Interactions between Pgp, Pin, and Aux/lax Auxin Transport Proteins from Arabidopsis

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polar growth. Natural auxins, primarily indole 3-acetic acid (IAA), are ampipathic weak acids that diffuse into membrane bilayers when protonated, suggesting that polar auxin movement is primarily established at the point of cellular efflux. However, recent evidence indicates that proton-coupled uptake transporters also play a role in polar auxin movement, particularly in roots (Swarup et al, 2005). Cellular auxin transport mediated by members of the PGP, PIN, and AUX/LAX protein families has recently been conclusively demonstrated in plant and heterologous expression systems (Chen et al, 1998; Geisler , Blakeslee et al, 2005; Bouchard et al, 2006; Terasaka et al, 2005; Petrasek et al, 2006; Yang et al, 2006). Members of the PIN subfamily of ion-coupled major facilitator proteins and P-glycoprotein (PGP) ABC transporters (Sieberer and Leyser, 2006; Geisler and Murphy, 2006) have been shown to independently mediate auxin export, while AUX/LAX proteins and at least one Arabidopsis PGP, PGP4, mediate auxin uptake (Swarup et al, 2005; Yang et al, 2006; Terasaka et al, 2005). In those heterologous expression studies, AUX1 exhibited strict substrate and inhibitor specificities similar to what is seen in planta. PGPs and PINs did not. Here we characterize PGP interactions with PINs and AUX1 to ascertain whether such interactions in planta influence rates and specificity of auxin transport. PIN proteins, named for the pinformed inflorescence exhibited by pin1, are plasma membrane proteins and constitute a unique subfamily of the ion-coupled major facilitator efflux superfamily (Gälweiler et al., 1998). PIN proteins have been shown to be essential to establishing the basal levels of auxin transport required for normal plant development, as they align with the auxin transport vector and are essential for both programmed and plastic polar development (reviewed in Leyser, 2005). Mutations in PIN genes result in aberrant organogenesis and/or auxindependent tropic responses (reviewed in Benjamins et al., 2005). PINs exhibit tissue-specific expression patterns that correspond exactly with patterns of auxin flux in discrete tissues. Because of this characteristic, PIN1 has become a valuable subcellular marker for studies of practically every aspect of plant development. However, PINs may also be involved in apolar auxin export from cells, as some PINs exhibit tissue-specific nonpolar membrane orientations (Heisler et al., 2005; Reinhardt et al., 2003; this report). 1 Department of Horticulture, Purdue University, West Lafayette, Indiana 47907-2010 Center for Plant Molecular Biology, University of Tübingen, Tübingen, Germany Institute of Plant Biology, Basel-Zurich Plant Science Center, University of Zurich, CH-8007 Zurich, Switzerland Purdue Discovery Park, West Lafayette, Indiana 47907-2010 Proceedings 33 PGRSA Annual Meeting 43 The direct auxin transport catalyzed by PINs was recently demonstrated by heterologous expression in plant, yeast, and mammalian cells (Chen et al., 1998; Petrasek et al., 2006). PINmediated transport was also seen to be sensitive to inhibitors of organic anion transporters (OATs) in mammals and yeast, suggesting activation of other transport processes, as is the case with other major facilitator proteins (Wang et al., 2005; Campbell et al., 2004; Figure S1b). However, when expressed in non-plant cells, Arabidopsis PINs exhibited reduced sensitivity to the non-competitive auxin transport inhibitor 1-naphthylphthalamic acid (NPA) and reduced substrate specificity, demonstrated by substantial export of benzoic acid, a nontransported analog of IAA. Further, PIN1 expression resulted in increased transport only when expressed in Arabidopsis cell cultures, but not in tobacco cells, yeast, or mammalian cells. These results suggest that interactions with other proteins may confer the transport specificities observed in intact plants. Multiple Drug Resistance P-glycoproteins (ABCB/MDR/PGPs, hereafter referred to as PGPs) are integral membrane ATP binding cassette (ABC) transporter proteins consisting of two homologous halves, each containing a nucleotide-binding fold/ATP-hydrolysis site and six transmembrane helices, joined by a flexible linker region (Ambudkar et al., 1999). Although originally identified in human multiple drug resistance to chemotherapeutics, PGPs exhibit greater specificities for ampipathic anions than other drug resistance proteins and are better characterized as ATP-activated, amphipathic anion transporters. PGPs bind membraneembedded ampipathic substrates and move them to the surface of the opposing membrane leaflet in an ATP-dependent fashion (Ambudkar et al., 2005; Blakeslee et al., 2005b). PGP-like ABC transporters are thought to share similar substrate binding domain structures and exhibit mechanistic similarities with drug-activated, ion-coupled major facilitator proteins (Yin et al., 2006; Pasrija et al., 2005; Venter et al., 2005). In