Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae.

Multidrug resistance (MDR) to different cytotoxic compounds in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 (Sts1, Ydr1, or Lem1) ATP-binding cassette (ABC) multidrug transporter. We have raised polyclonal antibodies recognizing the yeast Pdr5 ABC transporter to study its biogenesis and to analyze the molecular mechanisms underlying MDR development. Subcellular fractionation and indirect immunofluorescence experiments showed that Pdr5 is localized in the plasma membrane. In addition, pulse-chase radiolabeling of cells and immunoprecipitation indicated that Pdr5 is a short-lived membrane protein with a half-life of about 60 to 90 min. A dramatic metabolic stabilization of Pdr5 was observed in delta pep4 mutant cells defective in vacuolar proteinases, and indirect immunofluorescence showed that Pdr5 accumulates in vacuoles of stationary-phase delta pep4 mutant cells, demonstrating that Pdr5 turnover requires vacuolar proteolysis. However, Pdr5 turnover does not require a functional proteasome, since the half-life of Pdr5 was unaffected in either pre1-1 or pre1-1 pre2-1 mutants defective in the multicatalytic cytoplasmic proteasome that is essential for cytoplasmic protein degradation. Immunofluorescence analysis revealed that vacuolar delivery of Pdr5 is blocked in conditional end4 endocytosis mutants at the restrictive temperature, showing that endocytosis delivers Pdr5 from the plasma membrane to the vacuole.     

The multidrug transporter Pdr5: a molecular diode?

A subset of the family of ATP-binding cassette (ABC) transporters has been in focus owing to their involvement in conferring multidrug resistance in cancer cells and among immune compromised individuals. Saccharomyces cerevisiae is protected against xenobiotics by similar machineries that are part of the pleitropic drug resistance (PDR) network. The ABC transporter Pdr5 is an important member of this PDR network in yeast and is involved in cellular detoxification by the efflux of a wide variety of drugs and substrates. In this review, we focus on the aspects of detergent effects and the degeneracy in conserved sequences that is observed in the nucleotide binding domains of Pdr5 and discuss their functional relevance.     

The yeast Pdr15p ATP-binding cassette (ABC) protein is a general stress response factor implicated in cellular detoxification

ATP-binding cassette (ABC) transporters play important roles in drug efflux, but some may also function in cellular detoxification. The Pdr15p ABC protein is the closest homologue of the multidrug efflux transporter Pdr5p, which mediates pleiotropic drug resistance to hundreds of unrelated compounds. In this study, we show that the plasma membrane protein Pdr15p displays limited drug transport capacity, mediating chloramphenicol and detergent tolerance. Interestingly, Pdr15p becomes most abundant when cells exit the exponential growth phase, whereas its closest homologue, Pdr5p, disappears after exponential growth. Furthermore, in contrast to Pdr5p, Pdr15p is strongly induced by various stress conditions including heat shock, low pH, weak acids, or high osmolarity. PDR15 induction bypasses the Pdr1p/Pdr3p regulators but requires the general stress regulator Msn2p, which directly decorates the stress response elements in the PDR15 promoter. Remarkably, however, Pdr15p induction bypasses upstream components of the high osmolarity glycerol (HOG) pathway including the Hog1p and Pbs2p kinases as well as the dedicated HOG cell surface sensors. Our data provide evidence for a novel upstream branch of the general stress response pathway activating Msn2p. In addition, the results demonstrate a cross-talk between stress response and the pleiotropic drug resistance network.     

Three-dimensional structure by cryo-electron microscopy of YvcC, an homodimeric ATP-binding cassette transporter from Bacillus subtilis

