FEMS Yeast Research Advance Access published June 5, 2015

ABC Transporter Cdr1p Harbors Charged Residues in the Intracellular

Loop and Nucleotide-Binding Domain Critical for Protein Trafficking

and Drug Resistance.

Abdul Haseeb Shah, Atanu Banerjee, Manpreet Kaur Rawal, Ajay Kumar

Saxena, Alok Kumar Mondal and Rajendra Prasad*,

School of Life Sciences, Jawaharlal Nehru University, New Delhi

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Key Words: ABC transporter, Cdr1p, multidrug resistance, Candida albicans,

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nucleotide binding domains, intracellular loops

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Running Title: Role of ICL-NBD interaction in Cdr1p trafficking

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To whom correspondence should be addressed: Rajendra Prasad,

Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru
University, New Delhi 110067, India. Tel.: 91-11-26704509; Fax: 91-1126741081; E-mail: rp47jnu@gmail.com.
*

Present Address: Amity University Haryana, Amity Education Valley,

Gurgaon 122413, India
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Abstract

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The ABC transporter Cdr1 protein of Candida albicans, which plays a major

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role in antifungal resistance, has two transmembrane domains (TMDs) and

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two nucleotide binding domains (NBDs). The twelve transmembrane helices

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of TMDs that are interconnected by extracellular and intracellular loops

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(ICLs) mainly harbor substrate recognition sites where drug binds while

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cytoplasmic NBDs hydrolyze ATP which power drug efflux. The coupling of

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ATP hydrolysis to drug transport requires proper communication between

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NBDs and TMDs which is expected to be accomplished by ICLs. This study

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examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the

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critical residues on the basis of their inter-atomic distances. Among nine

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pairs that fall within a proximity of <4 , an ion pair between K577 of ICL1

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and E315 of NBD1 was found to be critical. The substitution, swapping and

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changing the length or charge of K577 or E315 by directed mutagenesis led

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to a misfolded, non-rescuable protein entrapped in intracellular structures.

32

Furthermore, the equipositional ionic pair forming residues from ICL3 and

33

NBD2 (R1260 and E1014) did not impact protein trafficking. These results

34

point to a new role for ICL/NBD interacting residues in PDR ABC

35

transporters in protein folding and trafficking.

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Introduction

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The transporters belonging to the ATP Binding Cassette (ABC) superfamily

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translocate a diverse spectrum of solutes across membranes driven by ATP

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binding and hydrolysis and are involved in a wide variety of physiological

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processes (Jones et al., 2009; Jones and George 2013). Apart from their

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physiological roles, ABC proteins also impact human diseases (Dawson and

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Locher 2006; Gottesman and Ambudkar 2001; Lankat-Buttgereit and

47

Tamp 2002). ABC full transporters consist of two cytoplasmic nucleotide-

48

binding domains (NBDs) and two transmembrane domains (TMDs).

49

Although NBDs possess well-conserved sub-domains, such as Walker A,

50

Walker B, Q loop, etc each TMD typically consists of six transmembrane

51

helices (TMHs) interconnected by intracellular (ICLs) and extracellular loops

52

(ECLs) (Dawson and Locher 2006).

53

The crystal structure of bacterial ABC transporter Sav1866 revealed that the

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ICLs mediate the communication between NBDs and TMDs through specific

55

coupling helices (Dawson and Locher 2006). Similar interactions between

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the X-loop in the NBD domain and cytoplasmic helices connecting the

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various TMHs are also important for peptide transport in the TAP1/2

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antigen processing heterodimeric transporter complex (Oancea et al., 2009).

59

There are other biochemical studies from higher eukaryotic transporters

60

that also point to close interactions between ICLs and NBDs, which impact

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protein folding and trafficking. For instance, the mutations in ICL3 in

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human MRP transporter ABCC4 lead to defects in expression and

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membrane localization, mostly by loss of protein helicity and disruption of

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salt bridges with interacting residues from the NBD (Cheepala et al., 2013).

