Article Figures & Data
Figures
- FIG 1
Graphical illustration of C. albicans Cdr1 indicating the location of the cysteine residues. The center panel indicates the presumed topology of Cdr1; N, N terminus; C, C terminus. The positions (dashed arrows) of the 10 cytosolic cysteines (i.e., cysteines 1 to 5 of the N-terminal NBD N1 and cysteines 10 to 14 of the C-terminal NBD N2) are shown above, and the positions of the 13 cysteines of the TMD regions (i.e., cysteines 6 to 9 in T1 and 15 to 23 in T2) are indicated as yellow circles underneath the center panel. Each cysteine was given a unique identifier from N to C terminus of Cdr1 (red numbers 1 to 23). The Walker A1, Walker A2, Walker B1, and Walker B2 motifs, the helical domains HD1 and HD2, and the ABC1 (C1) and ABC2 (C2) signature motifs are shown as blue boxes, and the Q-loop, D-loop, and H-loop regions of NBD1 (Q1, D1, H1) and NBD2 (Q2, D2, H2) are shown as black boxes (top). The TMD topology of Cdr1 at the bottom is drawn approximately to scale. It shows individual TMSs (magenta rectangles) numbered from 1 to 12 and the PDR motifs (11) (orange boxes; PDRA [A] and PDRB [B] and EL6 motif [6M] and EL6 helix [6H]) near the N termini of EL3 and EL6, respectively. Each pair of PDR motifs is separated by a conserved three-residue proline-kink. Plasma membrane boundaries are indicated with dashed gray lines.
- FIG 2
SDS-PAGE and in-gel fluorescence of plasma membrane samples isolated from ADΔΔ cells overexpressing 18 CDR1PC-GFP variants. SDS-PAGE (7% polyacrylamide) of crude plasma membrane samples (10 μg protein) isolated from ADΔΔ (−), ADΔΔ-CaCDR1P-GFP (+), and ADΔΔ cells overexpressing the CDR1PC-GFP variants: N-terminal variants N1 (1), TS1 (2), EL3 (3), T1 (4), NTS1 (5), and NT1 (6); C-terminal variants N2 (7), TS2 (8), EL6 (9), T2 (10), NTS2 (11), and NT2 (12); and variants of the indicated N- and C-terminal subdomain combinations N12 (13), TS12 (14), EL36 (15), T12 (16), NTS12 (17), and NTS12-S1 (18). In-gel fluorescence images of the same polyacrylamide gels before Coomassie blue staining are presented underneath; the fluorescence signals were used to quantify the Cdr1 expression levels (Table 2).
- FIG 3
Confocal microscopy of ADΔΔ cells overexpressing Cdr1PC-GFP variants. ADΔΔ/CaCDR1P-GFP (wt; bottom right) was included as a positive control. Brightfield and fluorescent images of cells expressing the N-terminal variants are at the top, images of cells expressing the C-terminal variants are displayed underneath, and variants of N- and C-terminal subdomain combinations are shown at the bottom; see text for further details. MICFLC values (mg/liter) are indicated in yellow. To allow fair comparisons, all images were taken with identical confocal microscope settings.
- FIG 4
Plasma membrane protein profiles of ADΔΔ cells overexpressing cysteine-deficient Cdr1PC-GFP variants. −, ADΔΔ; +, ADΔΔ-CDR1P-GFP; variant numbers (1 to 18) are the same as in Fig. 2 and Table 2. The major plasma membrane proton pump, Pma1 (∼110 kDa) and Cdr1-GFP (∼200 kDa), bands are indicated with black arrows. Some lanes (7, 11, 12, 13, 17, and 18; N2, NTS2, NT2, N12, NTS12, and NTS12-S1 [i.e., CID], respectively) had a noticeably upregulated ∼70-kDa protein band (Ssa2; green arrow; see Table S2 in the supplemental material). M, Precision Plus protein marker.
- FIG 5
Confocal microscopy of ADΔΔ-CDR1P-GFP (wt), ADΔΔ-CDR1PC-NTS12-GFP, and ADΔΔ-CDR1-CID-GFP. The point mutation S1106I (arrow) recovered plasma membrane localization and function in CDR1PC-NTS12-S1-GFP (i.e., CID). MICFLC values (mg/liter) are shown in yellow.
