hemenway C.S. Heitman J. J. Biol. Chem. 1996; 271: 18527-18534Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and single colonies were grown in SD-UT (synthetic minimal medium without uracil and tryptophan, supplemented with 2% glucose and 100 μm CuCl2). Cells co-expressing PGP1-YFP and TWD1-CFP (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) grown in the presence of 10 μm drugs or solvent control to an A600 around 0.8 were washed and incubated in mounting media containing 4,6-diamidino-2-phenylindole, and fluorescence pictures were collected by confocal laser scanning microscopy (Leica, DMIRE2) equipped with argon (488 nm) and UV lasers (410 nm). Fluorescence and DIC (differential interference contrast) images were processed using Adobe Photoshop 7.0. Vector controls showed no detectable fluorescence. Plasma membrane fractions were separated via continuous sucrose gradient centrifugation (34Geisler M. Girin M. Brandt S. Vincenzetti V. Plaza S. Paris N. Kobae Y. Maeshima M. Billion K. Kolukisaoglu U.H. Schulz B. Martinoia E. Mol. Biol. Cell. 2004; 15: 3393-3405Crossref PubMed Scopus (81) Google Scholar) and subjected to 4-20% PAGE (Long Life Gels, Life Therapeutics), and Western blots were immunoprobed using anti-GFP (Roche Applied Science) and anti-Renilla luciferase (rLuc; Chemicon Int.). For auxin analogue detoxification assays (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 38Luschnig C. Gaxiola R.A. Grisafi P. Fink G.R. Genes Dev. 1998; 12: 2175-2187Crossref PubMed Scopus (649) Google Scholar), transformants grown in SD-UT to an A600 of ∼0.8 were washed and adjusted to an A600 of 1.0 in water. Cells were diluted 10× five times, and each 5 μl was spotted on minimal media plates supplemented with 750 μm 5-fluoroindole (Sigma). Growth at 30 °C was assessed after 3-5 days. Assays were performed with three independent transformants. BRET Constructs and Assays—Renilla luciferase (rLuc, Gen-Bank™ accession number AY189980) was amplified by PCR from plasmid pRL-null (Promega) and inserted in-frame into AscI sites generated in the coding regions of pRS314CUP-FKBD and pRS314CUP-TWD1 (64 bp) using the QuikChange XL site-directed mutagenesis kit (Stratagene). In this way, rLuc was inserted into the very N terminus of TWD1. Single colonies co-expressing PGP1-YFP (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) and TWD1-rLuc were grown in selective synthetic minimal medium (SD-UT) in the presence of inhibitors or adequate amounts of solvents. 200 ml of overnight cultures were harvested at A600 = 1 by centrifugation for 10 min at 1500 × g and washed two times with ice-cold milliQ water. The resulting pellet was suspended in 4 ml of ice-cold lysis buffer (50 mm Tris, 750 mm NaCl, 10 mm EDTA) supplemented with proteases inhibitors (Complete tablets, Roche Diagnostics) and an equivalent volume of acid-washed glass beads (inner diameter, 0.5 mm, Biospec Products Inc.) was added. Cells were broken by vortexing 10 times for 1 min, with 1-min intervals on ice for cooling. The supernatant was decanted, and the beads were washed four times with 10 ml of ice-cold lysis buffer. Supernatants were centrifuged at 4,500 × g (S1) and 12,000 × g (S2) for 10 min at 4 °C to remove unbroken cells and other debris. Membranes were collected by centrifugation at 100,000 × g (P3) for 1 h at 4°C. The membrane pellet was homogenized using a pestle in 300 μl of ice-cold STED10 buffer (10% sucrose, 50 mm Tris, 1 mm EDTA, 1 mm dithiothreitol) supplemented with proteases inhibitors to give a suspension of about 3 mg of proteins/ml as measured using the Bradford assay (Bio-Rad). All preparations were stored at -80 °C for subsequent use. In vitro measurement of the BRET signal was performed using 200 μl of yeast membrane suspension (∼600 μg of proteins) in a white 96-well microplate (OptiPlate-96, PerkinElmer Life Sciences). 5 μm coelenterazine (Biotium Inc.) was added, and sequential light emission acquisition in the 410 ± 80 nm and 515 ± 30 nm windows was started after 1 min using the Fusion microplate analyzer (PMT = 1100 V, gain = 1, reading time = 1 s; PerkinElmer Life Sciences). BRET ratios were calculated as described (39Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3684-3689PubMed Google Scholar) as follows: [(emission at 515 ± 30 nm) - (emission at 410 ± 80 nm) × Cf]/(emission at 410 ± 80 nm), where Cf corresponds to (emission at 515 ± 30 nm)/(emission at 410 ± 80 nm) for the rLuc fusion protein expressed alone in the same experimental conditions. The results are the average of 10 readings collected every minute; presented are average values from 6-10 independent experiments with four replicates each. Yeast Auxin Transport Assays—IAA transport experiments were performed as in (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In short, S. cerevisiae strains JK93dα (37Hemenway C.S. Heitman J. J. Biol. Chem. 1996; 271: 18527-18534Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) were grown as described above, loaded with [3H]-IAA (20 Ci/mmol; American Radiolabeled Chemicals Inc.) for 20 min on ice, washed twice with cold water, and resuspended in 15 ml of SD, pH 5.5. 