65

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR),

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which results in impaired localization and hampered chloride transport

67

leading to cystic fibrosis, maps to the cytoplasmic loop regions (Seibert et al.,

68

1996; Xie et al., 1995). In human P-glycoprotein, the mutation of ICL

69

residues also leads to maturation and processing defects where the protein

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remains underglycosylated and trapped within the endoplasmic reticulum

71

(ER) (Loo and Clarke 1994; Kapoor et al., 2013). Moreover, protein function

72

and maturation defects in the NBD2 mutant of Pgp could be suppressed by

73

a second site mutation in ICL2, once again emphasizing the importance of

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NBD-ICL interactions in protein function and maturation (Loo et al., 2013).

75

The functional role of ICLs and ECLs of fungal ABC transporters are not well

76

resolved. Considering the importance of ICLs as an important

77

communicating interface between NBDs and TMDs, this study evaluates

78

their role in the functioning of the multidrug ABC transporter Cdr1 protein

79

(Cdr1p) of pathogenic yeast, Candida albicans. For this, we employed a

80

rational approach and mutagenized residues based on homology model

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(Niimi et al., 2012) that were predicted to be in close proximity between ICLs

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and conserved sub-domains of NBDs.

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Material and Methods

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Materials

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Rhodamine 6G (R6G), ketoconazole (KTC), anisomycin (ANI), miconazole

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(MIC), clotrimazole (CTR), cycloheximide (CYH), itraconazole (ITC), adenosine

87

triphosphate (ATP), oligomycin (OM), trypsin, phenylmethanesulfonyl

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fluoride (PMSF), p-tosyl-L-lysine chloromethyl ketone (TLCK), and tosyl

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phenylalanyl chloromethyl ketone (TPCK) were procured from Sigma

90

Chemical Co. (St. Louis, MO). Fluconazole (FLC) was generously provided by

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Ranbaxy, India. Ascorbic acid (AA) was purchased from SRL (Mumbai,

92

India). Oligonucleotides used in this study were commercially procured from

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Sigma Genosys, India and are listed in Table S1. ER-TrackerTM Red Dye (ER-

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TR) and vacuolar membrane staining dye (FM4-64) were purchased from

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Molecular Probes. Anti-GFP monoclonal antibody and anti-mouse secondary

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antibody were purchased from Santa Cruz Biotechnology Inc. (Texas, USA)

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and were used at 1:5000 dilution in PBST. The anti-Pma1 (PM ATPase)

98

polyclonal antibody was a gift from Professor Ramon Serrano (Universidad

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Politecnica de Valencia-CSIC, Valencia, Spain) and was used at 1:5000

100

dilution in PBST.

101

Strains and media components

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The yeast strains used in the study are listed in Table S2. Plasmids were

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maintained in E. coli Dh5 strain cultured in Luria-Bertani medium (Difco,

104

BD Biosciences, MD, USA) to which ampicillin was added (0.1 mg/ml). WT

105

and CDR1 mutant strains were cultured either in YEPD broth or on YEPD

106

agar plates. Media chemicals were obtained either from Difco (Detroit, MI) or

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HiMedia (Mumbai, India). SD-ura- dropout medium (0.67% yeast nitrogen

108

base, 0.2% dropout mix and 2% glucose) containing 2.5% (w/v) agar was

109

used for growth and selection of mutants after yeast integration.

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Drug susceptibility assays

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Susceptibility to various drugs was evaluated either by broth micro-dilution

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or serial dilution spot assay. Cell suspensions were fivefold serially diluted
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in saline (0.9% NaCl) solution, and a 4 l aliquot of each dilution was

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spotted on YEPD or YEPD-drug plates as described previously

115

(Mukhopadhyay et al., 2002; Shah et al., 2014).

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Site directed mutagenesis

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The introduction of mutations at a particular position was achieved by PCR

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amplification of the pPSCDR1-GFP plasmid containing the CDR1 gene with

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pre-designed primers containing the mutation of interest. Mutagenesis was

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performed using a quick-change site directed mutagenesis kit from Agilent

121

technologies following manufacturers instructions. Positive plasmid clones

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were used for the transformation of the Saccharomyces cerevisiae strain

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AD1-8u- using the lithium acetate transformation protocol after linearization

124

with XbaI as described previously (Shah et al., 2014).

