2.A.69 The Auxin Efflux Carrier (AEC) Family
Plants possess tissue-specific, pmf-driven, cellular, auxin efflux systems. These carriers are saturable, auxin-specific, and localized to the basal ends of auxin transport-competent cells. They may be found in various plant tissues including vascular tissues and roots. They are responsible for the polar (downwards) transport of auxins from the leaves to the roots. They also function in gravitropism. In fact, gravity-dependent relocation of auxin efflux carriers has been demonstrated (Ottenschläger et al., 2003). A single plant such as Arabidopsis thaliana possesses at least six such systems. Two isoforms in A. thaliana, one in vascular tissue (PIN1) and one in roots (REH1 or EIR1) have been functionally characterized as have homologues from Oryza sativa. These plant proteins are 600-700 amino acyl residues long and exhibit 8-12 transmembrane spanners. Expression analysis of PIN family genes in Chinese hickory revealed their potential roles during grafting and salt stress (Yang et al. 2022).
The rate of auxin transport across the plasma membrane is regulated by the Auxin Binding Protein 1, ABP1, which influcences PIN activity at the plasma membrane (Čovanová et al. 2013). This highlights the relevance of ABP1 for the formation of developmentally important, PIN-dependent auxin gradients. Genome-wide analysis of the PIN gene family in common wheat (Triticum aestivum) identified 44 TaPIN genes and characterized them to understand their structures, functions, and distribution across various tissues (Kumar et al. 2021). Phylogenetic analyses led to the classification of TaPIN genes into seven groups, providing evidence of an evolutionary relationship with Arabidopsis thaliana and Oryza sativa. The physical and biochemical properties, conserved motifs, chromosomal, subcellular localization, transmembrane domains, and 3D structures were also examined. Expression patterns of the TaPIN genes were different in different tissues and developmental stages. Several members of the TaPIN family were induced during biotic and abiotic stress (Kumar et al. 2021).
Morphogenesis and adaptive tropic growth in plants depends on gradients of the phytohormone auxin, mediated by PIN auxin transporters. PINs localize to a particular side of the plasma membrane (PM) or to the endoplasmic reticulum (ER) to directionally transport auxin and maintain intercellular and intracellular auxin homeostasis, respectively. Zhang et al. 2020 swapped the domains between ER- and PM-localized PIN proteins, as well as between apical- and basal PM-localized PINs from Arabidopsis thaliana, to shed light on why PIN family members with similar topological structures reside at different membrane compartments within cells. The N- and C-terminal TMSs and central hydrophilic loop contribute to their differential subcellular localizations and cellular polarities, but the pairwise-matched N- and C-terminal TMSs, resulting from intramolecular domain-domain co-evolution, are also crucial for their divergent patterns of localization (Zhang et al. 2020).
Homologues of the AEC family are found in bacteria (E. coli, Klebsiella pneumoniae, Synechocystis, Aquifex aeolicus, Bacillus subtilis and Rickettsia prowazekii) as well as in archaea (Methanococcus jannaschii and Methanobacterium thermoautotrophicum.) The K. pneumoniae homologues (MdcF, 319 aas) has been implicated in malonate uptake. The O. oeni homologue, MleP, is a malate permease. The bacterial proteins are 300-400 aas in length (Young et al. 1999).
Yeast also possess homologues of the AEC family. Saccharomyces cerevisiae has three functionally uncharacterized AEC members (YL52, spP54072, 64.0 kDa; YNJ5, spP53930, 71.2 kDa; and YB8B, spP38355, 47.5 kDa), and Schizosaccharomyces pombe also has a sequenced homologue. It is thus clear that members of the AEC family are widespread, being found in Gram-negative, Gram-positive and cyanobacteria, in archaea, and in both fungi and plants. C. elegans, however, appears to lack identifiable homologues of the AEC family (Young et al. 1999).
