Subcellular localization of Sf9 cells infected with CT- and/or TM domain deletion mutants of UGT2B7. (A) Schematic sequences of UGT2B7 deletion mutants. The numbers represent the residue position of WT UGT2B7 counted from the N-terminus. SP, signal peptide. (B) Immunoblotting to confirm the expression of WT UGT2B7 and its mutants in insect cells. Sf9 cells, transfected with recombinant bacmids to obtain P1 viruses, were lysed and analyzed. After the collection of P1 viruses, the pelleted cells were resuspended in PBS, and an aliquot of the suspension was mixed with 2× SDS-PAGE sample buffer to prepare whole-cell lysates. HLM and Mock represent HLM (5 μg protein) and baculosomes prepared from Sf9 cells infected with control viruses (10 μg), respectively. Anti-mouse UGT antibody was used as a primary antibody (Mackenzie et al., 1984). (C) Fractionations of Sf9 cells to determine localization of the UGT2B7 mutants. UGT2B7 WT/mutants were simultaneously overexpressed with CNX in Sf9 cells. Sf9 homogenates were fractionated by sequential centrifugation. The fractions were analyzed by immunoblotting to determine the UGT2B7 WT/mutant subcellular localization. Protein amounts of loaded fractions are indicated below: homogenate (Homo), 10 μg; nuclei & mitochondria (MT), 2.5 μg; microsomes (MS), 5 μg; cytosol (Cyt), 7 μg. Anti-mouse UGT antibody and anti-CNX antibody were used as primary antibodies. Each band was quantified with ImageJ software, and was shown as the percentage of the total.

Localization of the CT and/or TM domain–deleted mutants of UGT2B7 and the CD4-UGT chimeric protein in COS-1 cells. COS-1 cells transfected with UGT2B7 WT or the mutants were fixed and stained 24 hours after transfection. (A) Localization of UGT2B7 and UGT2B7-HA (UGT2B7 with a C-terminal HA-tag) were compared with anti-UGT2B7 antibody (green). Cells were also stained red with an antibody to endogenous CNX, a marker protein of the ER. In the merged panels, nuclei stained by DAPI are shown in cyan. (B) Localization of the C-terminal–deleted mutants of UGT2B7. The sequences of the mutants are shown in Fig. 2A. An HA-tag was conjugated to their C-terminus for immunochemical detection. Cells were stained with anti-HA antibody (green) and anti-CNX (red). (C) Colocalization of UGT2B7 WT/mutants and CNX were quantified, and the mean ± S.D. values are shown. (D) Schematic sequences of CD4 and its chimeric proteins whose cytoplasmic domains were replaced with that of UGT2B7. The numbers represent the residue position of WT CD4 counted from the N-terminus. The DM of UGT2B7 is highlighted in the C-terminus of each chimera. SP, signal peptide. (E) Localization of CD4 chimeric proteins. CD4-UGT represents a CD4 chimera whose cytoplasmic domain was replaced with that of UGT2B7. Cells were stained with anti-CD4 antibody, which recognizes the extracellular domain of CD4 (green) and anti-CNX (red). In merged panels, the nuclei are shown in cyan. Scale bar, 10 μm.

Subcellular localization of a series of C-terminal–truncated mutants of UGT2B7 in Sf9 infected cells. (A) Schematic sequences of C-terminal–truncated UGT2B7 mutants. The numbers represent the residue position of WT UGT2B7 counted from the N-terminus. An HA-tag was conjugated to the C-terminus for detection by immunochemical methods. SP, signal peptide. (B) Immunoblotting to detect UGT2B7 mutants in insect cells. Sf9 cells transfected with recombinant bacmids to obtain P1 viruses were lysed and analyzed. After the collection of P1 viruses, the pelleted cells were resuspended in PBS, and an aliquot of the suspension was mixed with 2× SDS-PAGE sample buffer to prepare whole-cell lysates. WT-HA and Mock represent UGT2B7-HA microsomes (5 μg) and control microsomes (10 μg), respectively. Anti–HA-tag antibody was used as a primary antibody. (C) Fractionations of Sf9 cells to determine the localization of the UGT2B7 mutants. Human CNX was also transfected in cells expressing UGT2B7 deletion mutants. Sf9 homogenates were fractionated by sequential centrifugation. Protein amounts of loaded fractions were as follows: homogenate (Homo), 15 μg; nuclei & mitochondria (Mt), 6 μg; microsomes (MS), 5 μg; cytosol (Cyt), 10 μg. Anti-HA antibody and anti-CNX antibody were used as primary antibodies. Each band was quantified with ImageJ software and was shown as a percentage of the total.