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Immunodetection and Confocal microscopy

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Plasma membrane (PM) was prepared and used for the immunodetection of

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the Cdr1 protein in WT and mutant strains as described previously (Shukla

128

et al., 2003). The immunodetection of GFP-tagged Cdr1 protein was

129

performed using an HRP-labeled anti-GFP antibody. GFP-tagged proteins

130

were imaged using an Olympus FluoView FV1000 laser confocal

131

microscope (PA, USA) with an 100X oil immersion objective lens.

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Endoplasmic reticulum (ER) and vacuolar staining was performed in live

133

yeast cells using ER-TR and vacuolar staining FM4-64 dyes respectively as

134

per manufacturers protocol. Briefly, cells grown overnight were washed

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three times with PBS and treated either with ER-tracker dye (0.5 M) for half

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an hour or with vacuole staining dye FM4-64 (1 M) for two hours, washed

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again with PBS and visualized under confocal microscope. The cells were

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imaged under an oil immersion objective at 100X magnification using an

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Olympus FluoView FV1000 laser confocal microscope (PA, USA).

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Substrate transport assays

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R6G efflux was determined essentially as described previously (Shukla et al.,

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2003). Briefly, log phase cells were washed and resuspended as a 2%

143

suspension in a PBS solution and incubated for 2 h in the presence of R6G

144

at a 10 M final concentration. After washing, the cells were resuspended in

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PBS with 2% glucose. After 40 min, a 1 ml aliquot was centrifuged, and the

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absorbance of the supernatant was measured at 527 nm.

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OM-sensitive ATPase assay

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ATPase activity of WT and mutant Cdr1p was monitored by the oligomycin-

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sensitive release of inorganic phosphate as described previously (Shukla et

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al., 2003). Ten micrograms protein from WT and each mutant strain PM

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fraction was used to determine the Cdr1p ATPase activity in an Invitro

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assay. The reaction was started by adding 5 mM ATP to the reaction mixture

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and was terminated after 30 min of incubation at 30C by addition of 1 ml of

154

stop solution containing 0.5% SDS.

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Results

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Rational Mutagenesis of interfacial NBD and ICL residues

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We adopted a rational mutagenesis strategy to get insight into

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communication between conserved motifs of NBDs and ICLs of Cdr1p. The

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interatomic distances and hydrogen-bonding network between conserved

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residues of NBDs and ICLs were calculated using Cdr1p homology models

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(Niimi et al., 2012) to identify the interacting residues of Cdr1p using the

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CONTACT program of the CCP4 suite (Winn et al., 2011). All other TMD-

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NBD interfacial interactions within Cdr1p were analyzed using the protein

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interfaces, surfaces and assemblies service (PISA) at the European

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Bioinformatics Institute (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html)

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(Krissinel and Henrick 2007). The intra molecular interactions of the IC1

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loop (567-597 residues), IC2 loop (645-657 residues), IC3 loop (1248-1279)

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and IC4 loop (1337-1345) with NBD residues were analyzed in both closed

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and open putative conformations (Winn et al., 2011). The data revealed a set

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of close interactive residues between ICLs and NBDs of Cdr1p, which are

171

depicted in Table 1. It is evident that between NBDs and ICLs, a total of nine

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putative residue pairs lay within 4 of inter atomic interacting distances.

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The predicted conserved NBD residues E315 and E1014 fall in signature

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sequence motifs of NBD1 and NBD2, respectively, whereas N297 falls in an

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X-loop of NBD1. Few other interacting residues fall near the Q-loop/H loop

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of NBD domains as shown in Table 1. Of note, the CONTACT program of the

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CCP4 suite did not predict any residue from ICL2 and ICL4, which could be

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within close proximity to NBD motifs.

