The ubiquitin-protein ligase MIEL1 localizes to peroxisomes to promote seedling oleosin degradation and lipid droplet mobilization.

Lipid droplets are organelles conserved across eukaryotes that store and release neutral lipids to regulate energy homeostasis. In oilseed plants, fats stored in seed lipid droplets provide fixed carbon for seedling growth before photosynthesis begins. As fatty acids released from lipid droplet triacylglycerol are catabolized in peroxisomes, lipid droplet coat proteins are ubiquitinated, extracted, and degraded. In Arabidopsis seeds, the predominant lipid droplet coat protein is OLEOSIN1 (OLE1). To identify genes modulating lipid droplet dynamics, we mutagenized a line expressing mNeonGreen-tagged OLE1 expressed from the OLE1 promoter and isolated mutants with delayed oleosin degradation. From this screen, we identified four miel1 mutant alleles. MIEL1 (MYB30-interacting E3 ligase 1) targets specific MYB transcription factors for degradation during hormone and pathogen responses [D. Marino et al., Nat. Commun. 4, 1476 (2013); H. G. Lee and P. J. Seo, Nat. Commun. 7, 12525 (2016)] but had not been implicated in lipid droplet dynamics. OLE1 transcript levels were unchanged in miel1 mutants, indicating that MIEL1 modulates oleosin levels posttranscriptionally. When overexpressed, fluorescently tagged MIEL1 reduced oleosin levels, causing very large lipid droplets. Unexpectedly, fluorescently tagged MIEL1 localized to peroxisomes. Our data suggest that MIEL1 ubiquitinates peroxisome-proximal seed oleosins, targeting them for degradation during seedling lipid mobilization. The human MIEL1 homolog (PIRH2; p53-induced protein with a RING-H2 domain) targets p53 and other proteins for degradation and promotes tumorigenesis [A. Daks et al., Cells 11, 1515 (2022)]. When expressed in Arabidopsis, human PIRH2 also localized to peroxisomes, hinting at a previously unexplored role for PIRH2 in lipid catabolism and peroxisome biology in mammals.

An Arabidopsis pre-RNA processing8a (prp8a) missense allele restores splicing of a subset of mis-spliced mRNAs.

Eukaryotic precursor mRNAs often harbor noncoding introns that must be removed prior to translation. Accurate splicing of precursor messenger RNA depends on placement and assembly of small nuclear ribonucleoprotein (snRNP) sub-complexes of the spliceosome. Yeast (Saccharomyces cerevisiae) studies established a role in splice-site selection for PRE-RNA PROCESSING8 (PRP8), a conserved spliceosome scaffolding protein of the U5 snRNP. However, analogous splice-site selection studies in multicellular eukaryotes are lacking. Such studies are crucial for a comprehensive understanding of alternative splicing, which is extensive in plants and animals but limited in yeast. In this work, we describe an Arabidopsis (Arabidopsis thaliana) prp8a mutant that modulates splice-site selection. We isolated prp8a-14 from a screen for suppressors of pex14-6, which carries a splice-site mutation in the PEROXIN14 (PEX14) peroxisome biogenesis gene. To elucidate Arabidopsis PRP8A function in spliceosome fidelity, we combined prp8a-14 with various pex14 splice-site mutations and monitored the double mutants for physiological and molecular consequences of dysfunctional and functional peroxisomes that correspond to impaired and recovered splicing, respectively. prp8a-14 restored splicing and PEX14 function to alleles with mutations in the exonic guanine of the 5'-splice site but did not restore splicing or function to alleles with mutations in the intronic guanine of 5'- or 3'-splice sites. We used RNA-seq to reveal the systemic impact of prp8a-14 and found hundreds of differentially spliced transcripts and thousands of transcripts with significantly altered levels. Among differentially spliced transcripts, prp8a-14 significantly altered 5'- and 3'-splice-site utilization to favor sites resulting in shorter introns. This study provides a genetic platform for probing splicing in plants and hints at a role for plant PRP8 in splice-site selection.

Proteaphagy-Selective Autophagy of Inactive Proteasomes.

In this issue of Molecular Cell, Marshall et al. (2015) report that proteasomes, the ATP-dependent protease complexes that execute ubiquitin-dependent protein degradation in eukaryotes, can be degraded by a newly described form of selective autophagy, termed proteaphagy.

A pex1 missense mutation improves peroxisome function in a subset of Arabidopsis pex6 mutants without restoring PEX5 recycling.

