Supplementary figure 1 (S1): Sequence logo depicting the position of cysteines in Cdr1p and their conservation among 22 fungal ABC transporters having the same topology as Cdr1p. NBD1 Residues TM4 ECL3 NBD2 Residues TM 9 T M 10 11 ECL6

Supplementary figure 2 (S2): A E C1056S C1091S A E STRAIN FLC ITR KTC MIC CYH ANI R6G Control 0.5 0.015 0.12 0.25 Cdr1p-GFP 64 4 2 1 32 C1056S C1091S C1106S C1294S C1336S C193S C1106S C1294S B C1336S C193S C1056S C1091S C1106S C1294S C1336S C193S YPD FLC ITR KTC MIC CYH ANI Cdr1p-GFP Contol R6G F Cdr1p-GFP Control C1056S C1091S C1336S C1294S C1106S C193S C D

Supplementary figure 2 (S2) legend: The drug-resistance profiles and functional assays of the mutants in which critical cysteines were replaced with serines. A, B: Microtitre (MIC80 in μg/ml) and spot assays showing the drug-resistance profile of susceptible cysteine mutants of Cdr1p-GFP in which cysteine was changed to serine. The microtitre and spot assays were performed with different drugs as described in Fig. 1. C, D: The R6G efflux and ATPase assays of the drug-sensitive mutant variants of Cdr1p-GFP in which cysteine was changed to serine. The ATPase and R6G efflux assays were performed as described previously [8]. E, F: Confocal imaging and immunoblot of GFP-tagged Cdr1p and its sensitive cysteine-mutant variants in which cysteines were replaced with serine [8]. Bar graphs with error bars (showing standard error of mean) in panel F are the average of percentage values of expression (compared to Cdr1p-GFP which is taken as 100%) from two independent experiments (n=2).

Cdr1p+drug Cysless- Cdr1p-GFP Cysless Cdr1p-GFP +10ng/ml cyclo Cysless Cdr1p-GFP +20ng/ml cyclo Cysless Cdr1p-GFP +30ng/ml cyclo Cysless Cdr1p-GFP +50ng/ml cyclo Cysless Cdr1p-GFP +60ng/ml cyclo Cysless Cdr1p-GFP +40ng/ml cyclo Supplementary figure 3 (S3): Confocal microscopy showing membrane localization of Cdr1p-GFP & Cysless Cdr1p-GFP 8 HOURS 10 HOURS 12 HOURS 14 HOURS 16 HOURS 18 HOURS A. Membrane localisation in presence of sub MIC80 values of cycloheximide B. Membrane localisation of cysless Cdr1p-GFP at different time points of growth as observed by confocal microscopy 4

Supplementary table 1 (ST-1): List of oligonucleotides used in this study Residue No. Location Primer Sequence C193A NBD1 5’ GGGAGACCCGGTGCTGGTGCATCCACATTGTTAAAGACC 3' C325A 5' GGTGCTAATATCCAAGCCTGGGATAATGCCACTAGAGGG 3' C363A 5' CCTTTAATTGCTATTTATCAAGCCTCTCAAGATGCCTATGATTTA 3’ C402A 5' TTTGAAAAGATGGGTTGGAAAGCCCCTCAAAGACAAACTACT3’ C462A 5' GATGAATATTTTGTTGAAGCCGAACGATCCAACACCAGG 3’ C632A TMD1 5' CTATTGGTTAATGGCTATTTGGTGTACATTTG 3' C635A 5' GGTTAATGTGTATTTGGGCTACATTTGTTATGTCCC 3’ C712A 5' CGGTCGTGAATTCCAAGCTGCTCAATATGTTCC 3‘ C732A 5' CGTTCAAATCAAGTGGCTACTGCAGTGGGGTC 3’ C907A NBD2 5' GACTACATTGTTGAATGCTTTATCTGAAAGAGTCAC 3‘ C1041A 5' CTGCCTGGTCGATTGCTAAATTGATGAGAAAG 3' C1056A 5' GGTCAAGCCATTTTGGCTACTATCCATCAACC 3' C1091A 5' GAATTAGGTGAAAACGCTCAAACCATGATC 3' C1106A 5' GGTGCTGATCCAGCTCCTAAGGAAGC 3' C1294A TMD2 5' CCATGGGTCAATTAGCTATGTCTTTCAGTG 3' C1336A 5' GTACCATAGCATTTTTCGCTTGGTATTATCCATTAG 3' C1357A 5' GTTATTTACTATGGCTTTGAATTTCTGTGGTG 3‘ C1361A 5' GTGTTTGAATTTCGCTGGTGTTTTAGCTG 3' C1380A 5' GGATTTTCATGTACAGAGCTAATCCATTCACTTATTTGG 3 C1402A 5' GCTAACACTTTTGTTAAAGCTGCTGAACGTGAATATG 3' C1418A 5' CCACCAAATGGTGAATCTGCTTCTACTTATTTGGATCC 3' C1441A 5' CTAGAAATGATGGATCTGCTGCATTCTGTCAAATGAG 3' C1444A 5’ GGATCTTGTGCATTCGCTCAAATGAGTTCCACC 3' CDR1F 5' GCTACACTAGTATGTCAGATTCTAAGATGTCGTCG 3’ HR2 5'CTGACACTAGTTTAGTGATGGTGATGATGGTGATGCGCTGCTTTCTTATTTTTTTTCTCTCTGTTAC 3’

Supplementary table 2 (ST-2): List of yeast strains used in this study. Description Rrference AD1-8u- PSCDR1-GFP C193A-GFP C325A-GFP C363A-GFP C402A-GFP C462A-GFP C632A-GFP C635A-GFP C712A-GFP C732A-GFP C907A-GFP C1041A-GFP C1056A-GFP C1091A-GFP C1106A-GFP C1294A-GFP C1336A-GFP C1357A-GFP C1361A-GFP C1380A-GFP C1402A-GFP C1418A-GFP C1441A-GFP C1444A-GFP C193S-GFP C1056S-GFP C1091S-GFP C1106S-GFP C1294S-GFP C1336S-GFP Cysless CDR1-GFP Cysless CDR1-His MATa pdr1-3 hisG ura3 Äyor1::hisG Äsnq2::hisG Äpdr10::hisG Äpdr11::hisG Äycf1::hisG Äpdr15::hisG MATa pdr1-3 hisG ura3 Äyor1::hisG Äsnq2::hisG Äpdr10:: PDR5PROM-CDR1-GFP-GFPSTOP Äpdr11::hisG Äycf1::hisG Äpdr15::hisG CDR1-GFP cells carrying C193A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C325A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C363A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C402A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C462A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C632A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C635A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C712A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C732A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C907A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1041A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1056A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1091A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1106A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1294A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1336A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1357A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1361A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1380A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1402A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1418A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1441A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1444A mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C193S mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1056S mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1091S mutation in CDR1 ORF and integrated atPDR5 locus CDR1-GFP cells carrying C1106S mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1294S mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells carrying C1336S mutation in CDR1 ORF and integrated at PDR5 locus CDR1-GFP cells with all native cysteines converted to alanine except C193, C1056, C1091, C1106, C1294, C1336 which are converted to serine integrated at PDR5 locus CDR1-His cells with all native cysteines converted to alanine except C193, C1056, C1091, C1106, C1294, C1336 which are converted to serine integrated at PDR5 locus [5] [8] This Study