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Phenotypic analysis reveals the mislocalization of E315A and K577A

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variants of Cdr1 protein

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To evaluate whether the probable interactions between NBDs/ICLs on the

182

basis of their close proximity (as depicted in Table1) are functionally

183

relevant, we replaced all of the nine predicted NBD/ICL residues individually

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with an alanine by site directed mutagenesis. The drug susceptibility spot

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(Fig. 1A) and broth micro-dilution assays (Table S3) revealed that, among

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NBD residues, the E315A (which lay in signature sequence of NBD1)

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variant-expressing cells became highly susceptible to all of the tested drugs.

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In contrast, K198, F254 and E1014 replacement to alanine resulted in a

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decrease in resistance to a few selected drugs. The other NBD residues

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Q159, N297, Q934, L1180 and Q1189 did not impact the functioning of the

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protein (Fig. 1A). Among the nine ICL counterpart residues, only I574A and

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K577A mutant variants of ICL1 showed enhanced susceptibility to selected

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drugs, whereas the rest of the ICL mutant variants E576A, Y584A, S587A,

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V1251A, Y1257A, R1260A and F1270A showed phenotypes similar to the

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WT Cdr1 protein (Fig. 1B).

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To exclude the possibility of poor localization or expression of the Cdr1

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variant proteins resulting in the observed high susceptibility to drugs, we

198

evaluated the localization of the GFP tagged-Cdr1p mutant variant-

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expressing cells. The confocal images displayed in Fig. 2A showed that the

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localization of the mutant variant E315A to PM was highly abrogated and

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led to an increased accumulation within the intracellular structures. The

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putative pair mutant K577A from ICL1 also showed decreased membrane

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localization that was less severe compared with E315A. On the other hand,

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selectively susceptible NBD variants K198A, F254A, E1014A and ICL variant

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I574A -expressing cells exhibited proper localization, which was evident

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from their typical surface rimmed appearance of GFP fluorescence (Fig. 2A).

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The membrane localization was further confirmed by western analysis. The

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pure PM fractions from the individual variant protein expressing cells were

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subjected to immuno-blotting by employing monoclonal antibody against

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GFP. The expression levels of Cdr1p detected in western blots confirmed

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that all of the drug susceptible mutants, except E315A and K577A, were

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properly expressed and localized to the PM (Fig. 2B).

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Owing to poor localization, expectedly, E315A displayed severely abrogated

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ATPase activity, which was collaterally reflected in the reduced efflux

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activity. For example, the ATPase activity and efflux of R6G, a well-known

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substrate of Cdr1p, was more that 70% reduced in E315A expressing cells

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(Fig. 3 A & B). Notably, ATPase and efflux activities of E315A were

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comparable to native protein after the normalization of mutant protein

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activities to its membrane protein expression level compared to that of WT

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levels (Fig. 3 C, D & E). The other mutant variant proteins of K198A, F254A,

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I574A, K577A and E1014A elicited greater than 65% of native protein

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ATPase activity, which apparently was sufficient to support >70% of efflux of

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R6G (Fig. 3 A & B).

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E315 of NBD1 and K577 of ICL1 form an ionic pair

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According to the 3D model, E315 may be in close proximity to K577 of the

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ICL1 residue (2.80 apart, Table 1), suggesting that they may interact and

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form an ionic pair. We explored the nature of the E315/K577 interactions by

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exchanging and swapping these two residues. In the first instance, we

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replaced E315 with a lysine (E315K) and K577 with a glutamate (K577E).

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Notably, this exchange resulted in the E315K variant as a non-functional

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protein, whereas the K577E version displayed resistance to selective drugs

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(Fig. 4). Interestingly, the swapping of the two residues in the same strain

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(E315K/K577E) was not tolerated, and cells expressing these variants

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became highly susceptible to drugs. This implies that, not only the presence

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of individual charged residues, but their positioning/environment is equally

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important for maintaining the interaction between E315 and K577 (Fig. 4).

