Auxin binding protein:curiouser and curiouser

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1 586 Review Auxin binding protein:curiouser and curiouser Candace Timpte Auxin is implicated in a variety of plant developmental processes,yet the molecular mechanism of auxin response remains largely unknown.auxin binding protein 1 (ABP1) mediates cell expansion and might be involved in cell cycle control.structural modeling shows that it is a β-barrel dimer,with the C terminus free to interact with other proteins.we do not know where ABP1 performs its receptor function.most ABP1 is detected within the endoplasmic reticulum but the evidence indicates that it functions at the plasma membrane. ABP1 is established as a crucial component of auxin signaling,but its precise mechanism remains unclear. Candace Timpte Dept Biological Sciences, University of New Orleans, New Orleans, LA70148, USA. ctimpte@uno.edu Curiouser and curiouser! cried Alice. (As her body grew after swallowing a potion, she realized that she should give her now-distant feet a new pair of boots for Christmas). Alice went on planning to herself how she would manage it. They must go by the carrier, she thought; and how funny it will seem, sending presents to one s own feet! And how odd the directions will look! Through the Looking Glass(Lewis Carrol) The plant hormone auxin (indole-3-acetic acid, or IAA) is central to diverse plant growth and developmental responses. Some of the bestcharacterized examples are tropic growth responses (such as to gravity or light), stem elongation, lateral branching of roots and shoots, and vascular development 1. These whole-plant responses are the result of changes at the cellular level that include elongation, division or differentiation. However, the mechanisms of auxin perception and response are understood poorly. Some responses are rapid and others occur after a lag period, complicating the situation further. The first step in a classic hormone response pathway is a receptor binding a hormone. Many investigators have sought auxin receptors and several good candidates have been isolated 2. However, as well as binding auxin, the receptor must also transduce the auxin stimulus into the known responses. Collecting evidence that the auxin receptor interaction causes direct changes in the cell has been difficult. The immediate short-term auxin responses include changes in protoplast electrophysiology, guard-cell gating and early-response-gene induction. Longerterm responses include cell elongation, cell division and phenotypic changes in the whole plant. The choice of assay is the key to establishing an auxin receptor interaction; one must remember that more than one pathway might be activated by one receptor, and direct cause-and-effect relations must be established. Auxin-binding protein Twenty years ago, an auxin-binding activity was purified from maize coleoptiles by several groups 2,3. This auxin-binding protein, ABP1, was shown byphotoaffinity labeling to bind auxin 4 (its characterization is summarized in Ref. 2). The maize ABP1 cdnaencodes a 201 amino acid protein, with a 38 residue signal sequence. The unglycosylated protein is 20 kda, whereas the mature protein is 22 kda, containing a high-mannose-type oligosaccharide 2. ABP1 was the first plant protein discovered with a C-terminal KDELsequence, which is an endoplasmic reticulum (ER) retention signal 5. ABP1 has no hydrophobic regions. Thus, to function as a receptor, it probably associates with a membrane-bound docking protein. ABP1 bears no resemblance to well-known hormone receptors from animal systems and does not have substantial similarity to any mammalian gene. Yet, ABP1 has been identified from many plant species including maize, Arabidopsis, tobacco and radish 2. In spite of excellent research efforts, important questions need to be answered if ABP1 is to be established as the auxin receptor. First, does ABP1 ligand binding have biological relevance? The auxin receptor must bind auxin but also must evoke changes in the cell. Second, what is the structure of this protein, and how does this structure relate to its signaling mechanism? Third, where does ABP1 reside in the cell? Typically, a mammalian hormone binds the target ligand at the plasma membrane, although one exception is the steroid hormone receptor. Paradoxically, the KDEL sequence of ABP1 suggests an ER, not a plasma membrane, location. Could it be elsewhere in the cell? ABP1 is crucial for embryogenesis Recent genetic studies provide strong evidence for ABP1 mediating responses leading to cell elongation and embryogenesis. Arabidopsishas a single gene encoding ABP1 (Ref. 6) and disruption of this gene is expected to affect auxin signaling processes and to reveal ABP1 s role in plant development. A knockout plant harboring a T-DNA insertion in the first exon of the ABP1gene has been identified 7. Homozygous individuals were not recovered from this plant line, strongly indicating that disruption of the ABP1gene is lethal. About 25% of the seeds in transgenic siliques were white and nonviable, clear evidence of segregation of a lethal homozygous phenotype /01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S (01)

