Missense mutations in ABCG5 and ABCG8 disrupt heterodimerization and trafficking

Mutations in ABCG5 (G5) or ABCG8 (G8) cause sitosterolemia, an autosomal recessive disease characterized by sterol accumulation and premature atherosclerosis. G5 and G8 are ATP-binding cassette (ABC) half-transporters that must heterodimerize to move to the apical surface of cells. We examined the role of N-linked glycans in the formation of the G5/G8 heterodimer to gain insight into the determinants of folding and trafficking of these proteins. Site-directed mutagenesis revealed that two asparagine residues (Asn(585) and Asn(592)) are glycosylated in G5 and that G8 has a single N-linked glycan attached to Asn(619). N-Linked glycosylation of G8 was required for efficient trafficking of the G5/G8 heterodimer, but mutations that abolished glycosylation of G5 did not prevent trafficking of the heterodimer. Both G5 and G8 are bound by the lectin chaperone, calnexin, suggesting that the calnexin cycle may facilitate folding of the G5/G8 heterodimer. To determine the effects of 13 disease-causing missense mutations in G5 and G8 on formation and trafficking of the G5/G8 heterodimer, mutant forms of the half-transporters were expressed in CHO-K1 cells. All 13 mutations reduced trafficking of the G5/G8 heterodimer from the endoplasmic reticulum to the Golgi complex, and most prevented the formation of stable heterodimers between G5 and G8. We conclude that the majority of the molecular defects in G5 and G8 that cause sitosterolemia impair transport of the sterol transporter to the cell surface.     

Structure of the Human Cholesterol Transporter ABCG1

ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport.     

ABCG transporters: structure, substrate specificities and physiological roles

The ATP-binding cassette (ABC) transporter superfamily is one of the largest protein families with representatives in all kingdoms of life. Members of this superfamily are involved in a wide variety of transport processes with substrates ranging from small ions to relatively large polypeptides and polysaccharides. The G subfamily of ABC transporters consists of half-transporters, which oligomerise to form the functional transporter. While ABCG1, ABCG4 and ABCG5/8 are involved in the ATP-dependent translocation of steroids and, possibly, other lipids, ABCG2 (also termed the breast cancer resistance protein) has been identified as a multidrug transporter that confers resistance on tumor cells. Evidence will be summarized suggesting that ABCG2 can also mediate the binding/transport of non-drug substrates, including free and conjugated steroids. The characterization of the substrate specificities of ABCG proteins at a molecular level might provide further clues about their potential physiological role(s), and create new opportunities for the modulation of their activities in relation to human disease.     

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2′,5′-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2′,5′-dihydroxychalcone (2′,5′-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 μm), indicating competitive inhibition. In contrast, 2′,5′-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.     

The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding

Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding.     

半分子转运蛋白ABCG2的肿瘤多药耐药性研究及其应用

肿瘤常对临床上传统使用的多种化学治疗显示其内源性或获得性的药物耐受性即多药耐药性(multidrug resistance,MDR)．这种多药耐药性主要是由一类称为ABC(ATP-binding cassette)转运体蛋白超家族的跨膜蛋白引起的,它们结合并利用水解ATP提供的能量来转运药物,导致肿瘤细胞呈现抗药性．半分子转运蛋白ABCG2是近年来才发现的可归于ABC转运体大家族中的一个新成员,在肠、肝、胎盘和血脑屏障等部位大量表达,与全分子转运蛋白如P-gp (P-glycoprotein)和多药耐药蛋白(multi-drug resistance protein,MRP)相似,可以主动地把具有不同化学结构和作用于细胞内不同靶位点的化疗药物泵出胞外,从而引起肿瘤对多种抗癌药物(包括最新开发的药物)产生抗性．最近的一些十分有趣的研究还表明,ABCG2可能与干细胞分化状态和保护干细胞发育过程中免受周围环境的影响有关,而且还发现,它在侧群骨髓和神经干细胞内大量存在,因此,ABCG2可能在基因治疗中作为选择性的蛋白质标记正受到研究者们的极大关注．同时,ABCG2的单核苷酸多态性影响其结合并转运不同的底物/药物．鉴于ABCG2在肿瘤抗药性研究中的重要性以及它的一些新功能的初步研究表明,对ABCG2的生物学功能和作用机理以及在医学实践中的应用研究必将受到更大的关注．主要阐述了半分子ABC转运蛋白ABCG2的发现、重要的生化特性和生理功能及其相关的新研究进展和问题．     

ABCG2 Transports and Transfers Heme to Albumin through Its Large Extracellular Loop

ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hematopoietic progenitors. Due to its role in drug resistance, its expression has been correlated with a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin, a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX, and much less efficiently with pheophorbide a, but not with vitamin B12. K_d values are in the range 0.5–3.5 μm, with heme displaying the highest affinity. Nonporphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin, and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane translocation, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a K_d of about 3 μm.     