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Because the phenotypic defects in ICL1 mutant K577E were not equally as

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severe as those of its counterpart residue, E315K from NBD1, we

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rationalized that the next closest positively charged lysine residue (K580)

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could most likely also contribute to maintaining the interaction between

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NBD1 and ICL1. To test this, we mutated the lysine residue at position 580

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to glutamate (K580E). As seen in drug susceptibility spot assays, K580E

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mutant expressing cells remained highly resistant to drugs; however, the

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combined mutations of K580 and K577 to glutamate (K577E/K580E) led to

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phenotypic defects similar to that observed upon mutating their counterpart

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residue E315 from NBD1 to lysine (E315K) (Fig. 4). Notably, the mutation of

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either of the lysine residues to glutamate, together with the mutation of

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E315 to lysine in the same strain (E315K/K577E or E315K/K580E), led to

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highly drug susceptible mutants as was the case with the triple mutant

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(E315K/K577E/K580E). This reinforces the notion that E315 can also

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interact with K580 in the absence of K577 (Fig. 4).

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For Ionic pair E315/K577 charge is more critical than the size of side

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chain

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We further explored whether the size or the nature of amino acid residues at

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these critical positions is required for the normal functioning of Cdr1p. For
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this, we mutated the residues in the NBD or ICL positions with the residues

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of the same size or charge to check whether the wild type phenotype can be

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retained. To maintain the size of the residues at critical positions, we

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replaced E315 with glutamine and K577 with methionine. It is evident from

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the drug susceptibility profile that the mutant variant E315Q did not retain

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the wild type phenotype. The cells expressing the variant E315Q were highly

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susceptible to drugs, whereas the K577M variant showed partial restoration

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of WT phenotype. The replacement of K580 with a methionine (K580M) again

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retained the wild type phenotype; however, when each of K577 and K580

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were replaced with a methionine, the strain harboring the double mutant

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variant (K577M/K580M) remained highly susceptible to drugs. Notably, the

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replacement of K577 and K580 with a glutamate (K577E/K580E) yielded an

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identical drug susceptible phenotype (Fig. 4). However, the charge

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replacement gave mixed results. For example, the replacement of K577 with

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arginine (K577R) could maintain WT levels of drug resistance, whereas the

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replacement of E315 with aspartate (E325D) could only retain resistance to

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select drugs. Of note, the drug susceptibility of all mutants was also

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confirmed by broth micro-dilution assays (Table S4). The above results point

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to the fact that charge of amino acid residues at these critical NBD and ICL

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positions is more contributing to function than the size of their side chain.

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Discussion

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The molecular mechanism by which ATP hydrolysis is coupled to transport

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by fungal ABC transporters remains obscure. Taking analogy from other

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orthologs from higher eukaryotes, it is proposed that the ICLs of yeast ABC
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transporters are important for transporter function. However, the precise

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role of ICLs as an intra-domain communication interface or in protein

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trafficking and folding is poorly understood. This study explored the role of

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ICLs, particularly in terms of their interaction with conserved sub-domains

284

of NBDs. For this, we used a heterologous over-expression system where

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GFP-tagged Cdr1 protein and its mutant variants were expressed (Shukla et

286

al., 2003).

287

Based upon the close proximity of ICL residues with conserved motifs of

288

NBDs, all of the residues that lay within a <4 distance were subjected to

289

alanine scanning mutagenesis in which each amino acid residue was

290

replaced with an alanine. Although most of the residues yielded a neutral

291

version of variant protein upon replacement with alanine, the ICL1 residues

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K577 and E315 of NBD1 were the exceptions. Both of these residues, when

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replaced with alanine (E315A and K577A), resulted in variant proteins, with

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abrogated function as was evident from their highly drug susceptible

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phenotype. On closer examination, it was revealed that the E315A and

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K577A mutant proteins were not properly localized on to PM. The confocal

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images showed them entrapped within intracellular structures, which were

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also corroborated by western analysis. The K577 residue from ICL1 is highly

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conserved, as is the E315 from the extended signature C motif of NBD1 from

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the canonical ATP binding site. Our data point to the fact that E315, though

301

belonging to the extended signature C motif, is not directly involved in ATP

302

catalysis. This conclusion is supported by the fact that the abrogated

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ATPase activity of E315A was not due to a non-functional protein (activity

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after the normalization of protein expression levels is comparable to WT

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protein) but rather due to its poor localization to the PM.

