Human Organic Solute Transporter (hOST): protein interaction and membrane sorting process

Abstract

The human organic solute transporter (hOST) is a heterodimer composed of alpha and beta subunits. Physical association of hOSTα and β subunits is essential for their polarized basolateral plasma membrane localization and function in the export of bile acids and steroids. To understand the role of carboxyl- and amino-tails of OSTβ and mechanisms underlying membrane localization of hOST, the effects of tail deletion of the hOSTβ subunit and biological reagents on membrane distribution and transport function of hOST were investigated in stably transfected MDCK cells. After deletion of 35 amino acids from the amino-tail of hOSTβ, the efflux transport activity and polarized membrane distribution of the truncated hOSTβ was abolished. A co-immunoprecipitation study verified that the amino-tail of hOSTβ is essential for the association with hOSTα subunit. Treatments with acytochalasin D (interrupting ctin-filaments), bafilomycin A1 (inhibiting vacuolar H(+)-ATPase), brefeldin A (disrupting the Golgi complex), and calphostin C (inhibiting protein kinase C), significantly disrupted the polarized membrane distribution of hOST and markedly reduced transport activity in stably transfected MDCK cells. In summary, the 35 amino acid amino-terminal fragment of hOSTβ contains critical information for interaction with the hOSTα subunit and subsequent trafficking to the plasma membrane. These studies suggest that the membrane sorting process of hOST is mediated by a bafilomycin A1-sensitive vesicular pathway that is associated with the actin-cytoskeleton network. The membrane localization of hOST is also partially mediated through a brefeldin A sensitive mechanism, which controls its transit from the ER to Golgi and is regulated by PKC.

Keywords: Human OST; membrane trafficking; organic anion transporter; protein interaction.

Figure 1

Confocal microscopy and [³H]taurocholate (TC) efflux studies of human hOSTα and truncated β co-expressed in stably transfected MDCK cells. (A) Confocal fluorescence images: The cells were grown on glass coverslips and fixed with cold 100% methanol. In stably transfected MDCK cells, the hOSTα were co-expressed with a GFP fused wild type (β-GFP), C-terminal truncated (β-CT-GFP) or N-terminal truncated (β-NT-GFP) hOSTβ. TC transport activity of cells transfected with human organic solute transporter (hOST): Na⁺-independent [³H]TC efflux (B) and Na⁺-dependent [³H]TC uptake (C) were measured in stably transfected MDCK cells that were transfected with rat Asbt, human OSTα and wild type (β), or C-terminal truncated (β-CT) or N-terminal truncated (β-NT) hOSTβ cDNA. Data represent mean ±S.E. (bars) of triplicate determinations from at least three different cell culture preparations. Asterisks (*, p<0.05) indicate significant differences from wild type hOSTβ co-transfected cells. Messenger RNA levels of rAsbt, wild type and truncated hOSTα and β constructs in stably transfected MDCK cells were obtained using the reverse transcription-polymerase chain reaction (RT-PCR) (D) (see methods), and the reaction products were visualized by staining with ethidium bromide. Figure (C) and (D) indicate that these stably transfected MDCK cells have similar gene expression level for rAsbt, wild type and truncated hOSTα and β, and equally TC uptake activity.

Figure 2

Identification of the hOSTα and β proteins interacting domain. Co-Immunoprecipitation (co-IP) of N-end truncated hOSTβ and wild type hOSTα proteins in transiently co- transfected HEK293 cells. The Myc-tagged wild type (β) or N-terminal truncated hOSTβ (β-NT) were co-transfected with GFP-tagged wild type hOSTα constructs into HEK293 cells, respectively. The total protein extracted from transfected cells was immunoprecipitated by the polyclonal GFP (FL) antibody. The total protein extracted from the transfected HEK293 cells (cell lysate) and co-immunoprecipitated proteins (co-IP) were isolated by SDS-PAGE and analyzed by Western blot using a poly-Myc antibody to detect Myc tagged wild type or N-end truncated hOSTβ protein. The results demonstrated that only the Myc tagged hOSTβ protein (β) was co-immunoprecipitated by hOSTα, but not the amino-terminal truncated hOSTβ (β-NT). [β = wild type hOSTβ; β-NT= 35 amino acid of N-end fragment deleted hOSTβ; ΗΕΚ = Un-transfected HEK293 cells].

Figure 3

Effects of biological reagent treatments on cellular distribution and taurocholate (TC) efflux of hOST proteins. The GFP fused hOSTα (α-GFP) constructs were stably co-transfected with hOSTß and rat Asbt in MDCK cells. A: The membrane expression of hOSTα-GFP was detected by confocal fluorescence microscopy and photomicrograph of GFP-fused hOSTα was obtained on a confluent monolayer of the stably transfected MDCK cells cultured on glass coverslips. B: The Na⁺-independent efflux of taurocholate was examined in the MDCK cells stably transfected with rat Asbt, human OSTα and ß cDNAs. In this model system, the rAsbt acted as an apical bile acid loading pump in the stably transfected MDCK cells. The bile acid efflux activity from the basolateral domain by hOSTα and ß proteins was measured by radioactive scintillation counting. Data are presented as percentage (%) of total TC efflux and represent the mean value ± S.E. of three independent experiments performed in triplicate. Asterisk (*) for each stably transfected MDCK cells treated with drugs indicates significant difference (p<0.05) from drug untreated cells by unpaired t -test. The stably transfected MDCK cells were treated with 33μM nocodazole (Noc), 10μM colchicine (Col), 2μM cytochalasin D (Cyt D), 50nM bafilomycin A1(BA1), and 2μM brefeldin A (BFA), respectively, at 37°C for 16 hr. (Untreated = drug untreated stably transfected MDCK cells; rAsbt = MDCK cells transfected with rAsbt only).

Figure 4

Effects of protein kinase inhibitor treatment on membrane localization and taurocholate (TC) transport activity of hOST protein in stably transfected MDCK cells.The GFP fused hOSTα (α-GFP) constructs were stably co-transfected with hOSTβ and rat Asbt in MDCK cells. The stably transfected MDCK cells were pretreated with (+) or without H89 (a PKA inhibitor, 10μM), Calphostin C (Cal C, a PKC specific inhibitor, 1μM), and Protein Kinase G inhibitor (PKGi, 86μM), respectively, for 1 hr., at 37°C. A: confocal images, B: The Na⁺-independent bile acid transport activity by hOSTα and ß protein was measured by radioactive scintillation counting. Data are presented as percentage (%) of total TC efflux, and the mean value ± S.E. of two independent experiments performed in triplicate. Asterisk (*) for each stably transfected MDCK cells treated with protein kinase inhibitors indicates significant difference (p<0.05) from untreated cells by unpaired t -test. (Untreated = drug untreated stably transfected MDCK cells; rAsbt = MDCK cells transfected with rAsbt only).