Caveolin-1 interacts with ATP binding cassette transporter G1 (ABCG1) and regulates ABCG1-mediated cholesterol efflux

ATP-binding cassette transporter G1 (ABCG1) plays an important role in macrophage reverse cholesterol transport in vivo by promoting cholesterol efflux onto lipidated apoA-I. However, the underlying mechanism is unclear. Here, we found that ABCG1 co-immunoprecipitated with caveolin-1 (CAV1) but not with flotillin-1 and -2. Knockdown of CAV1 expression using siRNAs significantly reduced ABCG1-mediated cholesterol efflux without detectable effect on ABCA1-mediated cholesterol efflux. Disruption of the putative CAV1 binding site in ABCG1, through replacement of tyrosine residues at positions 487 and 489 or at positions 494 and 495 with alanine (Y487AY489A and Y494AY495A), impaired the interaction of ABCG1 with CAV1 and significantly decreased ABCG1-mediated cholesterol efflux. The substitution of Tyr494 and Tyr495 with Phe or Trp that resulted in an intact CAV1 binding site had no effect. Furthermore, Y494AY495A affected trafficking of ABCG1 to the cell surface. The mutant protein is mainly located intracellularly. Finally, we found that CAV1 co-immunoprecipitated with ABCG1 and regulated cholesterol efflux to reconstituted HDL in THP-1-derived macrophages upon the liver X receptor agonist treatment. These findings indicate that CAV1 interacts with ABCG1 and regulates ABCG1-mediated cholesterol efflux.     

ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cholesterol from cells to high density lipoprotein (HDL) but not to lipid-depleted apolipoprotein A-I. Here we show that human ABCG1 overexpressed in baby hamster kidney cells in the absence of lipoproteins traffics to the plasma membrane and redistributes membrane cholesterol to cell-surface domains accessible to treatment with the enzyme cholesterol oxidase. Cholesterol removed by HDL was largely derived from these domains in ABCG1 transfectants but not in cells lacking ABCG1. Overexpression of ABCG1 also increased cholesterol esterification, which was decreased by the addition of HDL, suggesting that a proportion of the cell-surface cholesterol not removed by HDL is transported to the intracellular esterifying enzyme acyl-CoA:cholesterol acyltransferase. A 638-amino acid ABCG1, which lacked the 40 N-terminal amino acids of the predicted full-length protein, was fully functional and of a similar size to ABCG1 expressed by cholesterol-loaded human monocyte-derived macrophages. Mutating an essential glycine residue in the Walker A motif abolished ABCG1-dependent cholesterol efflux and esterification and prevented localization of ABCG1 to the cell surface, indicating that the ATP binding domain in ABCG1 is essential for both lipid transport activity and protein trafficking. These studies show that ABCG1 redistributes cholesterol to cell-surface domains where it becomes accessible for removal by HDL, consistent with a direct role of ABCG1 in cellular cholesterol transport.     

ABCG2 membrane transporter in mature human erythrocytes is exclusively homodimer

The human ABCG2 protein, a member of ABC transporter family, was shown to transport anti-cancer drugs and normal cell metabolites. Earlier studies have demonstrated the expression of ABCG2 in hematopoietic stem cells and erythroid cells; however little is known about the expression and activity of ABCG2 in mature erythrocytes. In this report, we show that ABCG2 in mature human erythrocytes migrates with an apparent molecular mass of 140 kDa, under reducing conditions, on Fairbanks SDS gel system. In contrast, tumor cells expressing higher levels of ABCG2 show no detectable homodimers, when resolved under identical reducing conditions. Analysis of the same membrane extracts from tumor cells and human erythrocytes on Laemmli SDS gel system, where samples are boiled in the presence of increasing concentrations of disulfide reducing conditions and then analyzed, migrate with an apparent molecular mass of 70 kDa or a monomer. Drug transport studies using Pheophorbide A, a substrate of ABCG2, show the protein to be active in erythrocytes. Furthermore, Fumitremorgin C, a specific inhibitor of ABCG2 increases the accumulation of Pheophorbide A in erythrocytes and drug-resistant cells but not in the parental drug-sensitive cells. Given the ability of ABCG2 to transport protoprophyrin IX or heme, these findings may have implications on the normal function of erythrocytes.     