306

It became evident that E315 and K577 are indispensible to protein

307

localization because, when either of the two of ionic pairs is replaced with an

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opposite charge residue (E315K or K577E) to maintain a similar charge,

309

albeit in opposite positions, the protein variant could not reach the PM.

310

Interestingly, swapping the charge at two places in the same strain

311

(E315K/K577E) did not lead to the retention of PM localization, emphasizing

312

the critical nature of positioning in the native protein (Fig. 5). Unlike the

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E315/K577 ionic pair, which seems to be critical for Cdr1 protein

314

localization and function, a similar pair of ionizable, conserved amino acid

315

residues between ICL3 (E1014) and NBD2 (R1260) that lay at the equivalent

316

positions and within close proximity (3.27 apart) do not appear to be

317

critical (Fig. 6 & Table 1). The replacement of each with an alanine (E1014A,

318

R1260A) yielded a neutral variant protein (Fig. 1A & B). Moreover, few other

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ICL and NBD mutants within close proximity to each other although showed

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selective drug susceptibility, displayed normal localization to the PM and

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had unchanged ATPase and Rhodamine 6G efflux activities. Due to assay

322

limitations, efflux could only be measured with a single substrate

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Rhodamine 6G, whereas growth assays with other substrates show selective

324

changes in specificity profiles, likely resulting from decreased rate of

325

transport of these compounds. Together, the E315/K577 pair appears to be

326

most relevant for Cdr1 protein trafficking. The specific staining of cells

327

expressing the E315K/K577E mutant variants showed predominantly ER

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trapped protein (Fig. 7), which could not be rescued either by a chemical

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chaperone or by growing cells at low temperatures (Fig. S1). There was an

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asymmetric response between E315 and K577 with regard to their

331

restoration of either size or charge (E315Q, E315D and K577M, K577R). For

332

instance, when E315 was restored for size (E315Q) or charge (E315D), it did

333

not significantly improve the membrane localization of the protein (Fig 5).

334

The restoration of the size of K577 (K577M) led to partial restoration of

335

protein localization, whereas the restoration of the charge (K577R) could

336

maintain WT levels of protein expression (Fig 5). The replacement of ICL1

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residue K577 (K577E), compared with NBD residue E315 (E315K), only

338

triggered a partial impact on the localization and function of the protein.

339

However, the adjacent closest lysine (K580), together with K577, mimicked

340

the E315 phenotype. For example, though K580E or K580M individually did

341

not impact protein localization or function, the combined double or triple

342

mutations (K577E/K580E, K577M/K580M, E315K/K580E and

343

E315K/K577E/K580E) led to a severe protein localization defect (Fig. 5).

344

Charged residues from cytoplasmic loops (ICL5/ICL7) of human MRP1 are

345

also important for maintaining ionic interactions with its NBD2 region. The

346

mutant variants of these amino acid residues showed abrogated localization

347

associated with transport defects and entrapment within intracellular

348

structures, particularly in the ER (Iram and Cole 2011; Conseil et al., 2006;

349

Conseil et al., 2009; Iram and Cole 2012). The involvement of charged NBD

350

and ICL residues in inter-domain interaction appears to be conserved in

351

yeast ABC transporters as well. The well-known yeast multidrug

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transporters, Pdr5p and Yor1p, of S. cerevisiae have revealed

353

communication between NBD and TMD domains similar to that observed in

354

the bacterial ABC transporter, Sav1866 (Sauna et al., 2008;

355

Ananthaswamyet al., 2012; Pagant et al., 2008; Pagant et al., 2010).

356

Mutations in ICL1 and ICL4 of Yor1p results in susceptibility to oligomycin

357

and ER-trapped, misfolded protein (Pagant et al., 2008; Pagant et al., 2010).