- FIG 6
Architecture of the GX2[3]CPX3NPAD/E loop-helix motifs that are in contact with each other at the cytosolic apex between the two NBDs of Cdr1. (A) Model, viewed from the top, of the nucleotide-free conformation of the NBDs of Cdr1 (24) based on the ABCG5-G8 structure (18); the TMDs were removed for clarity. The N- (turquoise) and C-terminal (pink) halves are color coded. The characteristic signature motifs of the noncanonical CNBD1 (A1, B1, C2; for abbreviations see Fig. 1) and the catalytically active canonical CNBD2 (A2, B2, C1) are shown in blue and green, respectively, and the Q-, D-, and H-loops (Q1, Q2, D1, D2, H1, and H2) are in black and indicated with lines pointing toward their center. The N-terminal (4 and 5) and C-terminal (10 to 13) cysteines are shown as yellow dots; the N-terminal cysteines 1 to 3 that are part of A1, B1, and H1, respectively, are shown as sticks; and C1106 (14) is highlighted as a red dot near the center of the converging NBDs. The coupling helices (CH1 and CH2), unique ABCG transporter features which connect TMD1 and TMD2 with the E-helices (E1 and E2) of NBD1 and NBD2, respectively, are highlighted brown. The two GX2[3]CPX3NPAD/E loop-helix motifs providing tight contact between the two NBDs at the cytosolic apex are highlighted red (NBD1; degenerate motif; TTAD1 helix) and magenta (NBD2; canonical motif; NPAE2 helix). The canonical NPAE2 helix on the edge of one half of the centrally located NBD1-NBD2 contact region is positioned right underneath the canonical CNBD2, and the noncanonical TTAD1 helix (red) on the edge of the other half of the NBD1-NBD2 contact region is positioned right underneath the noncanonical CNBD1. (B) Close-up side-on view of the peripheral contact region near the center of the NBDs delineated with dashed gray lines in panel A. To show how H1 and H2 interact closely with the conserved contact motifs underneath, the remainder of the NBDs were removed. The noncanonical H1-Y361 and the canonical H2-H1059 are shown as black sticks. The six conserved CP and G residues of the noncanonical NBD1 (red) and the canonical NBD2 (magenta) GX2[3]CPX3NPAD/E motifs are in black (C402, P403, P1107) with the two N-terminal G residues (G399 and G1102) and C1106 shown as sticks. Residues of the loop regions that are in close proximity (<3 Å) near the center of the two NBDs are also shown as sticks: Q404 is in close contact with N1111 (red dashed line), R405 forms a possible salt bridge (red dashed lines) with E1109, and C1106 is in close contact (green dashed lines) with P1112. C1106 is part of CP2, and N1111 and P1112 are part of the canonical NPAE2 helix. Close contacts (<3 Å) between H1 and H2 with E1114 of the NPAE2 motif are indicated with black (H1-H2) and blue (H1-E1114 and H2-E1114) dashed lines, respectively. The five H1 residues YQCSQ were numbered 1 to 5, and the five H2 residues HQPSAL were numbered 6 to 11, respectively. Further details of the architecture surrounding this region are provided in Fig. S5 in the supplemental material.
- FIG 7
Location of the 23 cysteines and key residues (red) critically important for recovering efflux pump function of Cdr1PC-GFP variants. Model of Cdr1 (24) based on the ABCG5-G8 structure (18). The N-terminal (turquoise) and C-terminal (pink) halves are color coded, and the 23 cysteines (1 to 23) are shown as yellow dots, apart from C1106 (14), which is red. The model is viewed from the front (A) or back (B). The coupling helices (CH1 and CH2), unique PDR transporter features that connect TMD1 and TMD2 with the E-helices (E1 and E2) of NBD1 and NBD2, respectively, are highlighted brown. The characteristic contact points at the bottom of the two NBDs are encircled with black dotted lines (A and B). Mutations of key contact residues that could recover the function of various Cys-less Cdr1 variants are shown in red.
Tables
- TABLE 1
Phenotypes of ADΔΔ strains overexpressing CDR1P, CDR1PC, and 18 CDR1PC-GFP variants with the cysteines of the indicated CDR1P subdomains replaced with serine or alanine
↵a Superscript S, SS, and SSS indicate 2-fold, 4-fold, and 8-fold synergy, respectively, and superscript A indicates an additive effect on FLC transport of the indicated subdomain combinations (see text for further details).
↵b Gray areas indicate the various Cdr1 subdomains in which the cysteines have been replaced with alanine or serine. The position of individual cysteines and the residue they were replaced with (A or S) are as follows: T1, C193S, C325A, C363A, C402A, C462A; TS1, C632S, C635S; EL3, C712A, C732S; N1, C907A, C1041A, C1056S, C1091S, C1106S; TS2, C1294S, C1336S, C1357A, C1361A; EL6, C1380A, C1402A, C1418A, C1441S, C1444S.
↵c The ADΔΔ-CaCDR1PC-NTS12-GFP suppressor mutant was renamed ADΔΔ-CaCDR1-CID-GFP. It contained the S1106 of ADΔΔ-CaCDR1PC-NTS12-GFP replaced with I1106.
