The leucotriene C4 binding sites in multidrug resistance protein 1 (ABCC1) include the first membrane multiple spanning domain

The multiple drug resistance protein 1 (MRP1 or ABCC1) transports anticancer drugs and normal cell metabolites. Leucotriene C(4) (LTC(4)) is one of the highest affinity substrates of MRP1. In this study, we have synthesized and characterized a novel photoreactive azido analogue of LTC(4) (AALTC(4)). The specificity of AALTC(4) binding to MRP1 was confirmed using an LTC(4)-specific monoclonal antibody. Moreover, binding with radioiodinated [(125)I]AALTC(4) (or IAALTC(4)) to MRP1 was dramatically competed with unmodified LTC(4) and to a lesser degree by glutathione (GSH). Oxidized glutathione (GSSG) slightly increased IAALTC(4) binding to MRP1, while MK571, verapamil, and vincristine inhibited IAALTC(4) binding to MRP1. Using AALTC(4) together with a panel of epitope-specific and LTC(4)-specific monoclonal antibodies, we identified LTC(4) binding sites in MRP1. Western blotting of large tryptic fragments of MRP1 with three well-characterized epitope-specific mAbs (MRPr1, QCRL1, and MRPm6) showed LTC(4) binding in both the N- and C-terminal halves of MRP1. Furthermore, a peptide corresponding to the N-terminal membrane-spanning domain of MRP1 (MSD0) was photoaffinity labeled by AALTC(4), indicating that MSD0 contains an LTC(4) binding site. Higher resolution mapping of additional LTC(4) binding sites was obtained using eight MRP1 variants with each containing hemaglutanin A (HA) epitopes at different sites (at amino acid 4, 163, 271, 574, 653, 938, 1001, or 1222). MRP1 variants were photoaffinity labeled with IAALTC(4) and digested with trypsin to isolate specific regions of MRP1 that interact with LTC(4). These results confirmed that sequences in MSD0 interact with IAALTC(4). Other regions that were photoaffinity labeled by IAALTC(4) include TM 10-11, TM 16-17, and TM 12, shown previously to encode MRP1 drug binding site(s). Together, our results show a high-resolution map of LTC(4) binding domains in MRP1 and provide the first direct evidence for LTC(4) binding within MSD0.     

Equilibrium ligand binding to the human erythrocyte sugar transporter. Evidence for two sugar-binding sites per carrier

Equilibrium [3H]cytochalasin B binding to class I sites of human red cell membranes (the sugar transporter) was examined in the presence and absence of intracellular or extracellular sugars known to interact with the transport system. D-Glucose, a transported sugar, is without effect on cytochalasin B binding when present in the extracellular medium but is an effective inhibitor of binding when present within the cell. Ethylidene glucose and maltose (reactive but nontransported sugars) inhibit cytochalasin B (CCB) binding when present either outside or inside the red cell. Inhibition by intracellular sugar (Si) is of the simple, linear competitive type. Inhibition by extracellular sugars (So) is more complex; the Kd(app) for cytochalasin B binding increases in a saturable fashion with [So]. These observations are compared with the predictions of the one-site, alternating conformer model and the two-site model for substrate binding to the sugar transporter, X. The experimental results are inconsistent with the one-site model but are explained by a two-site model in which the ternary complexes of So . X . Si or So . X . CCBi exist and where the binding sites for So and Si display negative cooperativity when occupied by nontransported substrate and little or no cooperativity when occupied by the transported species, D-glucose.     

Conformational change induced by ATP binding in the multidrug ATP-binding cassette transporter BmrA

ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075-1085], we show here that addition of ATP in the presence of Mg2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters.     

Candida drug resistance protein 1, a major multidrug ATP binding cassette transporter of Candida albicans, translocates fluorescent phospholipids in a reconstituted system

Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that approximately 70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With approximately 6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was approximately 15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function.     

Functional interaction between the two halves of the photoreceptor-specific ATP binding cassette protein ABCR (ABCA4). Evidence for a non-exchangeable ADP in the first nucleotide binding domain

