The Role of Dileucine in the Expression and Function of Human Organic Anion Transporter 1 (hOAT1)

Abstract

Human organic anion transporter hOAT1 plays a critical role in the body disposition of environmental toxins and clinically important drugs including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. In the current study, we investigated the role of dileucine (L6L7) at the amino terminus of hOAT1 in the expression and function of the transporter. We substituted L6L7 with alanine (A) simultaneously. The resulting mutant transporter L6A/L7A showed no transport activity due to its complete loss of expression at the cell surface. Such loss of surface expression of L6A/L7A was consistent with a complete loss of an 80 kDa mature form and a dramatic decrease in a 60 kDa immature form of the mutant transporter in the total cell lysates. Treatment of L6A/L7A-expressing cells with proteasomal inhibitor resulted in a significant increase in the immature form of hOAT1, but not its mature form, whereas treatment of these cells with lysosomal inhibitor had no effect on the expression of the mutant transporters, suggesting that the mutant transporter was degraded through proteasomal pathway. The accumulation of mutant transporter in the endoplasmic reticulum (ER) was confirmed by coimmunolocalization of L6L7 with calnexin, an ER marker. Furthermore, treatment of L6A/L7A-expressing cells with sodium 4-phenylbutyrate (4PBA) and glycerol, two chemical chaperones, could not promote the exit of the immature form of the mutant transporter from the ER. Our data suggest that L6L7 are critical for the stability and ER export of hOAT1.

Figure 1.

Predicted transmembrane topology of hOAT1. Twelve transmembrane domains are numbered from 1 to 12. Potential glycosylation sites are denoted by tree-like structures. Potential phosphorylation sites are labeled as P. Positions of the mutation is indicated by •.

Figure 2.

³H-labeled PAH uptake by hOAT1 wild type (Wt) and its alanine-substituted mutants L6A, L7A, and L6A/L7A. Transport of PAH (20 μM, 3 min) in COS-7 cells expressing hOAT1 Wt, L6A, L7A, and L6A/L7A was measured. Uptake activity was expressed as a percentage of the uptake measured in Wt. The results represent data from three experiments, with triplicate measurements for each mutant. Asterisks indicate values significantly different (p < 0.05) from that of Wt.

Figure 3.

Cell surface and total cell expression of hOAT1 Wt and its mutant L6A/L7A. A. Immunoblotting analysis of cell surface expression of hOAT1 Wt and its mutant L6A/L7A. Cells were biotinylated, and the labeled cell surface proteins were precipitated with streptavidin beads, separated by SDS-PAGE, followed by immunoblotting with antimyc antibody (1:100). B. Immunoblotting analysis of total cell expression of hOAT1 Wt and its mutant L6A/L7A. Cells were lysed, and their proteins were separated by SDS-PAGE, followed by immunoblotting with anti-myc antibody (1:100). Mature form (cell surface form) was shown as arrow and immature form (ER-resident form) was shown as arrowhead. C. Immunoblotting analysis of β–actin, a house-keeping protein, in cells transfected with hOAT1 Wt and its mutant L6A/L7A.

Figure 4.

Effect of protease inhibitors on the total expression of hOAT1 Wt and its mutant L6A/L7A. Immunoblot analysis of total cell expression of hOAT1 Wt and L6A/L7A in cells treated with or without lysosomal inhibitors leupeptin/pepstatin A (50 μg/ml for 16 hrs) or proteasomal inhibitor MG132 (10 μM for 6 hrs). Treated cells were then lysed, followed by immunoblotting using anti-myc antibody (1:100). Mature form (cell surface form) was shown as arrow and immature form (ER-resident form) was shown as arrowhead.

Figure 5.

Immunolocalization of L6A/L7A. L6A/L7A-transfected cells were immunostained for L6A/L7A, and an ER marker calnexin. A, Fluorescence image of L6A/L7A (green). B. Fluorescence image of calnexin (red). C. Merged image (yellow) of a and b. Bar = ~ 10 mm.

Figure 6.

Effect of chemical chaperones on the expression of L6A/L7A. A. Effect of 4PBA on the expression of L6A/L7A. L6A/L7A-expressng cells were treated with or without 4PBA (1 mM, 24h) in the presence of MG132 (10 μM). Treated cells were then lysed, followed by immunoblotting with anti-myc antibody (1:100). B. Effect of glycerol on the expression of L6A/L7A. L6A/L7A-expressng cells were treated with or without glycerol (5%, 24h) in the presence of MG132 (10 μM). Treated cells were then lysed, followed by immunoblotting with anti-myc antibody (1:100). Mature form (cell surface form) was shown as arrow and immature form (ER-resident form) was shown as arrowhead.