PLEIOTROPIC DRUG TRANSPORTERS in Fungi Andr Goffeau, Bruxelles, - PowerPoint PPT Presentation

PLEIOTROPIC DRUG TRANSPORTERS in Fungi André Goffeau, Bruxelles, 2012

PDR RANK & BECH-HANSEN MGG 1973

PDR1 BALZI, CHEN ULASZEWSKI, BOUTRY, DI PIETRO JBC 1987

PDR5 GOLIN 1992

PDR5 BALZI et al , JBC 1994

ASYMETRIC NBD’s in Pdr5p Linker region Walker A Walker B GNSGCGKST SGGQKQRIAIA LLLDE NBD2 NBD1 GRPGSGCTT SGGERKRVSIA QCWDN * * * *** *** ** *

The 16 "full - The 16 "full - size" ABC transporters size" ABC transporters PDR5 - PDR5 - - - like like PDR5 PDR5 like like MRP - - like MRP like MRP - - like MRP like Pdr12 Pdr12 Pdr10 Yor1 Ykr103/4 Pdr10 Yor1 Pdr10 Pdr10 Snq2 Snq2 Yor1 Ykr103/4 Snq2 Snq2 Yor1 Pdr15 Pdr15 Ynr070 Ynr070 Pdr15 Pdr15 Pdr5 Yhl035 Yhl035 Pdr5 Yhl035 Yhl035 Pdr5 Pdr5 Pdr11 Pdr11 Pdr11 Pdr11 Bat1 Bat1 Yor011 Yor011 Bpt1 Bpt1 Ycf1 Ycf1 Ycf1 Ycf1 Yol075 Yol075 MDR MDR - - - like like NEW NEW MDR MDR - like like NEW NEW (TM-NBF)2 (TM-NBF)2 (NBF-TM)2 (NBF-TM)2 Ste6 Ste6 Ste6 MULTIPLE disruptions MULTIPLE disruptions MULTIPLE disruptions SINGLE disruptions SINGLE disruptions NO disruptions yet

ATP ATP PDR-like: Pdr5p ATP ATP MRP/MDR-like: Yor1p

pdr1-3 CARVAJAL et al MGG 1997

The pdr1- -3 3 mutant mutant overexpresses overexpresses The pdr1 a new protein band in the plasma membrane a new protein band in the plasma membrane 160kDa Pma Decottignies et al , 1997

GENETIC PURIFICATION ON GENETIC PURIFICATI Snq2p Pdr5p Yor1p Snq2p Pdr5p Yor1p 160 kDa Pma1p Δ pdr5 Δ snq2 Δ pdr5, Δ snq2 Decottignies et al., 1997

The mutant pdr1 pdr1- -3 3 in the regulatory regulatory gene The mutant in the gene PDR 1 1 activates activates expression of PDR expression of several ABC transporters transporters several ABC PDR 1 PDR 5 SNQ 2 YOR 1 Others ?

Elements of the Elements of the PDR Network PDR Network responsive responsive to the pdr1 pdr1- -3 3 to the Transcription Transcription Regulator Regulator > De RISI et al, > De RISI et al, 2002 2002

GENE TARGETS OVEREXPRESSED GENE TARGETS OVEREXPRESSED BY THE pdr1- -3 DRUG 3 DRUG- -RESISTANCE REGULATORY MUTATION RESISTANCE REGULATORY MUTATION BY THE pdr1 1. ABC PDR5 / SNQ2 / YOR1 / PDR10/ PDR15 2. MFS TPO1 / RTA1 / YOR049c 3. LIPIDS PDR16 / IPT1 / YLR099c 4. WALLS YPL088w / YGL028c / YLR413w / YNR067c GRE2 / MET17 / FRE4 / REV1 / YGR243 w 5. STRESS YLR346c / YGR035c / COS1 6. UNKNOWN De Risi, van den Hazel et al., 1999