0.5 ml-aliquots were filtered prior to t = 0 min and after 10 min were incubated at 30 °C. PGP1-mediated IAA export is expressed as relative retention of initial (maximal) loading (t = 0 min), which is set to 100%. Presented are average values from 6-8 independent experiments with four replicates each. Drug Binding Assays—Whole yeast NPA binding assays were performed essentially as described previously (26Noh B. Murphy A.S. Spalding E.P. Plant Cell. 2001; 13: 2441-2454Crossref PubMed Scopus (445) Google Scholar). 10 ml of yeast cultures were grown as described above to an A600 of 1, and cells were resuspended in 10 ml of SD, pH 4.5. To 1-ml aliquots (for each experiment four replicates were used) 10 nm [3H]NPA (60 Ci/mmol) and 10 nm [14C]benzoic acid (BA, 58 mCi/mmol; all from American Radiolabeled Chemicals Inc.) were added in the presence and absence of 10 μm NPA (±10 μm BA for competition experiments; 1000-fold excess). Cells were incubated for 1 h at 4°C under shaking and washed with ice-cold MilliQ water, and the tips of centrifuge tubes including pellets were subjected to scintillation counting. Reported values are the means of specific binding ([3H]NPA bound in the absence of cold NPA (total) minus [3H]NPA bound in the presence of cold NPA (unspecific)) from 4-8 independent experiments with four replicates each. For PPIase Affi-Gel pulldown assays, 2 mg of the PPIase domain/FKBD of TWD11-180 expressed in Escherichia coli and purified as described previously (40Weiergraber O.H. Eckhoff A. Granzin J. FEBS Lett. 2006; 580: 251-255Crossref PubMed Scopus (19) Google Scholar) was coupled to 2 ml of Affi-Gel-15 beads (Bio-Rad). 50 μl of Affi-Gel-PPIase or empty Affi-Gel beads (50% slurry) was resuspended in 450 μl of phosphate-buffered saline, pH 7.4, and 5 μl of radiolabeled [7-14C]BA (58 mCi/mmol, 0.1 mCi/ml) and [3H]NPA (60 Ci/mmol, 1 mCi/ml; all from American Radiolabeled Chemicals Inc.), diluted 20× in phosphate-buffered saline was added to four replications of each. After shaking for 1 h at 4°C, the beads were filtered on nitrocellulose, and the filters washed three times with cold MilliQ water and finally subjected to scintillation counting. Reported values are the means of specific binding ([3H]NPA bound to Affi-Gel-PPIase minus [3H]NPA bound to empty Affi-Gel beads) per 1 μg of coupled protein as measured using the Bradford assay (Bio-Rad). Presented are average values from 4-8 independent experiments with four replicates each. For microsomes of Arabidopsis NPA binding assays, Arabidopsis seedlings were grown in liquid cultures, and microsomes were prepared as described elsewhere (34Geisler M. Girin M. Brandt S. Vincenzetti V. Plaza S. Paris N. Kobae Y. Maeshima M. Billion K. Kolukisaoglu U.H. Schulz B. Martinoia E. Mol. Biol. Cell. 2004; 15: 3393-3405Crossref PubMed Scopus (81) Google Scholar). Four replicates of each 20 μg (ProCaMV35S-TWD1-HA) or 100 μg of protein (wild-type and twd1) in STED10 were incubated with 10 nm [3H]NPA (60 Ci/mmol) and 10 nm [14C]BA (58 mCi/mmol) in the presence and absence of 10 μm NPA (±10 μm BA for competition experiments; 1000-fold excess). After 1 h at 4°C under shaking, membranes were filtered over nitrocellulose filters (MF 0.45 μm, Millipore) and washed three times with cold MilliQ water, and filters were subjected to scintillation counting. Reported values are the means of specific binding ([3H]NPA bound in the absence of cold NPA (total) minus [3H]NPA bound in the presence of cold NPA (unspecific)) from three independent experiments with four replicates each. Plant Growth Conditions and Quantitative Analysis of Root Gravitropism—Arabidopsis thaliana plants were grown as described previously (30Geisler M. Kolukisaoglu H.U. Bouchard R. Billion K. Berger J. Saal B. Frangne N. Koncz-Kalman Z. Koncz C. Dudler R. Blakeslee J.J. Murphy A.S. Martinoia E. Schulz B. Mol. Biol. Cell. 2003; 14: 4238-4249Crossref PubMed Scopus (200) Google Scholar). For quantification of gravitropism in the presence of light (Fig. 5A, supplemental Fig. S6A), wild type, pgp1 (At2g36910) and pgp19 (At3g28860), and pgp1/pgp19 and twd1 (At3g21640) mutants (all ecotype Wassilewskija (Ws Wt)), seeds were surface sterilized and grown on 0.5× Murashige and Skoog medium, 0.7% phytoagar (Invitrogen) under continuous light conditions in the presence or absence of 5 μm NPA as described previously (20Bouchard R. Bailly A. Blakeslee J.J. Oehring S.C. Vincenzetti V. Lee O.R. Paponov I. Palme K. Mancuso S. Murphy A.S. Schulz B. Geisler M. J. Biol. Chem. 2006; 281: 30603-30612Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The angle of root tips from the vertical plane was determined using Photoshop 7.0. (Adobe Systems, Mountain View, CA), and each gravistimulated root was assigned to one of twelve 30° sectors in the circular histograms; the length of each bar represents the percentage of seedlings showing the same direction of root growth. (Figs. 5 and 6, supplemental Fig. S6). Helical wheels were plotted using PolarBar software.FIGURE 6Overexpression of TWD1 promotes root gravitropism. A, gravitropic root responses of TWD1 loss- (twd1-3) and gain-of-function, ProCaMV35S-TWD1-HA (35S-TWD1-HA) alleles in comparison with wild type (Col. Wt). Triangle indicates direction of gravitropism (g); triangles mark time point of 90° plate rotation (upper panel). Root curvatures was assigned to one of twelve 30° sectors in the circular histograms; the length of each bar represents the percentage of seedlings showing the same direction of root growth. Data are means ± S.E. (n = 3 with each 72-96 seedlings). B, time series of root curvatur

Referência(s)