Fig 1.
Homodimer formation of ABCG4, ABCG1 and ABCG2.
(A) We have generated specific antibodies against ABCG1 (αG1) and ABCG4 (αG4). The specificity of the applied antibodies was confirmed by Western Blot technique performed with 40 μg of whole cell lysates from HEK cells expressing ABCG1 (G1), ABCG2 (G2), ABCG4 (G4), or myc-tagged ABCG5 along with HA-tagged ABCG8 (G5/G8). Parental HEK cells were used as a negative control. (B) Co-immunoprecipitation experiments were performed with HEK cells co-transfected with GFP-ABCG4 (GFP-G4) and ABCG4 (G4). Single transfected samples served as controls. Cells were lysed and an anti-GFP antibody was used for immunoprecipitation. Western blots of precipitates were developed by our anti-ABCG4 (αG4) antibody. The protein expressions were also verified (input). The presence of both untagged and GFP-tagged ABCG4 in the precipitate of co-transfected cells indicates dimer formation. Similar experiments were performed with GFP-ABCG1 (GFP-G1) and ABCG1 (G1) (C), or GFP-ABCG2 (GFP-G2) and ABCG2 (G2) pairs (D). Like ABCG2, both ABCG1 and ABCG4 can form homodimers.
Fig 2.
ABCG4 forms heterodimer with either isoform of ABCG1.
(A) The full-length isoform of ABCG1 (G1) or its inactive mutant variant (G1KM) was co-expressed with ABCG4 (G4) in HEK293 cells. Cells were lysed, the protein expression was verified (input) and co-immunoprecipitation was performed using our anti-ABCG4 (αG4) antibody. Both wt ABCG1 and its inactive mutant were co-immunoprecipitated with ABCG4, indicating heterodimer formation between ABCG1 and ABCG4. (B) Similar results were obtained with cells co-expressing the short isoform of ABCG1 (G1S) or its non-functional mutant version (G1SKM). (C) In contrast, when cells were co-transfected with ABCG4 and wild type or the inactive variant of ABCG2 (G2 or G2KM), only ABCG4 was detected in the precipitate, demonstrating that no heterodimer formation occurs between ABCG2 and ABCG4.
Fig 3.
Subcellular localization of ABCG4.
The wild type (G4) or the inactive mutant form (G4KM) of ABCG4 was expressed in HEK293 cells, immunostained with anti-ABCG4 antibody (αG4) and visualized by confocal microscopy. The subcellular compartments were identified by using specific markers for the plasma membrane (cadherin), the Golgi complex (giantin), and the endoplasmic reticulum (calnexin). (A) The wild type ABCG4 protein was predominantly localized to the plasma membrane, however, some intracellular staining was also observed, which was co-localized with the Golgi marker. (B) The mutant form (G4KM) exhibited even more pronounced plasma membrane localization. Scale bars—10 μm.
Fig 4.
Growth rate and expression levels in ABCG1- and ABCG4-transfected cells.
(A) HEK293 cells were transiently transfected with ABCG4 (G4), ABCG1 (G1), ABCG2 (G2), or with catalytic site mutant variants of ABCG4 and ABCG1 (G4KM, G1KM). The cell growth of these cultures was monitored for 72 hours. Cells expressing the wild type ABCG4 or ABCG1 protein showed inhibited growth capacity as compared to cells expressing the inactive mutants or the wild type ABCG2 (G2). (B) The expression level of ABCG proteins was also assessed by Western analysis. The total expression of the transgene rapidly declined in the cultures transfected with the wild type ABCG4 or ABCG1, whereas stable expression levels were observed in cells expressing the inactive mutants or the wild type ABCG2. Numbers at the top indicate the elapsed time in hours. (C) Following a three week long selection with G418, no ABCG4 expression was detected in cultures transfected with wild type protein (G4), whereas expression of the inactive mutant (G4KM) persisted. For loading control Na+K+ ATPase was used.
Fig 5.
(A) HEK293 cells were transfected with wild type (wt) ABCG4 or its inactive mutant (G4 or G4KM, respectively). ABCG2-expressing cells served as a negative control (G2). Apoptotic cells in cultures were visualized by fluorescently labeled Annexin V (green). Lower panels depict Hoechst 33342 nuclear staining (blue) in the same fields of view. Similar experiments with the ABCG1 isoforms and their inactive mutant variants are shown in S3 Fig. (B) The quantitative results are expressed as the percentage of apoptotic cells of total cells. The mean values ± S.E.M. obtained from at least 3 independent transfections are shown. The fraction of apoptotic cells was significantly larger in cultures transfected with the wt ABCG4 or ABCG1 than in cultures expressing the inactive mutants (KM), or the wild type ABCG2. Asterisks indicate significant differences as compared to wild type expressing cells (p < 0.01). (C) Co-staining of ABCG4 expression (green) and Annexin V binding (red) in cultures transfected with wt G4 or G4KM demonstrates the connection between the functional expression of ABCG4 and apoptosis. (D) Parallel detection of Annexin V binding (green) and caspase-3 activity (red) in cell cultures transfected with wt or inactive mutant ABCG4 confirmed that the functional ABCG4-induced apoptosis. DIC images on the lower panels demonstrate the presence of cell in the same fields of view.
Fig 6.
Functional interaction between ABCG4 and ABCG1.
(A) To test the functional interaction between ABCG proteins, the wild type ABCG1 (G1, upper panels) or the wild type ABCG4 (G4, lower panels) was co-expressed with inactive mutant variants of the studied ABCG proteins (G1KM, G4KM, or G2KM) as indicated at the top. The apoptotic cells were identified by Annexin V binding (green). (B) The co-expression of the proteins was confirmed by dual immunostaining wherever isotype difference of the specific antibodies allowed. ABCG1 expression is shown in green, whereas the partners indicated at the top are depicted in red. (C) Quantitative evaluation of Annexin V binding experiments revealed that the fraction of the apoptotic cells were significantly reduced by the co-expression with the inactive mutant variant of ABCG1 (either isoform) or ABCG4, but not with that of ABCG2, as compared to the cultures expressing wild type (wt) ABCG1 alone. Similar results were obtained with the wild type short isoform of ABCG1, or with wt ABCG4. Asterisks represent significant differences as compared to the cultures expressing the wild type protein alone (p < 0.01).