YvcC, a multidrug transporter from Bacillus subtilis, is a member of the ATP-binding cassette superfamily, highly homologous to each half of human multidrug-resistance P-glycoprotein and to several other bacterial half-ABC transporters. Here, the purified recombinant histidine-tagged YvcC has been reconstituted into a lipid bilayer. Controlled and partial detergent removal from YvcC-lipid micelles allowed the production of particularly interesting lipid-detergent-YvcC ring-shaped particles, about 40 nm in diameter, well suited for single particle analysis by cryo-electron microscopy. Furthermore, binding of these histidine-tagged ring-shaped particles to lipid layers functionalized with a Ni(2+)-chelating head group generated a preferential perpendicular orientation, eliminating the missing cone in the final three-dimensional reconstruction. From such analysis, a computed volume has been determined to 2.5 nm resolution giving a detailed insight into the structural organization of this half-ABC transporter within a membrane. The repetitive unit in the ring-shaped particles is consistent with a homodimeric organization of YvcC. Each subunit was composed of three domains: a 5 nm height transmembrane region, a stalk of about 4 nm in height and 2 nm in diameter, and a cytoplasmic lobe of about 5-6 nm in diameter. The latest domain, which fitted with the reported X-ray structure of HisP, was identified as the nucleotide-binding domain (NBD). The 3D reconstruction of the YvcC homodimer well compared with the very recent X-ray crystallographic data on the MsbA homodimer from Escherichia coli, supporting the existence of a central open chamber between the two subunits constituting the homodimer. In addition, the 3D reconstruction of YvcC embedded in a membrane revealed an asymmetric organization of the two NBDs sites within the homodimer, as well as a dimeric interaction between two homodimers.     

The Nucleotide-Free State of the Multidrug Resistance ABC Transporter LmrA: Sulfhydryl Cross-Linking Supports a Constant Contact,Head-to-Tail Configuration of the Nucleotide-Binding Domains

ABC transporters are integral membrane pumps that are responsible for the import or export of a diverse range of molecules across cell membranes. ABC transporters have been implicated in many phenomena of medical importance, including cystic fibrosis and multidrug resistance in humans. The molecular architecture of ABC transporters comprises two transmembrane domains and two ATP-binding cassettes, or nucleotide-binding domains (NBDs), which are highly conserved and contain motifs that are crucial to ATP binding and hydrolysis. Despite the improved clarity of recent structural, biophysical, and biochemical data, the seemingly simple process of ATP binding and hydrolysis remains controversial, with a major unresolved issue being whether the NBD protomers separate during the catalytic cycle. Here chemical cross-linking data is presented for the bacterial ABC multidrug resistance (MDR) transporter LmrA. These indicate that in the absence of nucleotide or substrate, the NBDs come into contact to a significant extent, even at 4°C, where ATPase activity is abrogated. The data are clearly not in accord with an inward-closed conformation akin to that observed in a crystal structure of V. cholerae MsbA. Rather, they suggest a head-to-tail configuration ‘sandwich’ dimer similar to that observed in crystal structures of nucleotide-bound ABC NBDs. We argue the data are more readily reconciled with the notion that the NBDs are in proximity while undergoing intra-domain motions, than with an NBD ‘Switch’ mechanism in which the NBD monomers separate in between ATP hydrolysis cycles.     

Toward understanding the mechanism of action of the yeast multidrug resistance transporter Pdr5p: a molecular modeling study

Pleotropic drug resistant protein 5 (Pdr5p) is a plasma membrane ATP-binding cassette (ABC) transporter and the major drug efflux pump in Saccharomyces cerevisiae. The Pdr5p family of fungal transporters possesses a number of structural features significantly different from other modeled or crystallized ABC transporters, which include a reverse topology, an atypical ATP-binding site, a very low sequence similarity in the transmembrane section and long linkers between domains. These features present a considerable hurdle in molecular modeling studies of these important transporters. Here, we report the creation of an atomic model of Pdr5p based on a combination of homology modeling and ab initio methods, incorporating information from consensus transmembrane segment prediction, residue lipophilicity, and sequence entropy. Reported mutations in the transmembrane substrate-binding pocket that altered drug-resistance were used to validate the model, and one mutation that changed the communication pattern between transmembrane and nucleotide-binding domains was used in model improvement. The predictive power of the model was demonstrated experimentally by the increased sensitivity of yeast mutants to clotrimazole having alanine substitutions for Thr1213 and Gln1253, which are predicted to be in the substrate-binding pocket, without reducing the amount of Pdr5p in the plasma membrane. The quality and reliability of our model are discussed in the context of various approaches used for modeling different parts of the structure.     