2 Review 587 Table 1. C-terminal sequences tested for hyperpolarization Peptide Sequence Hyperpolarization? Consensus WDE.C...KEDL Not known Maize ABP1 WDEDCFEAA..KDEL Yes Nicotiana tabacum ABP1 WDEECYQTTSWKDEL Yes Pz DEDCFEAA..KDEL Yes Nt-C15 WDEECYQTTSWKDEL Yes Nt-C12..ECYQTTSW.KDEL No Mutation targets WDEECYQTTSWKDEL a No Deletion WDEECYQTTSW... No a Text in bold indicates target residues for mutation. Addition of a transgenic, functional copy of ABP1 rescued the embryonic-lethal phenotype, suggesting that normal embryo development requires at least one copy of ABP1. Examination of the nonviable embryos revealed that ABP1 is required early in plant development: embryos arrested after the globular stage. Newly formed cross walls between cells were wrongly oriented and cells failed to elongate, leading to embryo death 7. These results provide direct evidence that ABP1 plays a crucial role in embryonic morphogenesis. Whether the role is in cell elongation, embryo polarity establishment or individual cell polarity could not be determined. To address the polarity versus elongation issue, antisense suppression was used to create an ABP1 loss-offunction mutation in the BY-2 tobacco cell line. The results enabled the two types of expansion commonly observed in cultured plant cells to be differentiated 8. Auxin-induced elongation to increase cell volume beyond that of the divided cell was abolished in these transgenic cells 7. However, cell expansion to replace cell volume following division was not affected in the ABP1-antisense lines. Thus, elongation growth is the crucial auxin response in cultured cells and failure to elongate is the probable cause of embryo lethality in the transgenic knockout plants. ABP1 mediates cell expansion The complementary approach, overproducing ABP1, confirms the role of ABP1 in auxin-mediated cell expansion. Tobacco was transformed with ABP1 under the control of an inducible promoter 9. In control plants, auxin only induced growth at the leaf tips, whereas, in overproducing transgenic plants, it induced growth throughout the leaf. Regions that are not normally auxin responsive acquired inducible growth that was strictly dependent on the presence of auxin; a structurally similar inactive auxin did not stimulate growth. Thus, overproducing ABP1 extended the range of auxin sensitivity in mature leaf tissue 9. A meticulous analysis of individual cells from ABP1-overproducing plants reveals that auxin-inducible cell expansion is a component of this growth 10. The abundance of ABP1 in each cell correlates with the extent of auxin-induced cell expansion and with cell size in transgenic plants. Evidence from cultured cells supports a role for ABP1 in cell expansion. Cultured maize cells overproducing ABP1 expanded in an auxindependent fashion and were greater in volume than control cells 9. Antisense-suppressed ABP1 tobacco BY-2 cells had undetectable levels of ABP1 protein and lacked auxin-induced cell expansion when compared with wild-type cells 10. Auxin-induced cell division might involve ABP1 Auxin-mediated growth might also have a division component. Cells from ABP1-overproducing tobacco leaves were examined for nuclear division stage 10. The proportion of nuclei in G2 stage was double that of the wild type. By sequential analysis of cells in developing leaves, cell expansion was found to precede the G2 advance in the cycle. The premature G2 advance is probably an indirect effect of the increased cell volume of transgenic plants 10. A conditional ABP1 knockout mutation has been constructed by producing a transgenic ABP1 antibody in the tobacco BY2 cell line (C. Perrot-Rechenmann, pers. commun.). This transgenic antibody presumably binds ABP1 in planta and limits its activity within the cell. These knockout cells showed no significant change in cell volume but arrested at the G1 phase of the cell cycle. Thus, ABP1 might play a crucial role in the regulation G1 and G2/M phases of the cell cycle. Although the conclusions from these two transgenic studies differ, the results indicate a crucial role for ABP1 in plant cells. Furthermore, either knocking out or overproducing ABP1 provides crucial evidence that ABP1 mediates perception of auxin in cultured cells and that disruption of this signal causes changes in the cell cycle. ABP1 triggers a plasma membrane electrical response Hyperpolarization of the cell membrane occurs within minutes after applying biologically active auxin, providing a convenient assay for evaluating auxin response at the outer face of the plasma membrane. ABP1 has been implicated in this response in many studies 11. Synthetic peptides corresponding to the C terminus of ABP1 were tested in the hyperpolarization assay 12,13 (Table 1). Peptide Pz is a maize-derived sequence. Two others are tobacco-derived peptides: the first, Nt-C15, is most similar to the wild-type sequence whereas the second, Nt-C12, lacks three conserved residues. The maize and Nt-C15 peptides all induce hyperpolarization, much as auxin does when applied to tobacco protoplasts. The truncated Nt-C12 peptide fails to induce the hyperpolarization response. This study confirms previous results that exogenous peptides derived from ABP1 can elicit an electrical response. These results confirm that the homologous system is more efficient than the heterologous system, because peptides and membranes were derived from the same species 13.