↵d +2 means twofold increased.
- TABLE 2
Cdr1 expression levels and ATPase activities of plasma membrane preparations isolated from ADΔΔ (negative control) and from ADΔΔ strains overexpressing CDR1P-GFP (positive control) and the CDR1PC-GFP Cys-deficient variants
↵a The strain symbols are the same as in Table 1.
↵b The ATPase activities were corrected for the oligomycin-sensitive ADΔΔ background (22 ± 10 nmol/min/mg); values are the means (±SDs) of two technical replicates measured three times.
↵c The ATPase activities with a − sign were below the detection limit of the assay.
↵d Cdr1 expression levels (% relative to wild-type Cdr1P-GFP).
↵e Cdr1 ATPase activities and MICFLCs (% relative to wild-type Cdr1P-GFP) normalized with respect to the different expression levels.
↵f Heat shock protein SSA2 was upregulated in these strains (Fig. 4 and text give further details).
↵g Significant differences between the Cdr1 ATPase activities were determined with the Student t test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
- TABLE 3
Drug susceptibilities of ADΔΔ-CaCDR1PC-NT1 and -NTS12 suppressor mutants
↵a All CDR1 variants had a C-terminal GFP tag.
↵b The MICs for the sensitive control strain ADΔΔ were 0.25 (ANI), 0.015 (CHX), 1 (FLC), 0.002 (CLT), 0.5 (R6G), 0.008 (KTC), and 0.25 (NIG) mg/liter, respectively.
↵c ND, not determined.
↵d This strain was renamed ADΔΔ-CaCDR1-CID-GFP.
↵e Deletion of “GA” causing frameshift and premature termination of Cdr1.
↵f Insertion of “A” causing frameshift and premature termination of Cdr1.
- TABLE 4
S. cerevisiae strains used in this study
- TABLE 5
Plasmids used in this study
↵a These plasmids contain the wild-type (P) (6) or the Cys-less (PC) (30) C. albicans CDR1 alleles cloned into the PacI/NotI restriction sites of either plasmid pABC3 or pABC3-GFP (51).
FIG S1
Conserved extracellular loop cysteines (Cys) are critical for Cdr1 folding and/or function. All Cys of NBD1 (N1; 5), TMD1 (TS1; 2), or EL3 (E3; 2) or of NBD2 (N2; 5), TMD2 (TS2; 5), or EL6 (E6; 5) were replaced with Ala or Ser. The domains in which Cys have been replaced with Ala or Ser are indicated in gray, and their MICFLCs are underneath each cartoon of the individual ADΔΔ-CaCDR1PC-GFP variants. NT1 and NTS12 variants were used to select for natural suppressor mutations (red dots) that could partially, or fully, recover Cdr1 function. The mutations are in critically important contact points for the opening and closing of the transporter. Download FIG S1, TIFF file, 2.0 MB.
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FIG S2
Confocal microscopy of ADΔΔ-CaCdr1PC-GFP variants shown in Fig. S1. MICFLCs (mg/liter) are shown in yellow. Download FIG S2, TIFF file, 2.7 MB.
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FIG S3
Sequence alignments of the GX2[3]CPX3NPAD/E loop-helix motif(s). The figure shows the alignment of the highly conserved GX2CPX3NPAD/E loop-helix motif of ABCG/WBC half transporters (top three panels) with the corresponding N-terminal (NBD1; left panels) and C-terminal (NBD2; right panels) regions of full-size ABCG transporters. The ABCG transporters are grouped into plant, fungal, and human ABCG/WBC half transporters (the fungal ABCG half transporters are C. albicans 19.3120 orthologs). Full-size ABCG transporters are also commonly known as PDR transporters. Fungal cluster F PDR transporters (FF; i.e., S. cerevisiae YOL075C orthologs) are the common ancestor of all plant and fungal PDR transporters (PDRanc). Representative plant and fungal PDR transporter subclusters FA, FB, FC, FD, FG, FH1, and FH2 (11) are shown. Fungal cluster E (FE) PDR transporters, which are cholesterol importers, were excluded from the alignment. The alignments were created with ClustalW (default settings), and the ClustalX color scheme of the JalView sequence editing software was chosen to display sequence conservation. Plant PDR transporters had one additional residue inserted just before the conserved G residue of the GX2[3]CPX3NPAD/E motif (see NBD2 alignments above). Numbers to the left denote the first residue number of individual sequences. The types of residues conserved in individual positions of the GX2[3]CPX3NPAD/E motif are displayed separately underneath their alignments for each group of ABCG transporters. A magenta helix indicates a highly conserved NPAD/E helix motif whereas red helices emphasize the noncanonical, degenerate, nature of the “NBD1-NPAD/E” helices of plant and fungal PDR transporters. The lines and numbers between the conserved G, CP, and NPAD/E motifs represent the numbers (2 or 3) of residues inserted between them for the various groups of ABCG transporters. X = any, and h = small hydrophobic, amino acids (see text for further details). The phylogenetic relationship and a list of the individual sequences with species abbreviations can be found in Text S1. Download FIG S3, TIFF file, 2.7 MB.