Peroxisomes are eukaryotic organelles critical for plant and human development because they house essential metabolic functions, such as fatty acid ß-oxidation. The interacting ATPases PEX1 and PEX6 contribute to peroxisome function by recycling PEX5, a cytosolic receptor needed to import proteins targeted to the peroxisomal matrix. Arabidopsis pex6 mutants exhibit low PEX5 levels and defects in peroxisomal matrix protein import, oil body utilization, peroxisomal metabolism, and seedling growth. These defects are hypothesized to stem from impaired PEX5 retrotranslocation leading to PEX5 polyubiquitination and consequent degradation of PEX5 via the proteasome or of the entire organelle via autophagy. We recovered a pex1 missense mutation in a screen for second-site suppressors that restore growth to the pex6-1 mutant. Surprisingly, this pex1-1 mutation ameliorated the metabolic and physiological defects of pex6-1 without restoring PEX5 levels. Similarly, preventing autophagy by introducing an atg7-null allele partially rescued pex6-1 physiological defects without restoring PEX5 levels. atg7 synergistically improved matrix protein import in pex1-1 pex6-1, implying that pex1-1 improves peroxisome function in pex6-1 without impeding autophagy of peroxisomes (i.e., pexophagy). pex1-1 differentially improved peroxisome function in various pex6 alleles but worsened the physiological and molecular defects of a pex26 mutant, which is defective in the tether anchoring the PEX1-PEX6 hexamer to the peroxisome. Our results support the hypothesis that, beyond PEX5 recycling, PEX1 and PEX6 have additional functions in peroxisome homeostasis and perhaps in oil body utilization.

PEX16 contributions to peroxisome import and metabolism revealed by viable Arabidopsis pex16 mutants.

Peroxisomes rely on peroxins (PEX proteins) for biogenesis, importing membrane and matrix proteins, and fission. PEX16, which is implicated in peroxisomal membrane protein targeting and forming nascent peroxisomes from the endoplasmic reticulum (ER), is unusual among peroxins because it is inserted co-translationally into the ER and localizes to both ER and peroxisomal membranes. PEX16 mutations in humans, yeast, and plants confer some common peroxisomal defects; however, apparent functional differences have impeded the development of a unified model for PEX16 action. The only reported pex16 mutant in plants, the Arabidopsis shrunken seed1 mutant, is inviable, complicating analysis of PEX16 function after embryogenesis. Here, we characterized two viable Arabidopsis pex16 alleles that accumulate negligible PEX16 protein levels. Both mutants displayed impaired peroxisome function - slowed consumption of stored oil bodies, decreased import of matrix proteins, and increased peroxisome size. Moreover, one pex16 allele exhibited reduced growth that could be alleviated by an external fixed carbon source, decreased responsiveness to peroxisomally processed hormone precursors, and worsened or improved peroxisome function in combination with other pex mutants. Because the mutations impact different regions of the PEX16 gene, these viable pex16 alleles allow assessment of the importance of Arabidopsis PEX16 and its functional domains.

Disparate peroxisome-related defects in Arabidopsis pex6 and pex26 mutants link peroxisomal retrotranslocation and oil body utilization.

Catabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail-anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling ß-oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by treatment with a proteasome inhibitor or by combining pex26 with peroxisome-associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex.

The PEX1 ATPase Stabilizes PEX6 and Plays Essential Roles in Peroxisome Biology.

A variety of metabolic pathways are sequestered in peroxisomes, conserved organelles that are essential for human and plant survival. Peroxin (PEX) proteins generate and maintain peroxisomes. The PEX1 ATPase facilitates recycling of the peroxisome matrix protein receptor PEX5 and is the most commonly affected peroxin in human peroxisome biogenesis disorders. Here, we describe the isolation and characterization of, to our knowledge, the first Arabidopsis (Arabidopsis thaliana) pex1 missense alleles: pex1-2 and pex1-3pex1-2 displayed peroxisome-related defects accompanied by reduced PEX1 and PEX6 levels. These pex1-2 defects were exacerbated by growth at high temperature and ameliorated by growth at low temperature or by PEX6 overexpression, suggesting that PEX1 enhances PEX6 stability and vice versa. pex1-3 conferred embryo lethality when homozygous, confirming that PEX1, like several other Arabidopsis peroxins, is essential for embryogenesis. pex1-3 displayed symptoms of peroxisome dysfunction when heterozygous; this semidominance is consistent with PEX1 forming a heterooligomer with PEX6 that is poisoned by pex1-3 subunits. Blocking autophagy partially rescued PEX1/pex1-3 defects, including the restoration of normal peroxisome size, suggesting that increasing peroxisome abundance can compensate for the deficiencies caused by pex1-3 and that the enlarged peroxisomes visible in PEX1/pex1-3 may represent autophagy intermediates. Overexpressing PEX1 in wild-type plants impaired growth, suggesting that excessive PEX1 can be detrimental. Our genetic, molecular, and physiological data support the heterohexamer model of PEX1-PEX6 function in plants.