3 588 Review Fig. 1. Conceptual model of ABP1. ABP1 is a β-barrel structure modeled on similarity with cupin families and concavalin A for dimerization. Residue W44 (red) and the cluster of residues forming the putative metal-binding site (blue) might form the platform for auxin binding. The purple ribbon indicates conserved β-turn anchor residues. Abbreviations: C-term, C-terminal; N-term, N-terminal. N-term C-term C-term N-term TRENDS in Plant Science The C-terminal charged residues in ABP1 were mutagenized and the entire protein was tested in the hyperpolarization assay 14. The charged residues were mutated to the cognate amine residues, singly and paired, and a KDEL deletion mutant protein was constructed (Table 1). The KDEL deletion evoked the same hyperpolarization response as the wild type when applied to cells. None of the charge-substituted mutant proteins evoked a hyperpolarization response 14. Thus, the substitution of charged residues causes ABP1 to fail to interact with the plasma membrane protein that affects hyperpolarization, implicating a charge charge interaction between the proteins. Alternatively, the mutated ABP1 might simply misfold and fail to interact with the plasma membrane protein. The electrical response of plant cells was affected by antibodies directed against ABP1. Several monoclonal antibodies induce hyperpolarization in tobacco cell protoplasts and act as auxin agonists 13. Three other monoclonal antibodies act as antagonists and block auxin action, either by recognizing the auxin-binding site as the epitope 13 or by immobilizing ABP1 in a nonfunctional conformation. Similarly, antibodies affected orchid cell stomatal opening. Both the D16 monoclonal antibody, raised against the putative auxin binding site of ABP1 (Ref. 15), and a monoclonal antibody against an ABP1 peptide, induced stomatal opening and acidification, similar to the effects of auxin 16. A monoclonal antibody that targets the C terminus of ABP1 and the peptide Pz stimulated stomatal closure and increased ph, similar to the mode of abscisic acid. A Pz peptide lacking the KDEL sequence had no effect. This result is curious because other data suggests that the KDEL is not required for hyperpolarization stimulation. However, small changes in a peptide can cause great changes in peptide structure. These immunological results indicate that ABP1 transduces the auxin signal to the plasma membrane to effect hyperpolarization, perhaps by interacting with another protein. The amount of ABP1 might be tightly regulated in the cell. As the evidence above indicates, increased production of ABP1 enhances auxin sensitivity 9,10,17,18. Examination at the molecular level reveals that transgenic overexpression of wild-type ABP1 generated a ~100-fold increase in expression by RNA blotting but only a 30% increase in detectable protein by immunoblotting 18. In antisense transgenic plants, maximal inhibition of ABP1 protein was merely 50%, indicating that complete inhibition of ABP1 might be detrimental to the plant 18. Structure of ABP1 The three-dimensional structure of ABP1 could give clues about the mechanism of signaling or potential protein protein interactions. The first model for the auxin-binding site of ABP1 was based on the structure and interaction of 45 different auxin analogs 19. This model proposed a planar, indole ringbinding platform, a charged carboxylic acid-binding site and a hydrophobic transition region. By photoaffinity labeling with azido IAA (Ref. 20) and immunology 21, two regions were implicated in auxin binding. Structure mapping studies using a panel of monoclonal antibodies further defined the identity of residues forming the auxin-binding platform and the carboxylic acid-binding site 13. β-barrel dimer Comparisons of amino acid sequences show that there are several highly conserved residues between auxin binding proteins in monocots and dicots 2,22. An augmented model has been proposed based on these and additional comparisons with the cupin and vicilin superfamily of proteins 23. The structural basis of this model relies on conserved residues corresponding to β-barrel turn anchors in the germin protein structure 24. The proposed structure is a β-barrel homodimer, containing β-sheets and no α-helix, consistent with circular dichroism spectra 25, and resembles the pseudodimer symmetry of a vicilin monomer 23. Recently, ABP1 was crystallized, and X-ray diffraction analysis to 1.9 Å resolution shows two glycosylated homodimers in asymmetric units 26. These crystal structure data are consistent with a β-barrel (Fig. 1). This level of