Characterization of drug transport,ATP hydrolysis,and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation

The overexpression of the human ATP-binding cassette half-transporter, ABCG2 (placenta-specific ABC transporter, mitoxantrone resistance-associated protein, breast cancer resistance protein), causes multidrug resistance in tumor cells. An altered drug resistance profile and substrate recognition were suggested for wild-type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. In order to characterize the different human ABCG2 transporters without possible endogenous dimerization partners, we expressed these proteins and a catalytic center mutant (K86M) in Sf9 insect cells. Transport activity was followed in intact cells, whereas the ATP binding and hydrolytic properties of ABCG2 were studied in isolated cell membranes. We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. In each case, ABCG2-dependent transport was blocked by the specific inhibitor, fumitremorgin C. A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. We found that ABCG2 is capable of a vanadate-dependent adenine nucleotide trapping. Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. These experiments document the applicability of the Sf9 expression system for parallel, quantitative examination of the specific transport and ATP hydrolytic properties of different ABCG2 proteins and demonstrate significant differences in their substrate interactions.     

Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2

ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/C608A), the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter- and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2.     

Oligomerization of the human ABC transporter ABCG2: evaluation of the native protein and chimeric dimers

Human ABCG2, a member of the ATP binding cassette (ABC) transporter superfamily, is overexpressed in numerous multidrug-resistant cells in culture. Localized to the plasma membrane, ABCG2 contains six transmembrane segments and one nucleotide binding domain (NBD) and is thought to function as a dimer or higher order oligomer. Chimeric fusion proteins containing two ABCG2 proteins joined either with or without a flexible linker peptide were expressed at the plasma membrane and maintained drug transport activity. Expression of an ABCG2 variant mutated in a conserved residue in the Walker B motif of the NBD (D210N) resulted in a non-functional protein expressed at the cell surface. Expression of an ABCG2 chimeric dimer containing the D210N mutation in the first ABCG2 resulted in a dominant-negative phenotype, as the protein was expressed at the surface but was not functional. Using a bifunctional photoaffinity nucleotide analogue and a non-membrane-permeable cysteine-specific chemical cross-linking agent, a dimer is the predominant form of oligomerized ABCG2 under our assay conditions. Furthermore, these experiments demonstrated that the dimer interface includes, but may not be limited to, interactions between residues in each monomeric NBD and separate disulfide interactions between the cysteines in the third extracellular loop of each monomer. By changing all three extracellular cysteines to alanine, we showed that although extracellular disulfide bonds may exist between monomers, they are not essential for ABCG2 localization, transport activity, or prazosin-stimulated ATPase activity. Together, these data suggest that ABCG2 functions as a dimer, but do not exclude functional higher order oligomers.     

ABCG transporters and disease

ATP-binding cassette (ABC) transporters form a large family of transmembrane proteins that facilitate the transport of specific substrates across membranes in an ATP-dependent manner. Transported substrates include lipids, lipopolysaccharides, amino acids, peptides, proteins, inorganic ions, sugars and xenobiotics. Despite this broad array of substrates, the physiological substrate of many ABC transporters has remained elusive. ABC transporters are divided into seven subfamilies, A-G, based on sequence similarity and domain organization. Here we review the role of members of the ABCG subfamily in human disease and how the identification of disease genes helped to determine physiological substrates for specific ABC transporters. We focus on the recent discovery of mutations in ABCG2 causing hyperuricemia and gout, which has led to the identification of urate as a physiological substrate for ABCG2.     

Dancing with the sterols: Critical roles for ABCG1, ABCA1, miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis

ATP binding cassette (ABC) transporters represent a large and diverse family of proteins that transport specific substrates across a membrane. The importance of these transporters is illustrated by the finding that inactivating mutations within 17 different family members are known to lead to specific human diseases. Clinical data from humans and/or studies with mice lacking functional transporters indicate that ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 are involved in cholesterol and/or phospholipid transport. This review discusses the multiple mechanisms that control cellular sterol homeostasis, including the roles of microRNAs, nuclear and cell surface receptors and ABC transporters, with particular emphasis on recent findings that have provided insights into the role(s) of ABCG1. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

An ATP-binding cassette gene (ABCG5) from the ABCG (White) gene subfamily maps to human chromosome 2p21 in the region of the Sitosterolemia locus.