Pexophagy and peroxisomal protein turnover in plants.

Peroxisomes are dynamic, vital organelles that sequester a variety of oxidative reactions and their toxic byproducts from the remainder of the cell. The oxidative nature of peroxisomal metabolism predisposes the organelle to self-inflicted damage, highlighting the need for a mechanism to dispose of damaged peroxisomes. In addition, the metabolic requirements of plant peroxisomes change during development, and obsolete peroxisomal proteins are degraded. Although pexophagy, the selective autophagy of peroxisomes, is an obvious mechanism for executing such degradation, pexophagy has only recently been described in plants. Several recent studies in the reference plant Arabidopsis thaliana implicate pexophagy in the turnover of peroxisomal proteins, both for quality control and during functional transitions of peroxisomal content. In this review, we describe our current understanding of the occurrence, roles, and mechanisms of pexophagy in plants.

Genetic Interactions between PEROXIN12 and Other Peroxisome-Associated Ubiquitination Components.

Most eukaryotic cells require peroxisomes, organelles housing fatty acid ß-oxidation and other critical metabolic reactions. Peroxisomal matrix proteins carry peroxisome-targeting signals that are recognized by one of two receptors, PEX5 or PEX7, in the cytosol. After delivering the matrix proteins to the organelle, these receptors are removed from the peroxisomal membrane or matrix. Receptor retrotranslocation not only facilitates further rounds of matrix protein import but also prevents deleterious PEX5 retention in the membrane. Three peroxisome-associated ubiquitin-protein ligases in the Really Interesting New Gene (RING) family, PEX2, PEX10, and PEX12, facilitate PEX5 retrotranslocation. However, the detailed mechanism of receptor retrotranslocation remains unclear in plants. We identified an Arabidopsis (Arabidopsis thaliana) pex12 Glu-to-Lys missense allele that conferred severe peroxisomal defects, including impaired ß-oxidation, inefficient matrix protein import, and decreased growth. We compared this pex12-1 mutant to other peroxisome-associated ubiquitination-related mutants and found that RING peroxin mutants displayed elevated PEX5 and PEX7 levels, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Also, we observed that disruption of any Arabidopsis RING peroxin led to decreased PEX10 levels, as seen in yeast and mammals. Peroxisomal defects were exacerbated in RING peroxin double mutants, suggesting distinct roles of individual RING peroxins. Finally, reducing function of the peroxisome-associated ubiquitin-conjugating enzyme PEX4 restored PEX10 levels and partially ameliorated the other molecular and physiological defects of the pex12-1 mutant. Future biochemical analyses will be needed to determine whether destabilization of the RING peroxin complex observed in pex12-1 stems from PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue.

Disrupting autophagy restores peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal matrix protein degradation.

Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.

Elevated growth temperature decreases levels of the PEX5 peroxisome-targeting signal receptor and ameliorates defects of Arabidopsis mutants with an impaired PEX4 ubiquitin-conjugating enzyme.