ABCR, also known as ABCA4, is a member of the superfamily of ATP binding cassette transporters that is believed to transport retinal or retinylidene-phosphatidylethanolamine across photoreceptor disk membranes. Mutations in the ABCR gene are responsible for Stargardt macular dystrophy and related retinal dystrophies that cause severe loss in vision. ABCR consists of two tandemly arranged halves each containing a membrane spanning segment followed by a large extracellular/lumen domain, a multi-spanning membrane domain, and a nucleotide binding domain (NBD). To define the role of each NBD, we examined the nucleotide binding and ATPase activities of the N and C halves of ABCR individually and co-expressed in COS-1 cells and derived from trypsin-cleaved ABCR in disk membranes. When disk membranes or membranes from co-transfected cells were photoaffinity labeled with 8-azido-ATP and 8-azido-ADP, only the NBD2 in the C-half bound and trapped the nucleotide. Co-expressed half-molecules displayed basal and retinal-stimulated ATPase activity similar to full-length ABCR. The individually expressed N-half displayed weak 8-azido-ATP labeling and low basal ATPase activity that was not stimulated by retinal, whereas the C-half did not bind ATP and exhibited little if any ATPase activity. Purified ABCR contained one tightly bound ADP, presumably in NBD1. Our results indicate that only NBD2 of ABCR binds and hydrolyzes ATP in the presence or absence of retinal. NBD1, containing a bound ADP, associates with NBD2 to play a crucial, non-catalytic role in ABCR function.     

Nonequivalent nucleotide trapping in the two nucleotide binding folds of the human multidrug resistance protein MRP1

Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadate-induced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[alpha-(32)P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.     

Investigation of ligand binding to the multidrug resistance protein EmrE by isothermal titration calorimetry

Escherichia coli multidrug resistance protein E (EmrE) is an integral membrane protein spanning the inner membrane of Escherichia coli that is responsible for this organism's resistance to a variety of lipophilic cations such as quaternary ammonium compounds (QACs) and interchelating dyes. EmrE is a 12-kDa protein of four transmembrane helices considered to be functional as a multimer. It is an efflux transporter that can bind and transport cytoplasmic QACs into the periplasm using the energy of the proton gradient across the inner membrane. Isothermal titration calorimetry provides information about the stoichiometry and thermodynamic properties of protein-ligand interactions, and can be used to monitor the binding of QACs to EmrE in different membrane mimetic environments. In this study the ligand binding to EmrE solubilized in dodecyl maltoside, sodium dodecyl sulfate and reconstituted into small unilamellar vesicles is examined by isothermal titration calorimetry. The binding stoichiometry of EmrE to drug was found to be 1:1, demonstrating that oligomerization of EmrE is not necessary for binding to drug. The binding of EmrE to drug was observed with the dissociation constant (K(D)) in the micromolar range for each of the drugs in any of the membrane mimetic environments. Thermodynamic properties demonstrated this interaction to be enthalpy-driven with similar enthalpies of 8-12 kcal/mol for each of the drugs in any of the membrane mimetics.     

Evidence for clustered mannose as a new ligand for hyaluronan- binding protein (HABP1) from human fibroblasts

We have earlier reported that overexpression of the gene encoding human hyaluronan-binding protein (HABP1) is functionally active, as it binds specifically with hyaluronan (HA). In this communication, we confirm the collapse of the filamentous and branched structure of HA by interaction with increasing concentrations of recombinant-HABP1 (rHABP1). HA is the reported ligand of rHABP1. Here, we show the affinity of rHABP1 towards D-mannosylated albumin (DMA) by overlay assay and purification using a DMA affinity column. Our data suggests that DMA is another ligand for HABP1. Furthermore, we have observed that DMA inhibits the binding of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity towards HA and DMA which depends on pH and ionic strength. These data suggest that affinity of rHABP1 towards different ligands is regulated by the microenvironment.     

Tissue-type plasminogen activator binding to human endothelial cells. Evidence for two distinct binding sites

The endothelium may contribute to fibrinolysis through the binding of plasminogen activators or plasminogen activator inhibitors to the cell surface. Using a solid-phase radioimmunoassay, we observed that antibodies to recombinant tissue-type plasminogen activator (rt-PA) and plasminogen activator inhibitor type 1 (PAI-1) bound to the surface of cultured human umbilical vein endothelial cells (HUVEC). HUVEC also specifically bound added radiolabeled rt-PA with apparent steady-state binding being reached by 1 h at 4 degrees C. When added at low concentrations (less than 5 nM), rt-PA bound with high affinity mainly via the catalytic site, forming a sodium dodecyl sulfate-stable 105-kDa complex which dissociates from the cell surface over time and which could be immunoprecipitated by a monoclonal antibody to PAI-1. rt-PA bound to this high affinity site retained less than 5% of its expected plasminogen activator activity. At higher concentrations, binding did not require the catalytic site and was rapidly reversible. rt-PA initially bound to this site retained plasminogen activator activity. These studies suggest that tissue-type plasminogen activator and PAI-1 are expressed on the surface of cultured HUVEC. HUVEC also express unoccupied binding sites for exogenous tissue-type plasminogen activator. The balance between the expression of plasminogen activator inhibitors and these unoccupied binding sites for plasminogen activators on the endothelial surface may contribute to the regulation of fibrinolysis.     