PDR3/YAP1/YRR1 • JULIUS SUBIC • CLAUDE JACQ • BERNARD TURCOTTE

PDR Network C. Jacq - F. Devaux – E.N.S

TRANSPORT SPECIFICITY of Pdr5p Kolaszkowski et al JBC 1996

THE YEAST ABC TRANSPORTER PDR5 MEDIATES RESISTANCE TO MANY DIFFERENT COMPOUNDS Steroids and their Herbicides Fluorescent dyes Anticancer drugs Fungicides sulfomethuron methyl structural rhodamine 6G chemosensitizers itraconazole fluometuron ketoconazole rhodamine 123 analogues tamoxifen chloroxuron econazole daunorubicin 5 β 3 α Detergents difenoxuron androstan ol 17 one miconazole doxorubicin 5 β 3 β chlorbromuron androstan ol 17 one triadimenol tetradecyltrimethyl- trifluoperazine 5 β 17 β diuron androstan ol 3 one bitertanol -ammonium bromide 5 α androstane 3,17 dione nuarimol Antibiotics hexadecyltrimethyl- 5 β androstane 3,17 dione Ionophores fenarimol -ammoniumbromide chloramphenicol 1,4 androstadiene 3,17 dione carbendazim zwittergent 3-10 valinomycin tetracycline 5 β pregnan 3 β ol 20 one diclobutrazol zwittergent 3-12 nigericin anisomycin 20 α ol 4 pregnen 3 one phenapronil zwittergent 3-14 monensin antimycin A1 trans dehydroandrosterone chlorothalonil brij 58 A23187 cycloheximide 4 androstene 3,17 dione cyprodinil Others brij 35 lincomycin epiandrosterone acetate dodine n octylglucoside oligomycin 2,3,5 triphenyltetrazoliumchloride epiandrosterone imazalil n dodecylglucoside tunicamycin 4 nitroquinoline n oxide estriol 16, 17 diacetate prochloraz n dodecylmaltoside thiolutin 8 hydroxyquinoline 17 α ethynyl estradiol azoxystrobin diphenylamine deoxycorticosterone flusilazol p aminodiphenylamine pregnenolone Δ PDR5 PDR5 tebuconazole n decylamine progesterone CGA64251 4 nitrophenol testosterone 2 mercapto- aniline diethylstilbestrol -benzothiazole anthron hexestrol krezoxim methyl tetranitrotetrazoliumchloride blue 4 hydroxytamoxifen nystatin resazurine soraphen A sporidesmin epoxiconazole staurosporine fluphenazine cerulenin Kolaczkowski et al., 1997 compactin

Membranes with overexpressed overexpressed Pdr5p Membranes with Pdr5p show ATP- -dependent dependent show ATP fluorescence quenching of Rhodamine Rhodamine 6G fluorescence quenching of 6G GWENAELLE CONSEIL,2003 GWENAELLE CONSEIL,2003 ATP PDR5 deleted fluorescence intensity 8 PDR5 overexpressed 6 4 oligomycin 2 10 20 30 time [min] Kolaczkowski et al., 1997

K i (chrysin) = 2.7 µM Competitive inhibition of Competitive inhibition of prenyl- -flavonoids flavonoids prenyl K i (galangin) = 1.1 µM on rhodamine 6G on rhodamine 6G interaction interaction K i (6,6-DMA-galangin) = 0.18 µM

Chrysin derivatives Partial and non competitive competitive Partial and non inhibition of the nucleotide nucleotide inhibition of the K i (6-DMA-galangin = 5.4 µM hydrolytic activity of hydrolytic activity of Pdr5p- -enriched enriched yeast Pdr5p yeast plasma membranes plasma membranes K i (6-DMA-galangin = 62 µM

HETEROLOGOUS EXPRESSION OF MEMBRANE PROTEINS A.DECOTTIGNIES/B.MONK

MULTIPLE DELETIONS OF YEAST MULTIPLE DELETIONS OF YEAST ABC TRANSPORTERS AND REGULATORS ABC TRANSPORTERS AND REGULATORS pdr1-3 Δ YOR1 AD1 AD1 Δ SNQ2 AD12 AD12 Δ PDR5 AD123 AD123 Δ PDR10 AD1234 AD1234 Δ YCF1 AD12346 AD12346 Δ PDR11 AD123456 AD123456 Δ PDR3 AD1234567 AD1234567 Δ PDR15 AD12345678 AD12345678 Δ PDR1 AD123456789 AD123456789