BACKGROUND: Peroxisomes house critical metabolic reactions. For example, fatty acid ß-oxidation enzymes, which are essential during early seedling development, are peroxisomal. Peroxins (PEX proteins) are needed to bring proteins into peroxisomes. Most matrix proteins are delivered to peroxisomes by PEX5, a receptor that forms transient pores to escort proteins across the peroxisomal membrane. After cargo delivery, a peroxisome-tethered ubiquitin-conjugating enzyme (PEX4) and peroxisomal ubiquitin-protein ligases mono- or polyubiquitinate PEX5 for recycling back to the cytosol or for degradation, respectively. Arabidopsis pex mutants ß-oxidize fatty acids inefficiently and therefore fail to germinate or grow less vigorously. These defects can be partially alleviated by providing a fixed carbon source, such as sucrose, in the growth medium. Despite extensive characterization of peroxisome biogenesis in Arabidopsis grown in non-challenged conditions, the effects of environmental stressors on peroxisome function and pex mutant dysfunction are largely unexplored. RESULTS: We surveyed the impact of growth temperature on a panel of pex mutants and found that elevated temperature ameliorated dependence on external sucrose and reduced PEX5 levels in the pex4-1 mutant. Conversely, growth at low temperature exacerbated pex4-1 physiological defects and increased PEX5 levels. Overexpressing PEX5 also worsened pex4-1 defects, implying that PEX5 lingering on the peroxisomal membrane when recycling is impaired impedes peroxisome function. Growth at elevated temperature did not reduce the fraction of membrane-associated PEX5 in pex4-1, suggesting that elevated temperature did not restore PEX4 enzymatic function in the mutant. Moreover, preventing autophagy in pex4-1 did not restore PEX5 levels at high temperature. In contrast, MG132 treatment increased PEX5 levels, implicating the proteasome in degrading PEX5, especially at high temperature. CONCLUSIONS: We conclude that growth at elevated temperature increases proteasomal degradation of PEX5 to reduce overall PEX5 levels and ameliorate pex4-1 physiological defects. Our results support the hypothesis that efficient retrotranslocation of PEX5 after cargo delivery is needed not only to make PEX5 available for further rounds of cargo delivery, but also to prevent the peroxisome dysfunction that results from PEX5 lingering in the peroxisomal membrane.

Peroxisomal ubiquitin-protein ligases peroxin2 and peroxin10 have distinct but synergistic roles in matrix protein import and peroxin5 retrotranslocation in Arabidopsis.

Peroxisomal matrix proteins carry peroxisomal targeting signals (PTSs), PTS1 or PTS2, and are imported into the organelle with the assistance of peroxin (PEX) proteins. From a microscopy-based screen to identify Arabidopsis (Arabidopsis thaliana) mutants defective in matrix protein degradation, we isolated unique mutations in PEX2 and PEX10, which encode ubiquitin-protein ligases anchored in the peroxisomal membrane. In yeast (Saccharomyces cerevisiae), PEX2, PEX10, and a third ligase, PEX12, ubiquitinate a peroxisome matrix protein receptor, PEX5, allowing the PEX1 and PEX6 ATP-hydrolyzing enzymes to retrotranslocate PEX5 out of the membrane after cargo delivery. We found that the pex2-1 and pex10-2 Arabidopsis mutants exhibited defects in peroxisomal physiology and matrix protein import. Moreover, the pex2-1 pex10-2 double mutant exhibited severely impaired growth and synergistic physiological defects, suggesting that PEX2 and PEX10 function cooperatively in the wild type. The pex2-1 lesion restored the unusually low PEX5 levels in the pex6-1 mutant, implicating PEX2 in PEX5 degradation when retrotranslocation is impaired. PEX5 overexpression altered pex10-2 but not pex2-1 defects, suggesting that PEX10 facilitates PEX5 retrotranslocation from the peroxisomal membrane. Although the pex2-1 pex10-2 double mutant displayed severe import defects of both PTS1 and PTS2 proteins into peroxisomes, both pex2-1 and pex10-2 single mutants exhibited clear import defects of PTS1 proteins but apparently normal PTS2 import. A similar PTS1-specific pattern was observed in the pex4-1 ubiquitin-conjugating enzyme mutant. Our results indicate that Arabidopsis PEX2 and PEX10 cooperate to support import of matrix proteins into plant peroxisomes and suggest that some PTS2 import can still occur when PEX5 retrotranslocation is slowed.

Plant peroxisomes: biogenesis and function.

Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of peroxisomal metabolism to meet the needs of a plant. Knowledge gained from plant peroxisomal research will be instrumental to fully understanding the organelle's dynamic behavior and defining peroxisomal metabolic networks, thus allowing the development of molecular strategies for rational engineering of plant metabolism, biomass production, stress tolerance, and pathogen defense.

A viable Arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes.

Peroxisomes are organelles that catabolize fatty acids and compartmentalize other oxidative metabolic processes in eukaryotes. Using a forward-genetic screen designed to recover severe peroxisome-defective mutants, we isolated a viable allele of the peroxisome biogenesis gene PEX13 with striking peroxisomal defects. The pex13-4 mutant requires an exogenous source of fixed carbon for pre-photosynthetic development and is resistant to the protoauxin indole-3-butyric acid. Delivery of peroxisome-targeted matrix proteins depends on the PEX5 receptor docking with PEX13 at the peroxisomal membrane, and we found severely reduced import of matrix proteins and less organelle-associated PEX5 in pex13-4 seedlings. Moreover, pex13-4 physiological and molecular defects were partially ameliorated when PEX5 was overexpressed, suggesting that PEX5 docking is partially compromised in this mutant and can be improved by increasing PEX5 levels. Because previously described Arabidopsis pex13 alleles either are lethal or confer only subtle defects, the pex13-4 mutant provides valuable insight into plant peroxisome receptor docking and matrix protein import.