Members of the AEC family are homologous to members of the BART superfamily (Mansour et al. 2007). Interestingly, the first halves of BASS family (TC# 2.A.28) members show extensive similarity with the second halves of AEC family members but not vice versa. Repeats of the basic 5 TMS element have not yet been demonstrated in members of the AEC family.
The transport reaction probably catalyzed by the auxin efflux carrier is:
Auxin (in) nH+ (out) → Auxin (out) nH+ (in)
Auxin efflux carrier, PIN-FORMED1 (PIN1) (Reinhardt et al., 2003; Carraro et al. 2012). Catalyzes auxin efflux without the participation of any other protein (Petrasek et al., 2006). PIN1 determines the direction of intercellular auxin flow (Wang et al. 2014). It consists of two TMS bundles, each of 5 TMSs at the N-terminus and the C-terminus of the protein (Nodzyński et al. 2016), confirming previous bioinformatic predictions (Mansour et al. 2007). Arabidopsis VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC) (TC# 8.A.175) is a plant-specific transmembrane protein that controls the development of veins in cotyledons. Yanagisawa et al. 2021 showed that the expression and localization of PIN1 is altered in vcc developing cotyledons, and that overexpression of PIN1-GFP partially rescues vascular defects of vcc mutants. Genetic analyses suggested that VCC and PINOID (PID), a kinase that regulates PIN1 polarity, are both required for PIN1-mediated control of vasculature development. VCC expression is upregulated by auxin, likely as part of a positive feedback loop for the progression of vascular development. VCC and PIN1 localized to the plasma membrane in pre-procambial cells but were actively redirected to vacuoles in procambial cells for degradation. In the vcc mutant, PIN1 failed to properly polarize in pre-procambial cells during the formation of basal strands, and instead, it was prematurely degraded in vacuoles. VCC plays a role in the localization and stability of PIN1, which is crucial for the transition of pre-procambial cells into procambial cells that are involved in the formation of basal lateral strands in embryonic cotyledons (Yanagisawa et al. 2021). Three inward-facing conformational structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) have been solved (Yang et al. 2022). The transmembrane domain of PIN1 shares a conserved NhaA fold. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site in an intracellular pocket, but with a much higher affinity (Yang et al. 2022). PIN-FORMED is required for shoot phototropism/gravitropism and facilitates meristem formation in Marchantia polymorpha (Fisher et al. 2023).
PIN1 of Arabidopsis thaliana
Auxin efflux facilitator PIN3: functions in auxin redistribution through the root cap in response to the gravity sensors, ARL2 (Q6XL73) and ARG1 (Q9ZSY2). Both ARG1 and ARL2 are DnaJ homologues and show regions homologous to translocation proteins, NPL1 and Sec63 (3.A.5.8.1 and 3.A.5.9.1, respectively). Cryo-EM structures of AtPIN3 in the apo state and in complex with its substrate indole-3-acetic acid (IAA) and the inhibitor N-1-naphthylphthalamic acid (NPA) at 2.6-3.0 Å resolution have been determined (Su et al. 2022). AtPIN3 exists as a homodimer, with TMSs 1, 2, and 7 in the scaffold domain involved in dimerization. The dimeric AtPIN3 forms a large, joint extracellular-facing cavity at the dimer interface while each subunit adopts an inward-facing conformation. The structural basis for the recognition of IAA and NPA were revealed and elucidated the molecular mechanism of NPA inhibition. The AtPIN3 structures support an elevator-like model for the transport of auxin, whereby the transport domains undergo up-down rigid-body motions and the dimerized scaffold domains remain static (Su et al. 2022). CslPIN3 is involved in the regulation of root growth and development as well as auxin accumulation in tea plants (Hu et al. 2023).
PIN3 of Arabidopsis thaliana
Auxin efflux carrier #10, PIN10/SLM1 (important for development; orthologous to A. thaliana PIN1 (Zhou et al., 2011)).
PIN10 of Medicago truncatula (Q673E5)
Auxin efflux carrier, PIN5. Regulates auxin homeostasis and metabolism. Mediates auxin transport across the endoplasmic reticular membrane, for the cytosol to the ER lumen (Mravec et al. 2009).