We characterized a new human ATP-binding cassette (ABC) transporter gene that is highly expressed in the liver. The gene, ABCG5, contains 13 exons and encodes a 651 amino acid protein. The predicted protein is closely related to the Drosophila white gene and a human gene, ABCG1, which is induced by cholesterol. This subfamily of genes all have a single ATP-binding domain at the N-terminus and a single C-terminal set of transmembrane segments. ABCG5 maps to human chromosome 2p21, between the markers D2S117 and D2S119. The abundant expression of this gene in the liver suggests that the protein product has an important role in transport of specific molecule(s) into or out of this tissue.     

The FLT3 Inhibitor Quizartinib Inhibits ABCG2 at Pharmacologically Relevant Concentrations,with Implications for Both Chemosensitization and Adverse Drug Interactions

The oral second-generation bis-aryl urea fms-like tyrosine kinase 3 (FLT3) inhibitor quizartinib (AC220) has favorable kinase selectivity and pharmacokinetics. It inhibits mutant and wild-type FLT3 in vivo at 0.1 and 0.5 µM, respectively, and has shown favorable activity and tolerability in phase I and II trials in acute myeloid leukemia, with QT prolongation as the dose-limiting toxicity. Co-administration with chemotherapy is planned. We characterized interactions of quizartinib with the ATP-binding cassette (ABC) proteins ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein). Its effects on uptake of fluorescent substrates and apoptosis were measured by flow cytometry, binding to ABCB1 and ABCG2 drug-binding sites by effects on [¹²⁵I]iodoarylazidoprazosin ([¹²⁵I]-IAAP) photolabeling and ATPase activity, and cell viability by the WST-1 colorimetric assay. Quizartinib inhibited transport of fluorescent ABCG2 and ABCB1 substrates in ABCG2- and ABCB1-overexpressing cells in a concentration-dependent manner, from 0.1 to 5 µM and from 0.5 to 10 µM, respectively, and inhibited [¹²⁵I]-IAAP photolabeling of ABCG2 and ABCB1 with IC₅₀ values of 0.07 and 3.3 µM, respectively. Quizartinib at higher concentrations decreased ABCG2, but not ABCB1, ATPase activity. Co-incubation with quizartinib at 0.1 to 1 µM sensitized ABCG2-overexpressing K562/ABCG2 and 8226/MR20 cells to ABCG2 substrate chemotherapy drugs in a concentration-dependent manner in cell viability and apoptosis assays. Additionally, quizartinib increased cellular uptake of the ABCG2 substrate fluoroquinolone antibiotic ciprofloxacin, which also prolongs the QT interval, in a concentration-dependent manner, predicting altered ciprofloxacin pharmacokinetics and pharmacodynamics when co-administered with quizartinib. Thus quizartinib inhibits ABCG2 at pharmacologically relevant concentrations, with implications for both chemosensitization and adverse drug interactions. These interactions should be considered in the design of treatment regimens combining quizartinib and chemotherapy drugs and in choice of concomitant medications to be administered with quizartinib.     

Expression of ATP binding cassette-transporter ABCG1 prevents cell death by transporting cytotoxic 7beta-hydroxycholesterol

Oxysterols result from cholesterol by enzymatic or oxidative processes. Some exert cytotoxic effects leading to necrosis or apoptosis. Detoxification of these compounds mainly occurs in the liver and requires transport from peripheral tissues towards it. Some ATP-binding cassette transporters are involved in export of cytotoxic compounds. In the current study, we investigated whether ABC transporter family member G1 (ABCG1) may be involved in oxysterol transport, since its gene expression is highly responsive to oxysterol loading. TetOff HeLa cells stably expressing ABCG1 showed decreased mass uptake of 7beta-hydroxycholesterol (7beta-HC) whereas that of other physiologically relevant oxysterols was unaffected. Application of 7beta-HC to ABCG1 expressing cells induced hyperpolarization of mitochondrial membrane potential and production of reactive oxygen species, indicating energy consumption by the ATP-binding cassette transporter when it is activated by its correct substrate. Our study points to detoxification as one of potential cellular functions of ABCG1. We assume that ABCG1 protects against 7beta-HC-induced cell death, an important role in prevention of neurodegenerative and cardiovascular disease.     

An ABCG/WBC-type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen

The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.