Multiple facets of Arabidopsis seedling development require indole-3-butyric acid-derived auxin.

Levels of auxin, which regulates both cell division and cell elongation in plant development, are controlled by synthesis, inactivation, transport, and the use of storage forms. However, the specific contributions of various inputs to the active auxin pool are not well understood. One auxin precursor is indole-3-butyric acid (IBA), which undergoes peroxisomal ß-oxidation to release free indole-3-acetic acid (IAA). We identified ENOYL-COA HYDRATASE2 (ECH2) as an enzyme required for IBA response. Combining the ech2 mutant with previously identified iba response mutants resulted in enhanced IBA resistance, diverse auxin-related developmental defects, decreased auxin-responsive reporter activity in both untreated and auxin-treated seedlings, and decreased free IAA levels. The decreased auxin levels and responsiveness, along with the associated developmental defects, uncover previously unappreciated roles for IBA-derived IAA during seedling development, establish IBA as an important auxin precursor, and suggest that IBA-to-IAA conversion contributes to the positive feedback that maintains root auxin levels.

A role for the root cap in root branching revealed by the non-auxin probe naxillin.

The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.

Hormonal control of underwater germination in rice.

The ability to germinate, develop, and thrive underwater is key to efficient rice cultivation. In this issue of Developmental Cell, Wang et al. (2024) illuminate a hormone synthesis and inactivation cascade that promotes germination of submerged rice seeds and may allow improved germination in the field.

Reducing PEX13 expression ameliorates physiological defects of late-acting peroxin mutants.

Proteins are targeted to the peroxisome matrix via processes that are mechanistically distinct from those used by other organelles. Protein entry into peroxisomes requires peroxin (PEX) proteins, including early-acting receptor (e.g. PEX5) and docking peroxins (e.g. PEX13 and PEX14) and late-acting PEX5-recycling peroxins (e.g. PEX4 and PEX6). We examined genetic interactions among Arabidopsis peroxin mutants and found that the weak pex13-1 allele had deleterious effects when combined with pex5-1 and pex14-2, which are defective in early-acting peroxins, as shown by reduced matrix protein import and enhanced physiological defects. In contrast, combining pex13-1 with pex4-1 or pex6-1, which are defective in late-acting peroxins, unexpectedly ameliorated mutant growth defects. Matrix protein import remained impaired in pex4-1 pex13-1 and pex6-1 pex13-1, suggesting that the partial suppression of pex4-1 and pex6-1 physiological defects by a weak pex13 allele may result from restoring the balance between import and export of PEX5 or other proteins that are retrotranslocated from the peroxisome with the assistance of PEX4 and PEX6. Our results suggest that symptoms caused by pex mutants defective in late-acting peroxins may result not only from defects in matrix protein import but also from inefficient removal of PEX5 from the peroxisomal membrane following cargo delivery.

Arabidopsis PIS1 encodes the ABCG37 transporter of auxinic compounds including the auxin precursor indole-3-butyric acid.

Differential distribution of the plant hormone auxin within tissues mediates a variety of developmental processes. Cellular auxin levels are determined by metabolic processes including synthesis, degradation, and (de)conjugation, as well as by auxin transport across the plasma membrane. Whereas transport of free auxins such as naturally occurring indole-3-acetic acid (IAA) is well characterized, little is known about the transport of auxin precursors and metabolites. Here, we identify a mutation in the ABCG37 gene of Arabidopsis that causes the polar auxin transport inhibitor sensitive1 (pis1) phenotype manifested by hypersensitivity to auxinic compounds. ABCG37 encodes the pleiotropic drug resistance transporter that transports a range of synthetic auxinic compounds as well as the endogenous auxin precursor indole-3-butyric acid (IBA), but not free IAA. ABCG37 and its homolog ABCG36 act redundantly at outermost root plasma membranes and, unlike established IAA transporters from the PIN and ABCB families, transport IBA out of the cells. Our findings explore possible novel modes of regulating auxin homeostasis and plant development by means of directional transport of the auxin precursor IBA and presumably also other auxin metabolites.