PIN5 of Arabidopsis thaliana
The auxin efflux carrier, AEC3 or PIN1, of 634 aas and 10 TMSs, plays a role in fruit development (Li et al. 2017).
AEC3 of Momordica charantia (bitter gourd)
Pin1B of 554 aas and 10 TMSs in a 5 + 5 TMS arrangement with a large hydrophilic loop between the two hydroophobic halves. This auxin transporter, OsPIN1b, is a regulator of leaf inclination in rice (Oryza sativa L.) (Zhang et al. 2023).
Pin1B of Oryza sativa L.
AEC family member of 416 aas and 10 TMSs in a 5 + 5 TMS arrangement, with a large hydrophilic loop between the two repeat units.
AEC family member of Trypanosoma cruzi (E7LII7)
Poorly characterized transporter YBR287w. Deletion of the gene leads to poor growth on glucose-minimal medium at 15 degrees C in the FY 1679 genetic background, but is not involved in mating or sporulation (Dueñas et al. 1999).
YBR287w of Saccharomyces cerevisiae
Auxin efflux carrier family member
AEC homologue of Aspergillus flavus (B8MZ51)
AEC family member
AEC family member of Entamoeba histolytica (C4MAS5)
Uncharacterized protein of 616 aas and 11 TMSs in a 5 + 6 arrangement
Rhodophyta (red algae)
UP of Cyanidioschyzon merolae
Uncharacterized transporter, nonessential for growth or sporulation, YLR152c, of 576 aas and 10 TMSs (Watson 2001).
UP of Saccharomyces cerevisiae
Uncharacterized protein of 442 aas and 10 TMSs in a 5 + 5 TMS arrangement.
UP of Citrus clementina
AEC homologue of Streptomyces coelicolor
Malate transporter (MleP) homologue
MleP homologue of Dickeya dadantii (E0SFK1)
Putative malonate transporter, MdcF
MdcF of Rhizobium meliloti
YfdV of Escherichia coli O6:H1
Putative MdcF malonate transporter of 316 aas and 10 TMSs.
MdcF homologue of Rhizobium loti
AEC family transporter, auxin efflux carrier, of 318 aas and 10 TMSs in a 5 + 5 TMS arrangement. The efflux mechanism of the substrate, indole 3-acetic acid, has been examined (Rai et al. 2021).
AEC of Bacillus licheniformis
GPR155 (GP155) of 870 aas and possibly 17 TMSs in a 15 + 2 TMS arrangement. The N-terminal 15 TMSs may consist of 3 repeats, each of 5 TMSs in a LSLSL (L = large; S = small peak of hydrophobicity). It may play a role in several types of cancer (Lee et al. 2022). GPR155 matches the first 10 TMSs (5 + 5 TMS arrangement while TMSs 6 - 10 are similar to the N-terminal half of the sodium bile transporter of the AsbT (SLC10) family. TMSs 11 - 17 are similar to GPCR proteins with 7 TMSs (Gyimesi and Hediger 2022). Lysosomal GPCR-like protein LYCHOS (GRP155) signals cholesterol sufficiency to mTORC1 (Shin et al. 2022).
GPR155 of Homo sapiens
Putative membrane permease-like protein. May belong to the BART superfamily (297aas; 10TMS)
permease-like protein of Chlorobium luteolum (Q3B5D8)
AEC homologue of Myxococcus xanthus
Malate permease, MleP (Labarre et al. 1996). Activated a few minutes after rehydration (Costantini et al. 2011).
MleP of Oenococcus (Leuconostoc) oeni
Putative auxin efflux carrier of 333 aas and 10 TMSs.
Auxin efflux carrier of Bifidobacterium longum
Uncharacterized protein of 308 aas and 10 TMSs (Hug et al. 2016).
UP of Candidatus Peribacter riflensis
Uncharacterized protein of 388 aas and 10 TMSs in a 5 + 5 TMS arrangement.
UP of Entamoeba histolytica