Clathrin-dependent internalization of OATP1A2 by PDZK1 and NHERF1.

<p>(A) Disruption of the clathrin-dependent pathway impairs OATP1A2-N-flag internalization. HEK-293 cells over-expressing OATP1A2-N-flag were treated with K⁺ depletion buffer or 10 mM acetic acid for 30 mins, relative to PBS-treated control. Internalization continued at 37°C for 5 or 10 mins as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094712#s2" target="_blank">Materials and Methods</a>,” followed by immunoblotting for OATP1A2-N-flag. (B) Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments). *: Different from control: <i>P</i><0.05. (C) <i>Top panel</i>: immunoblot analysis of OATP1A2-N-flag in HEK-293 cells containing co-expressed PDZK1-N-myc or NHERF1-N-myc after treatment with 10 mM acetic acid for 30 mins, followed by internalization at 37°C for 5 or 10 mins. <i>Bottom panel</i>: Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments). (D) <i>Top panel</i>: immunoblot analysis of OATP1A2-N-flag in HEK-293 cells containing co-expressed PDZK1-N-myc or NHERF1-N-myc after treatment with K⁺ depletion buffer for 30 mins, followed by internalization at 37°C for 5 or 10 mins. <i>Bottom panel</i>: Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments). Blotting for β-actin in separate aliquots of total lysates was used to confirm uniform protein loading.</p

Michaelis-Menten plot of E3S transport kinetics by OATP1A2 with or without co-transfected PDZK1 or NHERF1.

<p>E3S uptake was conducted over a 4-specific uptake by vector transfected cells and was also standardized to the amount of protein in each well. Kinetic parameters were calculated by nonlinear regression. Values are means ± S.E. (n = 3). **: Different from control: <i>P</i><0.01.</p

Association of OATP1A2 with PDZK1 and NHERF1.

<p>(A) co-immunoprecipitation of OATP1A2-N-flag and PDZK1-N-myc or NHERF1-N-myc. HEK-293 cells that co-expressed OATP1A2-N-flag and either PDZK1-N-myc or NHERF1-N-myc were lysed and subjected to immunoprecipitation with anti-flag antibody, followed by immunoblotting with anti-myc antibody. (B) Direct immunoblotting for N-Flag and N-myc tags, respectively, in the lysates of HEK-293 cells co-expressed with OATP1A2-N-flag and either PDZK1-N-myc or NHERF1-N-myc prior to immunoprecipitation.</p

Stability of OATP1A2 protein in HEK-293 cells in the presence or absence of co-expressed PDZK1 or NHERF1.

<p>(A) Western analysis of total cellular expression of OATP1A2-N-flag with or without co-expression of PDZK1 or NHERF1. <i>Top Panel</i>: HEK-293 cells were treated with 5 µg/ml puromycin for 24 and 48 h. Cells were harvested and lysate proteins were separated by SDS-polyacrylamide gel electrophoresis, followed by Western blotting with anti-flag antibody. <i>Bottom Panel</i>: After stripping, blots were reprobed with anti-β-actin antibody. (B) Densitometric analysis of the mature (∼95 KDa) isoform of OATP1A2-N-flag as a percentage of total OATP1A2 protein in the absence of puromycin treatment (means±S.E. of 3 individual experiments). Different from control: *<i>P</i><0.05; **<i>P</i><0.01.</p

Altered transport function and expression of OATP1A2 in HEK-293 cells in the presence and absence of co-transfected PDZK1 and NHERF1.

<p>(A) Transport of 300 nM [³H] E3S in OATP1A2-transfected HEK-293 cells with or without co-expressed PDZK1 or NHERF1 at 37°C. Values are means ± S.E. (<i>n</i> = 3). **: Different from control (OATP1A2 + vector): <i>P</i><0.01. (B) Western blot analysis of total cellular expression of OATP1A2-N-flag isoforms with or without co-expression of PDZK1 or NHERF1. <i>Top Panel</i>: Cells were lysed and proteins were separated by SDS-polyacrylamide gel electrophoresis, followed by Western blotting with anti-flag antibody. <i>Bottom Panel</i>: After stripping, the blot was reprobed with anti-β-actin antibody. (C) Western blot analysis of cell surface expression of OATP1A2-N-flag with or without co-expression of PDZK1 or NHERF1. Cells were biotinylated, and the labeled cell surface proteins were precipitated with streptavidin beads and separated by gel electrophoresis, followed by Western blotting with anti-flag antibody.</p

Scheme outlining the biotinylation-based internalization and recycling/membrane targeting assays.

<p>(A) Internalization assay. HEK-293 cells co-transfected with OATP1A2-N-flag and PDZK1-N-myc, NHERF1-N-myc or control vector were labeled with NHS-SS-biotin at 4°C. After quenching of residual NHS-SS-biotin with glycine (100 mM) the cells were warmed to 37°C to initialize internalization. At the end of incubations, sodium 2-mercaptoethanesulfonate (50 mM) was added to strip biotinylated, but non-internalized, proteins remaining at the cell surface. Cell lysates were prepared in lysis buffer and 500 µg protein from each sample was applied to streptavidin-agarose beads to capture biotinylated proteins. (B) Recycling/membrane targeting assay. HEK-293 cells co-transfected with OATP1A2-N-flag and PDZK1-N-myc, NHERF1-N-myc or control vector were labeled with NHS-SS-biotin at 4°C and then warmed to 37°C to initialize recycling/targeting for varying times; an additional aliquot of NHS-SS-biotin was applied to cells to optimize biotinylation of recycled/targeted proteins. At the end of incubations residual NHS-SS-biotin was quenched with glycine (100 mM) at 4°C. Cell lysates were prepared in lysis buffer and 500 µg protein from each sample was applied to streptavidin-agarose beads to capture biotinylated proteins. Electrophoresis and immunoblotting of lysate proteins bound to streptavidin/agarose beads was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094712#s2" target="_blank">Materials and Methods</a>.</p

Caveolin-dependent internalization of OATP1A2 by PDZK1 and NHERF1.

<p>(A) Disruption of the caveolin-dependent pathway impairs OATP1A2-N-flag internalization. HEK-293 cells over-expressing OATP1A2-N-flag were treated with filipin (5 µg/ml) or nystatin (25 µM) for 30 mins, relative to dimethylsulfoxide-treated control. The cells were allowed to internalize at 37°C for 5 or 10 mins as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094712#s2" target="_blank">Materials and Methods</a>” followed by immunoblotting for OATP1A2-N-flag. (B) Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments) *: Different from control: <i>P</i><0.05. (C) <i>Top panel</i>: immunoblot analysis of OATP1A2-N-flag in HEK-293 cells containing co-expressed PDZK1-N-myc or NHERF1-N-myc after treatment with filipin (5 µg/ml) for 30 mins, followed by internalization at 37°C for 5 or 10 mins. <i>Bottom panel</i>: Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments). *: Different from control: <i>P</i><0.05 (D) <i>Top panel</i>: immunoblot analysis of OATP1A2-N-flag in HEK-293 cells containing co-expressed PDZK1-N-myc or NHERF1-N-myc after treatment with nystatin (25 µM) for 30 mins, followed by internalization at 37°C for 5 or 10 mins. <i>Bottom panel</i>: Internalized OATP1A2-N-flag as a percentage of the total initial pool of biotinylated OATP1A2-N-flag at the cell surface (means±S.E. of 3 individual experiments). *: Different from control: <i>P</i><0.05. Blotting for β-actin in separate aliquots of total lysates was used to confirm uniform protein loading.</p