(A) Localization of C-terminal–truncated mutants of UGT2B7 in transfected COS-1 cells. Schematic sequences of the mutants are presented in Fig. 4A. All of the mutants were fused with an HA-tag at their C-termini for immunochemical detection. COS-1 cells transiently expressing the mutants were stained with anti-HA antibody (green). Endogenous CNX was also visualized with anti-CNX antibody (red) as an ER marker. Nuclei stained with DAPI are shown in cyan. Scale bar, 10 μm in the merge panels. (B) Colocalization of UGT2B7 mutants and CNX were quantified, and the mean ± S.D. values are shown.

Localization of internal deletion mutants of UGT2B7 in transfected COS-1 cells. (A) Schematic sequences of UGT2B7 mutants (Δ6-HA to Δ11-HA). (B) Immunoblotting to detect the UGT2B7 mutants in COS-1 cells. Cells were transfected with 2 μg of vector coding each UGT2B7 mutant and lysed 24 hours after transfection. The lysates (20 μg) were analyzed by immunoblotting. Anti-HA antibody was used as a primary antibody. (C) Immunofluorescence to show colocalizations of the UGT2B7 mutants with CNX. The mutants were transiently expressed in COS-1 cells and visualized with anti-HA antibody (green). Endogenous CNX was also stained with anti-CNX antibody (red) as an ER marker. Nuclei were stained with DAPI and are shown in cyan. Scale bar, 10 μm in the merge panel. (D) Colocalization of UGT2B7 mutants and CNX were quantified, and the mean ± S.D. values are shown.

Analysis of stability and secretion of the UGT2B7-HA mutants. (A) Triton X-100 treatment of microsomal fractions to detect aggregated forms of UGT2B7-HA and its mutants. Homogenates (Homo) were prepared from COS-1 cells with a syringe equipped with a 23-gauge needle followed by low-speed centrifugation. An aliquot of homogenate was further fractionated by ultracentrifugation, and supernatants and pellets were collected as cytosols (Cyt) and microsomes, respectively. Microsomes were solubilized in 1% Triton X-100, and were separated into soluble fraction (Sol.) and insoluble pellet (ppt) by another ultracentrifugation. Homogenate (15 μg) and equivalent volumes of the later fractions were analyzed by immunoblotting. Top and bottom panels show the results in short and long exposures, respectively. (B) Effect of treatment with proteasome inhibitor (MG132) and lysosome inhibitor cocktail (LIs) on expression levels of UGT2B7-HAs. COS-1 cells transfected with UGT2B7-HA and its deletion mutants (ΔCT and ΔTM) were treated with 1 μM MG132 or LIs, 10 μg/ml E-64d, 20 μg/ml leupeptin, and 10 μg/ml pepstatin A for 12 hours. Whole-cell lysates were prepared, and 20 μg were analyzed in immunoblotting. (C) The experiments with the inhibitors were performed and quantified three times. Each bar represents the mean ± S.D. of relative intensities as each control = 100%. (D) Secretion of UGT2B7-HA, CD4, and their deletion mutants were analyzed. COS-1 cells were transfected with each construct and were cultured in FBS-free medium for 12 hours. Cells were harvested for lysate preparation, and medium were concentrated with trichloroacetic acid. The samples were analyzed by immunoblotting with anti-HA antibody and anti-CD4 as primary antibodies.