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FIG S4
Maximum likelihood tree of representative plant, fungal, and human half ABCG/WBC transporters (A) and full-size plant and fungal PDR transporters (B). The maximum likelihood trees for representative plant, fungal, and human ABCG/WBC half transporters (A) and full-size plant and fungal PDR transporters (B) that were used in Fig. S3 are midpoint rooted. (A) Display of the phylogenetic tree for the selected plant (green), fungal (brown), and human (blue) ABCG/WBC half transporters. Although fungal ABCG half transporters separate into a clearly distinct clade, plant and human ABCG half transporters do not divide into distinct plant and human clades, indicating multiple common ancestors of ABCG half transporters that cross major species boundaries. (B) Full-size PDR transporters divide into three major types (100% bootstrap support) of symmetric fungal cluster F (FF; brown) PDR transporters (i.e., S. cerevisiae YOL075C orthologs; the common ancestor of all PDR transporters) and typical asymmetric plant (green) and fungal PDR transporters. Fungal sensu stricto PDR transporters separate into eight major clusters (FA [red], FB [purple], FC [orange], FD [magenta], FE [not shown], FG [blue], FH) with cluster H PDR transporters further dividing into two distinct subclusters H1 (light blue) and H2 (light green) (11). Arrows 1 to 3 point to times when the unique degenerate characteristics of the GX2[3]CPX3NPA[D/E] motifs evolved in plant and fungal PDR transporters (see text for further details). The scale bar indicates the number of amino acid substitutions per position. Bootstrap support for 100 replicates is shown above individual branches. Download FIG S4, TIFF file, 1.9 MB.
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FIG S5
Model of the GX2[3]CPX3NPAD/E loop-helix motifs and surrounding regions of the NBDs of Cdr1. Panels A and B are a front and rear view, respectively, of the model presented in Fig. 6 including the regions surrounding the NBDs of Cdr1 to show how these motifs interact with the NBDs. The helical regions (i.e., residues between Walker A and the ABC signature motif) above the ATP-binding site were removed for clarity, but the E-helices just after the Q-loop that are in close contact with the CHs of the TMDs were included to provide context. The color codes, important contact regions, lines, and descriptions are the same as in Fig. 6 (see text for further details). Download FIG S5, TIFF file, 2.6 MB.
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TABLE S1
DNA oligonucleotide primers used in this study. Download Table S1, DOCX file, 0.02 MB.
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TABLE S2
Mass spectrometry analysis of the ∼70-kDa protein band upregulated in N2 subdomain containing ADΔΔ-CaCDR1PC-GFP variants. The ∼70-kDa band was identified as S. cerevisiae heat shock protein Ssa2 (score: 2,156; database: Swiss-Prot). The identified peptides (bold) covered 43% of the protein. Download Table S2, DOCX file, 0.02 MB.
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FIG S6
Direct one-step multifragment cloning strategy to create ADΔΔ cells overexpressing CDR1PC-GFP variants. Up to four individual DNA fragments of the various CDR1 transformation cassettes that overlapped by 25 bp were amplified by PCR with the indicated forward (pro [pPDR5-pro] and P1- to P3-for) and reverse (ter [pPDR5-ter] and P1- to P3-rev) primers to amplify the indicated PCR fragments. The PDR5 promoter and downstream sequences, the gene of interest, the PGK1 terminator, and the URA3 selection marker are in blue, brown, green, and light blue, respectively. The homologous crossover events (2, 3, 4, or 5) that were necessary to integrate the entire transformation cassette in one piece (top), or 2, 3 or 4 pieces (underneath), into the genomic PDR5 locus are indicated with gray crosses. The yellow lines depict desired mutations that were introduced by primer design of the indicated CDR1-specific -for and -rev primer pairs. Correct transformants were verified by colony PCR with a primer pair (up [pPDR5-up] and down [pPDR5-down]) that binds ∼40 bp upstream and downstream of the chromosomal integration site, respectively. Download FIG S6, TIFF file, 1.1 MB.
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TEXT S1
FASTA file of select representative half and full-size ABCG transporters from plants and fungi including YOL075C orthologs of various fungi. Download Text S1, DOCX file, 0.06 MB.
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