HYPERSENSITIVITY TO DRUGS OF THE YEAST STRAIN AD1- HYPERSENSITIVITY TO DRUGS OF THE YEAST STRAIN AD1 -9, 9, DELETED OF MULTIPLE ABC TRANSPORTERS DELETED OF MULTIPLE ABC TRANSPORTERS INHIBITOR INHIBITOR WILD WILD- -TYPE TYPE AD1- -9 9 Fold- AD1 g/ml) Difference MIC (µ MIC ( µg/ml) MIC ( g/ml) MIC (µ µg/ml) Brefeldin A 100 6.25 16 Clotri mazole 1.56 0.0012 128 128 Crystal Violet 0.073 0.049 1.5 Cycloheximide 0.049 0.012 4.1 Fluconazole 12.5 0.391 32 Fluphenazine 12.5 6.25 2 8-Hydroxyquinoline 25 6.25 4 Imazalil 0.78 0.025 31 Ketokonazole 0.781 0.006 130 130 Miconazole 0.391 0.0031 126 126 Nocodazole 6.25 0.781 8 Propiconazole 1.56 0.003 5.2 Rhodamine 6G 25 0.098 255 255 Sanguinarine 3.13 0.781 4 Staurosporine 0.781 0.024 33 Sulfometuron methyl 6.25 1.56 4 Thiabendazole >100 14 7.2

STRAINS made by Anabelle Decottignies and Elvira Carvajal STRAINS curated by Stan Ulaszewski and Michel Ghislain michel.ghislain@uclouvain.bee

YEAST PDR AS A SYSTEM FOR YEAST PDR AS A SYSTEM FOR OVEREXPRESSION OF MEMBRANE PROTEINS OVEREXPRESSION OF MEMBRANE PROTEINS -Regulatory pdr1-3 pdr1 mutant -Membrane protein Yor1p gene under PDR5p YOR1 PDR5 promoter Pma1 -Host deleted of major contaminant ABC transporter genes ( PDR5, SNQ2, YOR1, PDR11 ) Decottignies et al., 1997

OVERPRODUCTION OF YOR1p OVERPRODUCTION OF YOR1p pdr1-3 pdr1-3 Δ ABC YOR1 PDR5 pr::YOR1 Yor1p Pma1p OLIGOMYCIN 0.1 4 30 µg/ml RESISTANCE Decottignies et al., 1997

MEMBRANE TRANSPORTERS OVEREXPRESSED IN S.CEREVISIAE ABC CaCDR1p • CaCDR2p • CgCDR1p • CgCDR2p • • ABCA1 Ben R p MFS •

SUPER CDR1 and Super PDR5 SUPER CDR1 and Super PDR5 in S.cerevisiae S.cerevisiae plasma membranes in plasma membranes SUPER PDR5 SUPER PDR5 SUPER CDR1 SUPER CDR1 MULTIDELETIONS MULTIDELETIONS B.MONK 2001

EM structural analysis of the S.cerevisiae Pdr5p MARCO/RIGAUD

PURIFIED Pdr5p PARTICLES

Pdr5p Particles Projection 50 Å

Dimeric Pdr5p A B A B B A A B

ATP binding requires two « head to tail » subunits subunit 1 B B C C A A Walker motifs A A C C B B subunit 2

Pgp structure, Chang et al ,2009

PHYLOGENY

NCU08056.2 NCU10009.2 Neurospora KLLA004PDR CAGL019PDR SNQ2 NCU09830.2 YALI024PDR PDR12 DEHA001PDR SCPO006YNR ASGO001PDR KLLA010PDR S Pombe YNR070W CAGL011PDR SCPO012YNR SNQ2 YALI005PDR PDR15 CAGL009PDR PDR5 YALI019PDR CAGL017PDR YALI007PDR PDR10 YALI009PDR KLLA017PDR NCU05591.2 DEHA025PDR DEHA013PDR KLLA011PDR DEHA024PDR DEHA019PDR ASGO002PDR Schizo pombe DEHA018PDR ASGO012PDR Neurospora crassa DEHA017PDR Yarrowia lipolytica 0.1 DEHA007PDR Debaryomyces hansenii Ashbya gossypii PDR5 Kluyveromyces lactis Candida glabrata Saccharomyces cerevisiae