resolution cannot confirm the auxinbinding site. A conserved region might be analogous to the metal-binding site of oxalate oxidase 23 and thus indicate that ABP1 has some unknown enzyme function. This speculation is intriguing, because no enzymatic activity has been reported for ABP1. Mobile C-terminus Experimental evidence suggests that binding auxin causes a conformational change involving the C-terminus 27. Interference mapping studies suggest that the C-terminus interacts with the auxin-binding site, perhaps through disulfide bonds 13. Two antibodies map to overlapping ABP1 regions but have opposite electrochemical effects, one agonistic and the other antagonistic to auxin action 14. In the presence of auxin, binding by one agonist antibody is completely abolished and the other antibody has a weaker interaction with ABP1 (Ref. 14). This result suggests

4 Review 589 ABP1 ER Docking protein Fig. 2. Model of ABP1 localization and action. Most ABP1 resides in the endoplasmic reticulum (ER) but it is also detectable in the Golgi and associated with the plasma membrane (PM). ABP1 probably associates with a transmembrane docking protein to propagate the auxin signal to the interior of the cell, or it could interact directly with ion channels. A conformational change is induced upon auxin binding. At the plasma membrane, auxin binding effects a hyperpolarization event, which also can be stimulated by ABP1- derived peptides. Because the ER does not provide the optimum ph for auxin binding, auxin-bound ABP1 in the Golgi might direct vesicle traffic of cell wall materials for expansion. Golgi Ion channel Cell wall Plasma membrane Hyperpolarization Vesicles to PM TRENDS in Plant Science that ABP1 undergoes a distinct conformational change when auxin is bound, changing the epitope. Circular dichroism data indicate a conformational change upon auxin binding 25. Although the C-terminus did not have sufficient similarity to the vicilin superfamily to model its location, a C-terminal tryptophan or WDE sequence might occupy the binding pocket in the absence of auxin 23. The experimental evidence presented above supports the importance of the conserved WDE residues. Conformational changes upon auxin binding might release the C-terminus for signal propagation and interaction with other proteins. Localization versus site of action remains perplexing The KDEL sequence of ABP1 appears to be effective at localizing ABP1 to the endoplasmic reticulum (ER). Paradoxically, the evidence indicates that ABP1 binds auxin with low affinity at the ph of the ER (Ref. 28). However, numerous visualization techniques show that most, but not all, ABP1 is localized to the ER, not the plasma membrane. ABP1 has been visualized at the plasma membrane of maize cultured cells by using immunogold labeling 29. ABP1 has also been detected throughout the Golgi and has been secreted into culture medium 29. A small population of ABP1 molecules has been detected at the plasma membrane of maize coleoptile protoplasts using silver-enhanced immunogold epipolarization microscopy 30. The number of ABP1 molecules at the surface was estimated to be as low as 1000, which represents only a small proportion of total cellular ABP1. A small number of cell surface receptors requires less hormone to achieve half-maximal occupancy. Physiologically, this would allow the cell to be sensitive to small amounts of hormone 29. Because antibodies are unlikely to enter intact cells, the evidence that several antibodies trigger the plasma membrane hyperpolarization response indicates that at least some portion of the ABP1 pool resides at the plasma membrane 11, Because the antibodies and peptides bind a protein in intact protoplasts that affects membrane conductivity, logically they must bind ABP1 localized to the plasma membrane. The C-terminal KDEL sequence was changed to examine its role in the localization of ABP1 (Ref. 17). KDEL was mutated to HDEL to enhance its retention in the ER or mutated to either KDELGL or KEQL to compromise its retention. As expected, the KDEL or HDEL proteins localized to the ER, whereas the KEQL and KDELGL proteins entered the Golgi stacks. However, there was no difference in the cell surface abundance of ABP1 in cells expressing mutant proteins as examined by electron microscopy or silver-enhanced immunogold epipolarization microscopy 17. Thus, even without the KDEL sequence, quantities of ABP1 do not localize massively to the plasma membrane. Similar results confirming the major ER localization were obtained by analysis of maize coleoptile rolled leaves 31. Neither