358

In conclusion, the interaction of amino acid residues from ICL1 and

359

signature C motif of canonical ATP binding of NBD1 (Prasad and Goffeau

360

2012) seems to form a critical ionic pair in Cdr1 protein that plays an

361

important additional role in protein trafficking. This study helps aid in

362

understanding the inter-domain communication that is important for

363

protein trafficking and drug extrusion in the pathogenic yeast, C. albicans.

364

Funding:

365

The work was supported in parts by grants to RP from the Department of

366

Biotechnology: DBT No.BT/01/CEIB/10/III/02; DBT

367

No.BT/PR13641/MED/29/175/2010 and DBT

368

No.BT/PR14879/BRB10/885/2010.

369

Acknowledgements

370

We acknowledge Prof. S.S. Komath, SLS, JNU & Prof. Suresh K. Ambudkar

371

NCI, NIH for providing useful suggestions during this work. We also

372

acknowledge Advanced Instrumentation Research Facility (AIRF) and

373

Central Instrumentation Facility (CIF), School of Life Sciences, Jawaharlal

374

Nehru University, India, for confocal microscopy. AHS acknowledges senior

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375

research fellowship from DBT project No.BT/PR14879/BRB10/885/2010

376

and Dr. D.S. Kothari Postdoctoral Fellowship from UGC, India.

377
378

References:

379

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380

yeast multidrug transporter Pdr5 adopts a cis conformation, and there are

381

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382

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Conseil G, Deeley RG, Cole SP. Functional importance of three basic

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residues clustered at the cytosolic interface of transmembrane helix 15 in

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the multidrug and organic anion transporter MRP1 (ABCC1). J Biol Chem.

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Conseil G, Rothnie AJ, Deeley RG et al. Multiple roles of charged amino

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acids in cytoplasmic loop 7 for expression and function of the multidrug and

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Dawson RJ, Locher KP. Structure of a bacterial multidrug ABC transporter.

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Decottignies A, Grant AM, Nichols JW, et al. ATPase and multidrug

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Gottesman MM, Ambudkar SV. Overview: ABC transporters and human

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Iram SH, Cole SP. Expression and function of human MRP1 (ABCC1) is

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causes differential misfolding in multiple domains of multidrug and organic

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Jones PM, George AM. Mechanism of the ABC transporter ATPase domains:

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Kapoor K, Bhatnagar J, Chufan EE et al. Mutations in intracellular loops 1

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Krissinel E, Henrick K. Inference of macromolecular assemblies from

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466

467

Table 1: Interacting residues between conserved motifs of NBDs and

468

ICLs predicted on the basis of their inter-atomic distances from Cdr1p

469

homology models.

470

interacting NBD residue. The inter-atomic distances shown represent the

471

distances between atoms mentioned in parentheses against each residue.

represents the approximate positioning of the

472

473
474
475

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475

476

Supporting Information:

477

Table S1: List of Oligonucleotides used in the study.

478

Table S2: List of strains used in the study.

479

Table S3: MIC80 (g) values of drug susceptible residues from predicted ICL-

480

NBD interacting partners obtained by broth micro dilution assay.

481

Table S4: MIC80 (g) values of different mutant variants of ICL-NBD

482

interacting ion pair obtained by broth micro dilution assay.

483

Fig S1: Cdr1p expression of WT and various ion-pair mutant variants in

484

presence of non inhibitory concentration of drug substrate cycloheximide

485

and lower temperature.

486

22

486

487

Figure Legends:

488

23

489

Figure 1: Drug susceptibility pattern of ICL-NBD interacting residues of

490

Cdr1p predicted on the basis of distance proximity. Drug susceptibility

491

profile of A) ICL interacting NBD residues from conserved NBD motifs B)

492

NBD interacting ICL residues, predicted on the basis of their inter-atomic

493

distances in the presence of different drugs as observed by spot assays. 4

494

microliters of 5-fold serial dilution of each strain were spotted on YEPD agar

495

plates containing different drugs at indicated concentrations. Spot assays

496

were repeated three times with fresh drug plates to ascertain the

497

reproducibility of results.