Rhodamine 123 binds to multiple sites in the multidrug resistance protein (MRP1)

The mechanisms of MRP1-drug binding and transport are not clear. In this study, we have characterized the interaction between MRP1 and rhodamine 123 (Rh123) using the photoreactive-iodinated analogue, [(125)I]iodoaryl azido-rhodamine 123 (or IAARh123). Photoaffinity labeling of plasma membranes from HeLa cells transfected with MRP1 cDNA (HeLa-MRP1) with IAARh123 shows the photolabeling of a 190 kDa polypeptide not labeled in HeLa cells transfected with the vector alone. Immunoprecipitation of a 190 kDa photolabeled protein with MRP1-sepcific monoclonal antibodies (QCRL-1, MRPr1, and MRPm6) confirmed the identity of this protein as MRP1. Analysis of MRP1-IAARh123 interactions showed that photolabeling of membranes from HeLa-MRP1 with increasing concentrations of IAARh123 was saturable, and was inhibited with excess of IAARh123. Furthermore, the photoaffinity labeling of MRP1 with IAARh123 was greatly reduced in the presence of excess Leukotreine C(4) or MK571, but to a lesser extent with excess doxorubicin, colchicine or chloroquine. Cell growth assays showed 5-fold and 14-fold increase in the IC(50) of HeLa-MRP1 to Rh123 and the Etoposide VP16 relative to HeLa cells, respectively. Analysis of Rh123 fluorescence in HeLa and HeLa-MRP1 cells with or without ATP suggests that cross-resistance to Rh123 is in part due to reduced drug accumulation in the cytosol of HeLa-MRP1 cells. Mild digestion of purified IAARh123-photolabeled MRP1 with trypsin showed two large polypeptides (approximately 111 and approximately 85 kDa) resulting from cleavage in the linker domain (L1) connecting the multiple-spanning domains MSD0 and MSD1 to MSD2. Exhaustive proteolysis of purified IAARh123-labeled 85 and 111 kDa polypeptides revealed one (6 kDa) and two (approximately 6 plus 4 kDa) photolabeled peptides, respectively. Resolution of total tryptic digest of IAARh123-labeled MRP1 by HPLC showed three radiolabeled peaks consistent with the three Staphylococcus aureus V8 cleaved peptides from the Cleveland maps. Together, the results of this study show direct binding of IAARh123 to three sites that localize to the N- and C-domains of MRP1. Moreover, IAARh123 provides a sensitive and specific probe to study MRP1-drug interactions.     

Structure of the human multidrug resistance protein 1 nucleotide binding domain 1 bound to Mg2+/ATP reveals a non-productive catalytic site

Human multidrug resistance protein 1 (MRP1) is a membrane protein that belongs to the ATP-binding cassette (ABC) superfamily of transport proteins. MRP1 contributes to chemotherapy failure by exporting a wide range of anti-cancer drugs when over expressed in the plasma membrane of cells. Here, we report the first high-resolution crystal structure of human MRP1-NBD1. Drug efflux requires energy resulting from hydrolysis of ATP by nucleotide binding domains (NBDs). Contrary to the prokaryotic NBDs, the extremely low intrinsic ATPase activity of isolated MRP1-NBDs allowed us to obtain the structure of wild-type NBD1 in complex with Mg2+/ATP. The structure shows that MRP1-NBD1 adopts a canonical fold, but reveals an unexpected non-productive conformation of the catalytic site, providing an explanation for the low intrinsic ATPase activity of NBD1 and new hypotheses on the cooperativity of ATPase activity between NBD1 and NBD2 upon heterodimer formation.     

Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The drug-binding pocket of P-gp is located in the transmembrane domains. Although occupation of the drug-binding pocket by one molecule is sufficient to activate the ATPase activity of P-gp, the drug-binding pocket may be large enough to accommodate two different substrates at the same time. In this study, we used cysteine-scanning mutagenesis to test whether P-gp could simultaneously interact with the thiol-reactive drug substrate, Tris-(2-maleimidoethyl)amine (TMEA) and a second drug substrate. TMEA is a cross-linker substrate of P-gp that allowed us to test for stimulation of cross-linking by a second substrate such as calcein-acetoxymethyl ester, colchicine, demecolcine, cyclosporin A, rhodamine B, progesterone, and verapamil. We report that verapamil induced TMEA cross-linking of mutant F343C(TM6)/V982C(TM12). By contrast, no cross-linked product was detected in mutants F343C(TM6), V982C(TM12), or F343C(TM6)/V982C(TM12) in the presence of TMEA alone. The verapamil-stimulated ATPase activity of mutant F343C(TM6)/V982C(TM12) in the presence of TMEA decreased with increased cross-linking of the mutant protein. These results show that binding of verapamil must induce changes in the drug-binding pocket (induced-fit mechanism) resulting in exposure of residues F343C(TM6)/V982C(TM12) to TMEA. The results also indicate that the common drug-binding pocket in P-gp is large enough to accommodate both verapamil and TMEA simultaneously and suggests that the substrates must occupy different regions in the common drug-binding pocket.     

Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2).

The human multidrug resistance protein 2 (MRP2, symbol ABCC2) is a polytopic membrane glycoprotein of 1545 amino acids which exports anionic conjugates across the apical membrane of polarized cells. A chimeric protein composed of C-proximal MRP2 and N-proximal MRP1 localized to the apical membrane of polarized Madin-Darby canine kidney cells (MDCKII) indicating involvement of the carboxy-proximal part of human MRP2 in apical sorting. When compared to other MRP family members, MRP2 has a seven-amino-acid extension at its C-terminus with the last three amino acids (TKF) comprising a PDZ-interacting motif. In order to analyze whether this extension is required for apical sorting of MRP2, we generated MRP2 constructs mutated and stepwise truncated at their C-termini. These constructs were fused via their N-termini to green fluorescent protein (GFP) and were transiently transfected into polarized, liver-derived human HepG2 cells. Quantitative analysis showed that full-length GFP-MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained on intracellular membranes in 27% of cells. Removal of the C-terminal TKF peptide and stepwise deletion of up to 11 amino acids did not change this predominant apical distribution. However, apical localization was largely impaired when GFP-MRP2 was C-terminally truncated by 15 or more amino acids. Thus, neither the PDZ-interacting TKF motif nor the full seven-amino-acid extension were necessary for apical sorting of MRP2. Instead, our data indicate that a deletion of at least 15 C-terminal amino acids impairs the localization of MRP2 to the apical membrane of polarized cells.     

Evidence for two nicotinamide binding sites on L-glutamate dehydrogenase

Circular dichroism saturation in the nicotinamide band of NADH, provides direct evidence for the binding of two nicotinamide rings per protomer of L-glutamate dehydrogenase. These two binding sites are titrated by NADH in the presence of both the substrate (L-glutamate) and an allosteric effector (GTP or Zn²⁺) while only one reacts in the absence of the effector. We suggest that the second binding site, not accessible to NADPH, is demasked by a conformational change of the protein induced by the allosteric effector.     

Suppression of cellular transformation by poly (A) binding protein interacting protein 2 (Paip2)

Controlling translation is crucial for the homeostasis of a cell. Its deregulation can facilitate the development and progression of many diseases including cancer. Poly (A) binding protein interacting protein 2 (Paip2) inhibits efficient initiation of translation by impairing formation of the necessary closed loop of mRNA. The over production of Paip2 in the presence of a constitutively active form of hRas(V12) can reduce colony formation in a semi-solid matrix and focus formation on a cell monolayer. The ability of Paip2 to bind to Pabp is required to suppress the transformed phenotype mediated by hRas(V12). These observations indicate that Paip2 is able to function as a tumor suppressor.     

Characterization of the 5'-flanking region of the human multidrug resistance protein 2 (MRP2) gene and its regulation in comparison withthe multidrug resistance protein 3 (MRP3) gene.

The multidrug resistance proteins MRP2 (symbol ABCC2) and MRP3 (symbol ABCC3) are conjugate export pumps expressed in hepatocytes. MRP2 is localized exclusively to the apical membrane and MRP3 to the basolateral membrane. MRP2 mRNA is expressed at a high level under normal conditions, whereas MRP3 mRNA expression is low and increases only when secretion across the apical membrane by MRP2 is impaired. We studied some of the regulatory properties of the two human genes using transient transfection assays with promoter-luciferase constructs in HepG2 cells and cloned fragments of 1229 nucleotides and 1287 nucleotides of the MRP2 and MRP3 5'-flanking regions, respectively. The sequence between nucleotides -517 and -197 was decisive for basal MRP2 expression. Basal promoter activity of MRP3 was only 4% of that measured for MRP2. At submicromolar concentrations, the histone deacetylase inhibitor trichostatin A reduced the MRP2 reporter gene activity and expression of the protein. Disruption of microtubules with nocodazole decreased gene and protein expression of MRP2 and increased MRP3 reporter gene activity. The genotoxic 2-acetylaminofluorene decreased the activity of the human MRP2 reporter gene construct, but increased MRP3 gene activity and enhanced the amounts of mRNA and protein of MRP2 and MRP3. Thus, regulation of the expression of these ATP-dependent conjugate export pumps is not co-ordinate, but in part inverse. The inverse regulation of the two MRP isoforms is consistent with their distinct localization, their different mRNA expression under normal and pathophysiological conditions, and their different directions of substrate transport in polarized cells.     