A mutation in intracellular loop 4 affects the drug-efflux activity of the yeast multidrug resistance ABC transporter Pdr5p

Multidrug resistance protein Pdr5p is a yeast ATP-binding cassette (ABC) transporter in the plasma membrane. It confers multidrug resistance by active efflux of intracellular drugs. However, the highly polymorphic Pdr5p from clinical strain YJM789 loses its ability to expel azole and cyclohexmide. To investigate the role of amino acid changes in this functional change, PDR5 chimeras were constructed by segmental replacement of homologous BY4741 PDR5 fragments. Functions of PDR5 chimeras were evaluated by fluconazole and cycloheximide resistance assays. Their expression, ATPase activity, and efflux efficiency for other substrates were also analyzed. Using multiple lines of evidence, we show that an alanine-to-methionine mutation at position 1352 located in the predicted short intracellular loop 4 significantly contributes to the observed transport deficiency. The degree of impairment is likely correlated to the size of the mutant residue.     

The transmembrane domain 10 of the yeast Pdr5p ABC antifungal efflux pump determines both substrate specificity and inhibitor susceptibility

We have previously shown that a S1360F mutation in transmembrane domain 10 (TMD10) of the Pdr5p ABC transporter modulates substrate specificity and simultaneously leads to a loss of FK506 inhibition. In this study, we have constructed and characterized the S1360F/A/T and T1364F/A/S mutations located in the hydrophilic face of the amphipatic Pdr5p TMD10. A T1364F mutation leads to a reduction in Pdr5p-mediated azole and rhodamine 6G resistance. Like S1360F, the T1364F and T1364A mutants were nearly non-responsive to FK506 inhibition. Most remarkably, however, the S1360A mutation increases FK506 inhibitor susceptibility, because Pdr5p-S1360A is hypersensitive to FK506 inhibition when compared with either wild-type Pdr5p or the non-responsive S1360F variant. Hence, the Pdr5p TMD10 determines both azole substrate specificity and susceptibility to reversal agents. This is the first demonstration of a eukaryotic ABC transporter where a single residue change causes either a loss or a gain in inhibitor susceptibility, depending on the nature of the mutational change. These results have important implications for the design of efficient reversal agents that could be used to overcome multidrug resistance mediated by ABC transporter overexpression.     

Complete inhibition of the Pdr5p multidrug efflux pump ATPase activity by its transport substrate clotrimazole suggests that GTP as well as ATP may be used as an energy source

The yeast Pdr5p transporter is a 160 kDa protein that effluxes a large variety of xenobiotic compounds. In this study, we characterize its ATPase activity and demonstrate that it has biochemical features reminiscent of those of other ATP-binding cassette multidrug transporters: a relatively high Km for ATP (1.9 mM), inhibition by orthovanadate, and the ability to specifically bind an azidoATP analogue at the nucleotide-binding domains. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is also known to be a potent transport substrate. Our results indicate, however, that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. Curiously, Pdr5p-mediated transport of clotrimazole continues at intracellular concentrations of substrate that should eliminate all ATPase activity. Significantly, however, we observed that the Pdr5p has GTPase and UTPase activities that are relatively resistant to clotrimazole. Furthermore, the Km(GTPase) roughly matches the intracellular concentrations of the nucleotide reported for yeast. Using purified plasma membrane vesicles, we demonstrate that Pdr5p can use GTP to fuel substrate transport. We propose that Pdr5p increases its multidrug transport substrate specificity by using more than one nucleotide as an energy source.     

ABC转运蛋白结构及在植物病原真菌中的功能研究进展

ABC (ATP-binding cassette)转运蛋白是最大的膜转运蛋白超家族之一,其主要功能是利用ATP水解产生的能量将底物进行逆浓度梯度运输.所有生物体都含有大量ABC蛋白. ABC蛋白位于细胞的不同空间,如细胞膜、液泡、线粒体和过氧化物酶体.通常,ABC转运蛋白由跨膜结构域(TMD)和核苷酸结合结构域(NBD)组成,分别与底物和ATP结合.NBD执行与ATP结合和水解,是ABC转运蛋白的动力引擎,TMD识别特异性配体.大多数ABC转运蛋白最初是通过研究生物体耐药性而被发现的,包括多效耐药(PDR)和多药耐药(MDR).本文对ABC转运蛋白的结构及作用机制,以及植物病原真菌中ABC转运蛋白功能的研究进展进行综述.     