immunofluorescence nor immunogold labeling detected ABP1 at the plasma membrane. Double-labeling experiments to detect conformational changes that might sequester the KDEL and allow transit to the plasma membrane were negative. Under conditions of auxin binding, ABP1 remained in the ER (Ref. 31). Carbohydrate analysis further confirmed the ER localization of ABP1. The ABP1 oligosaccharides are high mannose types, not the more complex carbohydrates expected if ABP1 traversed the Golgi stacks. Less than 2% of ABP, by glycan analysis, escaped the ER retention system 31. At this low level of escape, a special mechanism to avoid ER retention need not be invoked. Bulk flow or association with other proteins might be sufficient to allow this tiny amount of ABP1 to escape to the plasma membrane. The mechanism of KDEL-mediated ER retention and retrieval operates efficiently, such that an alternative delivery mechanism to the vacuole was proposed 32. A KDEL terminus was attached to a protein not normally localized to the ER. This construct circumvented the Golgi to reach the vacuole. Some such alternate mechanism might operate for ABP1. Because overproduction of ABP1 increases the sensitivity of cells to auxin, the presence of ABP1 at the plasma membrane might be tightly regulated. Never-ending story of auxin signaling ABP1 fits the criteria for a hormone receptor. Ample evidence indicates that ABP1 mediates auxin s effects on normal plant development. However, the molecular mechanism of ABP1 remains at best a skeletal model (Fig. 2). To reconcile the diverse effects of auxin, two sites of ABP1 activity are proposed.

5 590 Review Acknowledgements I am grateful to Barbara Triplett, Sarah Lingle, Hee-Jin Kim, Amy Herman and several anonymous reviewers for comments. Thanks to Jim Nolan for the structure figure. I thank Alan Jones and Catherine Perrot-Rechenmann for sharing unpublished results. Binding at higher or lower concentrations of auxin might temper the response. Although ABP1 has no hydrophobic regions, the auxin-binding signal must be transmitted to the cell. To this end, ABP1 might interact with a plasma membrane docking protein (yet to be identified) 3,33 or might interact directly with the ion channel. Auxin binding induces a conformational change in ABP1, enabling interaction with the docking protein, or alters the ABP1 dockingprotein complex to transmit the signal. The docking protein might be abundant at the plasma membrane; excess docking protein would then be available to interact with exogenously provided ABP1 or peptides in assays, and to mediate membrane hyperpolarization. Similarly, ER or Golgi-localized ABP1 might interact with a transmembrane protein to regulate the secretion of cell wall components to mediate cell expansion. Auxin-induced conformational changes in ABP1 might alter interactions with other membrane proteins, perhaps heterotrimeric G-proteins. Auxin is known to induce the transcription of several auxin-regulated genes that are repressed by the activation of a specific MAPK cascade 34. Furthermore, Arabidopsis cells overproducing the plant heterotrimeric Gα protein mimic the auxininduced increase in cell division 35. These results suggest that the auxin signaling pathway might involve the Gα protein to regulate cell cycle control. However, given the varied plant responses to auxin, there might be more than one type of auxin receptor in the cell. It has not been possible to cover all aspects of auxin signaling in this article. The importance of ABP1 in plant development is certain but more pieces of the puzzle remain to be identified. Refining the location and site of action of ABP1 might require the use of non-plant in vivo systems, such as the previously used COS cells 28. The proposed docking protein or other proteins that interact with ABP1 must be identified. Plant development, as the ultimate result of cell division and cell elongation, is affected by numerous hormone signals. The mechanism of integration of these signals and exactly where ABP1 fits into signaling cross-talk remain to be discovered. References 1 Davies, P.J., ed. (1995) Plant Hormones: Physiology, Biochemistry and Molecular Biology, Kluwer Academic Publishers 2 Jones, A.M. (1994) Auxin-binding proteins. Annu. Rev. Plant Physiol. 45, Klambt, D. (1990) A view about the function of auxin-binding proteins at plasma membranes. Plant Mol. Biol. 14, Jones, A.M. and Venis, M. (1989) Photoaffinity labeling of indole-3-acetic acid binding proteins in maize. Proc. Natl. Acad. Sci. U. S. A. 86, Pelham, H.R.B. 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