comparison to humans, where six PGP isoforms are found, the plant PGP subfamily is expanded (21 expressed members in Arabidopsis, 17 in rice) and exhibits more sequence diversity (Martinoia et al., 2002; Geisler and Murphy, 2006). Mutations in two clades of PGP genes in Arabidopsis, maize, and sorghum result in greater reductions in auxin transport than pin mutations, reduced elongation growth and shoot apical dominance, and altered tropic responses (Noh et al., 2001, 2003; Geisler et al., 2003, 2005; Multani et al., 2003; Terasaka et al, 2005; Santelia et al., 2005). However, to date, no loss in developmental polarity has been observed in pgp mutants, suggesting that PGPs function primarily in the loading and conductance of auxin through the long distance transport stream. As is the case with PINs, PGP genes exhibit tissue-specific expression and subcellular localization of their protein products. PGP1 expression is restricted to leaves, shoot apices, and roots; PGP4 expression is restricted to the root; MDR1/PGP19, which appears to play the greatest role in shoot to root transport, is expressed in shoots, leaves, and roots, with shoot expression enhanced in dark-grown seedlings (Noh et al, 2001, 2003; Geisler et al., 2003, 2005; Terasaka et al., 2005; Santelia et al., 2005; Petrasek et al., 2006; Bouchard et al., 2006; http://www.arabidopsis.org/info/expression/ATGenExpress.jsp; www.arexdb.org; https://www.genevestigator.ethz.ch/; this report). PGP1 and PGP4 exhibit apolar subcellular localizations in apical tissues, but polar localization consistent with the direction of transport in mature root tissues (Geisler et al., 2005; Terasaka et al., 2005). Proceedings 33 PGRSA Annual Meeting 44 Arabidopsis PGP1, PGP19, and PGP4 have been shown to mediate auxin-specific, energydependent cellular transport in plant cells, yeast and mammalian cells (Geisler et al., 2005; Terasaka et al., 2005; Santelia et al, 2005; Petrasek et al., 2006; Bouchard et al., 2006). However, as was seen with PINs, when Arabidopsis PGPs were expressed in yeast and mammalian cells, the resulting transport exhibited decreased substrate specificity and auxin transport inhibitor sensitivity, despite a lack of transport of any mammalian PGP substrates. Unlike PIN-mediated transport, no sensitivity to OAT transport inhibitors was observed. Taken together, these results suggest that, although no other transport proteins appear to be required for active transport of auxin by PGPs, other factors are required to confer the specificity and inhibitor sensitivity observed in PGP-mediated transport in planta. The AUX/LAX group of auxin permeases, best characterized by AUX1, belong to the aminoacyl permease subclass of the major facilitator superfamily and function in high affinity cellular auxin uptake (Marchant et al., 2002; Yang et al., 2006). AUX1 functions in auxin loading and has been shown to be the primary determinant of auxin redirection at the root tip, as is evidenced by the profound agravitropic behaviour of aux1 roots and the absolute requirement of AUX1 expression in Arabidopsis lateral root cap cells for restoration of gravitropic growth (Swarup et al., 2005). It has been suggested that PIN, PGP, and AUX/LAX auxin transport mechanisms may function antagonistically, additively, or synergistically in a tissue-specific manner (Blakeslee et al., 2005a, b). Precedents for synergistic interactions between ABC transporters and major facilitator proteins are seen in other organisms. In yeast, a genetic interaction between the pleiotropic drug resistance ABC transporter PDR5p and the hexose major facilitator protein transporters HXT11 and HXT9 has been demonstrated (Nourani et al., 1997). Interactions between major facilitator superfamily proteins and ABC transporters occur in membrane lipid rafts, and substrate binding for both classes occurs within the membrane leaflets (Yin et al., 2006; Pasrija et al., 2005; Venter et al., 2005). Although AUX/LAX-like permeases are members of the major facilitator superfamily, little evidence of interactions between AUX1-like permeases with PGPs or PIN-like major facilitator proteins is found in the literature; however the KNQ1 permease can compensate for PDR5-deficient yeast (Takacova et al., 2004). PGP interactions in planta may involve general stabilization of membrane transporters as is the case in animals (Lavie et al., 1998; Lavie and Liscovitch, 2000; Luker et al., 2000; Troost et al., 2004), as PIN1 was found to be delocalized in detergent-solubilized xylem parenchyma cells of hypertropic pgp19 hypocotyls and unaltered in pgp1 hypocotyls that exhibited relatively normal rates of tropic bending (Noh et al., 2003). The observed difference is presumably a result of the greater abundance of PGP19, but may reflect specific PIN-PGP interactions as well. In mammalian cells, PGPs are associated with sterol-rich, detergent-resistant microdomains (DRMs, sometimes referred to as “lipid rafts”) that are involved in the formation and stabilization of multi-protein complexes in the plasma membrane (Shogomori and Brown, 2003). DRMs containing PGPs have been characterized in plant cells (Mongrand et al., 2004; Borner et al., 2005) and appear to function in PIN-dependent auxin transport, as loss of STEROL METHYLTRANSFERASE1 (SMT1) function results in altered membrane sterol composition, auxin transport, and PIN localization (Willemsen et al., 2003). Methods utilized in purification of native PGP1, 2, 4, and 19 from Arabidopsis also suggest DRM localization (Noh et al., 2001; Murphy et al., 2002; Geisler et al., 2003; Terasaka et al., 2005). Proceedings 33 PGRSA Annual Meeting 45 In this report, we describe the use of mutant analyses, gene expression studies, subcellular protein localization, protein-protein interaction assays, and co-expression studies utilizing the best characterized members of the respective families (PIN1,2; PGP1,19,4; and AUX1) to elucidate PIN, PGP, and AUX/LAX auxin transport interactions Analysis of pin1 pgp19 and pin1 pgp1 pgp19 mutant phenotypes suggests both additive and synergistic functions. The double and triple mutants exhibit additive phenotypes in inflorescences and the shoot apical meristem and leaves, synergistic phenotypes in leaves, but epistatic phenotypes in the shoot apex when PGP1 function is lost, presumably as a result of localized ectopic auxin accumulations at the shoot apex. Defects in root basipetal auxin transport and gravitropism are seen in pgp4 pin2 double mutants, but are less than what is expected from individual phenotypes, suggesting some synergistic activity Overlapping and non-overlapping protein localizations are found with PIN, PGP, and AUX1 pairings. PIN1, PGP1, and, to a lesser extent, PGP19 appear to overlap in the shoot apex. In shoot tissues, PIN1 is restricted to basal plasma membranes of the vascular parenchyma, while PGP19 exhibits apolar localization on the plasma membrane of starch sheath cells. In the root, PIN1 and PGP19 localize to the bottom of vascular cells, and PGP19 exhibits apolar localization in endodemal cells, where AUX1 is abundant. PGP1 exhibits apolar localization in cortical cells below the distal elongation zone and polar localization at the top of cortical and epidermal cells above. PGP1 localization is reiterated in lateral roots. Interestingly, PGP localization in epidermal cells above the elongation zone is only seen in cells where PIN2 expression is weak or non-existent. PGP4 exhibits apolar localization in cortical and epidermal cells from the lateral root cap to the distal elongation zone, but, consistent with an uptake function, polar localization at the bottom of epidermal cells above the elongation zone. PGP4 colocalizes with AUX1 in cells within the lateral root cap. In a file of three cells at the border of the distal and proximal elongation zone, polar orientation of PGP4 and PGP1 is reversed. This reversal is hypothesized to function in trapping of basipetally-transported auxin to facilitate rapid gravitropic responses, as this region is the primary site of gravitropic root bending and functions as a border region past which auxin transport is rapidly reduced (Geisler et al, 2005). Interactions between the C-terminus of PGP19 and the central region of PIN1 and PIN2 were demonstrated by yeast two hybrid analysis. PGP-PIN interactions were further demonstrated with in vivo co-immunoprecipitations. However, in co-IPs, interactions between PGP1 and PIN1 were also seen, suggesting that such interactions may occur in planta, but are likely to involve another factor, most likely TWD1 ( Geisler et al, 2003). Interactions of PGP4 could not be successfully tested in either yeast two hybrid assays or co-IPs. No evidence of AUX1 interactions with PIN1, 2 or PGP1, 4, 19 could be seen in these systems. PIN1 and PGP19 were found to co-localize in detergent resistant “lipid raft” membrane microdomains. PGP19 appears to stabilize PIN1 in these structures, as PIN1 was preferentially detergent-solubilized from pgp19 membranes. PIN1 membrane localization is more readily perturbed in vascular tissues of the root tip where Pin1 and PGP19 co-localize. Proceedings 33 PGRSA Annual Meeting 46 Interactions with PIN proteins appear to modulate auxin transport activity as well as enhance substrate and inhibitor specificities. Co-expression of PIN1 with PGP1 and PGP19 in mammalian cells resulted in enhanced transport activity, substrate specificity, and enhanced auxin transport inhibitor sensitivity. Co-expression of PGP1 and PGP19 with PIN2, which does not co-localize with PGP1 or PGP19 in planta, results in decreased activity. Co-expression of PIN2 with PGP4 enhances auxin uptake, while co-expression of PGP4 and PIN1 results in reversal of transport activity. Co-expression of AUX1 with PINs and PGPs results in additive activity only, consistent with independent function of AUX1.These results suggest that PINs and PGPs can function as both independent and interactive efflux mechanisms in both localized and long-distance auxin transport. However, PINs appear to provide the dominant vectorial component of major polar auxin flows.