Further localization of binding sites for thrombin and protein C in human thrombomodulin

To elucidate the binding sites for thrombin and protein C in the six epidermal growth factor (EGF) domains of human thrombomodulin, recombinant mutant proteins were expressed in COS-1 cells. Mutant protein EGF456, which contains the fourth, fifth, and sixth EGF domains from the NH2 terminus of thrombomodulin, showed complete cofactor activity in thrombin-catalyzed protein C activation, as did intact thrombomodulin or elastase-digested thrombomodulin. EGF56, containing the fifth and sixth EGF domains, did not have cofactor activity; but EGF45, containing the fourth and fifth EGF domains, had about one-tenth of the cofactor activity of EGF456. Thrombin binding to attached recombinant thrombomodulin (D123) was inhibited by EGF45 as well as by EGF56. A synthetic peptide (ECPEGYILDDGFICTDIDE), corresponding to Glu-408 to Glu-426 in the fifth EGF domain, inhibited thrombin binding to attached thrombomodulin (D123) with an apparent Ki of 95 microM. At Ca2+ concentrations of 0.25-0.3 mM, intact protein C was maximally activated by thrombin in the presence of EGF45, EGF456, or EGF1-6, which contains the first to sixth EGF domains; but such maximum cofactor activity was not observed when gamma-carboxyglutamic acid-domainless protein C was used. These findings suggest that: 1) thrombin binds to the latter half of the fifth EGF domain; and 2) protein C binds to the fourth EGF domain of thrombomodulin through Ca2+ ions.     

Evidence for two distinct Mg2+ binding sites in G(s alpha) and G(i alpha1) proteins

The function of guanine nucleotide binding (G) proteins is Mg²⁺ dependent with guanine nucleotide exchange requiring higher metal ion concentration than guanosine 5′-triphosphate hydrolysis. It is unclear whether two Mg²⁺ binding sites are present or if one Mg²⁺ binding site exhibits different affinities for the inactive GDP-bound or the active GTP-bound conformations. We used furaptra, a Mg²⁺-specific fluorophore, to investigate Mg²⁺ binding to α subunits in both conformations of the stimulatory (G_sα) and inhibitory (G_iα1) regulators of adenylyl cyclase. Regardless of the conformation or α protein studied, we found that two distinct Mg²⁺ sites were present with dissimilar affinities. With the exception of G_sα in the active conformation, cooperativity between the two Mg²⁺ sites was also observed. Whereas the high affinity Mg²⁺ site corresponds to that observed in published X-ray structures of G proteins, the low affinity Mg²⁺ site may involve coordination to the terminal phosphate of the nucleotide.     

比较两种昆虫病原真菌中结构相似的ATP结合匣转运蛋白的抗氧化力与多药抗性

[目的]比较分析球孢白僵菌(Beauveria bassiana)BbT1和玫烟棒束孢(Isaria fumosorosea)IfT1两种结构相似的ATP结合匣转运蛋白的生物学功能.[方法]基于Bb2860野生株构建BbT1的敲除株和回补株,并将IFT1在BbT1敲除株中异源重组表达,比较各菌株的表型变化.[结果]与野生株、回补株及异源重组株相比,敲除株对20 - 40 mmol/L过氧化氢和2-8 mmoL/L甲萘醌氧化胁迫的抵抗力下降27％ -2.1倍,对多菌灵、伊曲康唑、菌核净、放线菌酮、乙嘧酚和4-硝基喹啉N-氧化物等不同类型化学药物的抗药性下降28％ -4.7倍,对斜纹夜蛾Spodoptera litura二龄幼虫的毒力下降20％左右,而野生株、回补株及异源重组株之间无任何表型的显著差异.[结论]BbT1和IfT1是结构相似且功能一致的转运蛋白,分别是两种生防真菌多药抗性的决定因子之一,因参与抗氧化反应而对毒力有所贡献.