The role of hydrogen bond acceptor groups in the interaction of substrates with Pdr5p, a major yeast drug transporter

The yeast ABC (ATP-binding cassette protein) multidrug transporter Pdr5p transports a broad spectrum of xenobiotic compounds, including antifungal and antitumor agents. Previously, we demonstrated that substrate size is an important factor in substrate-transporter interaction and that Pdr5p has at least three substrate-binding sites. In this study, we use a combination of whole cell transport assays and photoaffinity labeling of Pdr5p with [(125)I]iodoarylazidoprazosin in purified plasma membrane vesicles to study the behavior of two series of novel substrates: trityl (triphenylmethyl) and carbazole derivatives. The results indicate that site 2, defined initially by tritylimidazole efflux, requires at least a single hydrogen bond acceptor group (electron pair donor). In contrast, complete inhibition of rhodamine 6G efflux and [(125)I]iodoarylazidoprazosin binding at site 1 requires substrates with three electronegative groups. Carbazole and trityl substrates with two groups show saturating, incomplete inhibition at this site. This type of inhibition is frequently observed in bacterial multidrug-binding proteins that use a pocket with multiple binding sites. The presence of multiple sites with different requirements for substrate-Pdr5p interaction may explain the broad specificity of xenobiotic compounds transported by this protein.     

Protein kinase C effectors bind to multidrug ABC transporters and inhibit their activity

P-Glycoprotein and homologous multidrug transporters contain a phosphorylatable linker sequence that was proposed to control drug efflux on the basis that it was indeed phosphorylated in vitro and in vivo, and that inhibitors of protein kinase C (PKC) inhibited both P-glycoprotein phosphorylation and activity. However, site-directed mutagenesis of all phosphorylatable residues did not alter the drug resistance. The present work shows that PKC effectors are able to bind directly to multidrug transporters, from either cancer cells (mouse P-glycoprotein), yeast (Saccharomyces cerevisiae Pdr5p), or protozoan parasite (Leishmania tropica ltmdr1), and to inhibit their energy-dependent drug-efflux activity. The binding of staurosporine and derivatives such as CGP 41251 is prevented by preincubation with ATP, suggesting at least partial interaction at the ATP-binding site. In contrast, more hydrophobic compounds such as calphostin C and CGP 42700 bind outside the ATP-binding site and strongly interfere with drug interaction. A direct correlation is obtained between the efficiencies of PKC effectors to inhibit energy-dependent interaction of rhodamine 6G with yeast Pdr5p, to promote intracellular drug accumulation in various multidrug resistant cells, and to chemosensitize growth of resistant cells. The noncompetitive inhibition by PKC effectors of rhodamine 6G interaction with Pdr5p suggests that the binding might interfere with signal transduction between nucleotide hydrolysis and drug interaction. The overall results indicate that the multidrug transporters from different species display common features for interaction with PKC inhibitors. The hydrophobic derivative of staurosporine, CGP 42700, constitutes a potentially powerful modulator of P-glycoprotein-mediated multidrug resistance.     

Mutations define cross-talk between the N-terminal nucleotide-binding domain and transmembrane helix-2 of the yeast multidrug transporter Pdr5: possible conservation of a signaling interface for coupling ATP hydrolysis to drug transport

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable K(m)((ATP)) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport.     

The structure of the multidrug resistance protein 1 (MRP1/ABCC1). crystallization and single-particle analysis

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette (ABC) polytopic membrane transporter of considerable clinical importance that confers multidrug resistance on tumor cells by reducing drug accumulation by active efflux. MRP1 is also an efficient transporter of conjugated organic anions. Like other ABC proteins, including the drug resistance conferring 170-kDa P-glycoprotein (ABCB1), the 190-kDa MRP1 has a core structure consisting of two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD). However, unlike P-glycoprotein and most other ABC superfamily members, MRP1 contains a third MSD with five predicted transmembrane segments with an extracytosolic NH(2) terminus. Moreover, the two nucleotide-binding domains of MRP1 are considerably more divergent than those of P-glycoprotein. In the present study, the first structural details of MRP1 purified from drug-resistant lung cancer cells have been obtained by electron microscopy of negatively stained single particles and two-dimensional crystals formed after reconstitution of purified protein with lipids. The crystals display p2 symmetry with a single dimer of MRP1 in the unit cell. The overall dimensions of the MRP1 monomer are approximately 80 x 100 A. The MRP1 monomer shows some pseudo-2-fold symmetry in projection, and in some orientations of the detergent-solubilized particles, displays a stain filled depression (putative pore) appearing toward the center of the molecule, presumably to enable transport of substrates. These data represent the first structural information of this transporter to approximately 22-A resolution and provide direct structural evidence for a dimeric association of the transporter in a reconstituted lipid bilayer.