498

24

498
499

Figure 2: Membrane localization and expression of drug susceptible

500

mutant variants of ICL-NBD interacting partners of Cdr1p. Membrane

501

localization and expression of drug-susceptible ICL-NBD interacting mutant

502

variant proteins as observed by A) confocal microscopy and B) western blots.

503

For the expression of Cdr1p-GFP, an anti-GFP monoclonal antibody, and for

504

loading control, protein expression Pma1p an anti-Pma1 antibody was used.

505

A total of 20 g of PM protein fraction of each mutant was used for

506

immunoblotting. PM was prepared individually for three separate western

507

blotting experiments. Results shown are the representative of the individual

508

experiments.

509
25

509
510

Figure 3: Substrate transport and ATPase activity of drug-susceptible

511

ICL-NBD mutants of Cdr1p. A) Percentage efflux of R6G was measured and

512

compared with its efflux in WT Cdr1p, which was set at 100%. B) OM-

513

sensitive ATPase activity of the Cdr1p mutants performed using 10 g of

514

each protein. The released inorganic phosphate was detected colorimetrically

515

at 880 nm as described in the methods. C) Normalized membrane

516

expression of E315A mutant of Cdr1p to that of its expression in WT Cdr1p.

517

Normalized expression levels were calculated by comparing the expression of

518

mutant to WT expression levels which in turn was related to the expression

519

levels of membrane Pma1p taken as loading control. D) ATPase activity of

520

E315A mutant of Cdr1p normalized to its membrane protein expression

521

levels. E) R6G efflux activity of E315A mutant of Cdr1p normalized to its

26

522

membrane protein expression levels. Error bars indicate SD for 3 or more

523

independent experiments.

524

27

524
28

525

Figure 4: Drug resistance profile of different mutant variants of ionic

526

pair forming residues. Spot assay shows the drug susceptibility profile of

527

different mutant variants of ionic pair forming residues E315-K577 between

528

NBD and ICL. Different drugs where used in YEPD agar plates at indicated

529

concentrations and 4 microliters of 5-fold serial dilution of each strain was

530

spotted on agar plates. Spot assays were repeated three times with fresh

531

drug plates to ascertain the reproducibility of results.

532

29

532
533

Figure 5: Membrane localization and expression of different mutant

534

variants of ionic pair forming residues. Membrane localization and

535

expression of different mutant variants of ionic pair forming residues E315-

536

K577 between NBD and ICL as observed by A) confocal microscopy and B)

537

and western blots. For the expression of Cdr1p-GFP an anti-GFP

538

monoclonal antibody and for loading control protein expression Pma1p an

539

anti-Pma1 polyclonal antibody was used. 20 g of PM protein fraction of

540

each mutant was used for immunoblotting. PM was prepared individually for

541

three separate western blotting experiments. Results shown are the

542

representative of the individual experiments.

543

30

543
544

Figure 6: Interaction between ionic pair of residues. Different interacting

545

residues forming ionic pairs in ICL1-NBD1 interface (A, upper panel) and

546

ICL3-NBD2 interface (B, upper panel). Lower panel in A & B shows

547

conservation score of the ionic pair of residues involved in ICL-NBD

548

interactions depicted by web logo and arbitrary conservation values obtained

549

from alignment with 85 similar fungal transporters. Web logos were

550

generated online at http://weblogo.berkeley.edu/logo.cgi.

551

31

551
552

Figure 7: Intracellular localization of various mutant variants of ICL-

553

NBD ionic pair. Intracellular localization of various mutant strains as

554

observed by A) ER-tracker dye (as an example, ER regions are denoted by

555

arrow heads in two panels) or B) by vacuolar membrane staining dye FM4-

556

64 (as an example, vacuolar regions are denoted by arrow heads in two

557

panels). Cells were grown overnight, washed three times with PBS and

558

visualized under confocal microscope after staining as described in methods.

32