Reconstitution of cholesterol-transfer activity using mouse liver CMVs. CMVs were prepared from the livers of G5G8^−/− mice and from mice infected with adenoviruses that either express G5 and G8 or have no insert. Inside-out vesicles were made from the CMV- and ATP-dependent [³H]-cholesterol-transfer assays and were performed as described in the Experimental Procedures. (Inset) Immunblot analysis of G5 in the whole liver homogenate (25 µg), the postnuclear fraction (25 µg), and the CMVs (5 µg) of mice expressing G5 and G8 using a rabbit polyclonal antihuman G5 Ab (5). A total of 25 µg of protein from each fraction was subjected to immunoblotting using an anti-LDLR Ab (39). Alkaline phosphatase activity was measured as described in the Experimental Procedures using 25 µg of membrane protein.

Chemical cross-linking of the G5G8 complex. Partially purified native G5G8 (20 µg) that was obtained after fractionation over a Blue 2 column (see the Experimental Procedures) was incubated at room temperature for 15 min in the presence or absence of ATP (5 mM), MgCl₂ (2 mM), NaVO₄(1 mM), and BeF_x (1 mM/5 mM). The proteins were then chemically cross-linked using disuccinimidyl glutarate (DSG) according to the instructions of the manufacturer. The proteins were then analyzed by SDS–PAGE and Western blotting.

Reconstitution of purified G5G8 into proteoliposomes and measurement of ATP-dependent cholesterol transfer. (A) Native and recombinant G5 and G8 (both wild-type and mutant) were purified from the livers of G5G8^−/− mice by affinity chromatography as described in the Experimental Procedures. A total of 20 ng of protein was resolved on a 12.5% SDS--PAGE gel, and the gel was stained with silver as described in the Experimental Procedures. (B) ATP-dependent [³H]-cholesterol transfer was performed as described in the Experimental Procedures using proteoliposomes (15 ng) containing G5G8 purified from wild-type mice (native G5G8) or G5G8^−/− mice infected with adenoviruses expressing wild-type G5 and G8 or mutant forms of G5 and G8 (MG5 and MG8) that fail to hydrolyze ATP. The ATP-dependent [³H]-cholesterol-transfer activity was calculated from the difference between the signals measured in the presence of ATP ± addition of the inhibitor vanadate (VO₄).

Palmitoylation of G5. (A) Recombinant G5 and G8 were transiently expressed in CRL-1601 hepatocytes. A total of 48 h after transfection of the expression plasmids, the cells were metabolically labeled with [³H]-palmitic acid. The cells were solubilized in 1 mL of lysis buffer, and G5 and G8 were immunoprecipitated using anti-G5 and anti-G8 specific antibodies. Immunoprecipitated samples were separated by 8% SDS–PAGE followed by immunoblotting or X-ray film exposure (7 days). (B) Labeled cells were washed once with 20 mL of ice-cold PBS, broken by passing through a 25-gauge needle 20 times, and then treated with 1 M hydroxylamine (pH 7.0) or 1 M Tris-HCl (pH 7.0) for 2 h at 37 °C, followed by IP, SDS–PAGE, and Western blotting or X-ray film exposure. (C) Site-directed mutagenesis was used to substitute selected cysteines predicted to be located in the cytoplasmic regions of G5 with alanine. The effect of these substitutions on palmitoylation was assayed as described above.

Effects of C-terminal truncations of G5 and G8 on subcellular localization in polarized hepatoctyes. (A) Sequence comparison of the C-terminal tails of G5 and G8 from 16 different species. Regions that were deleted in these studies and had no consistent qualitative effect on protein localization are highlighted in blue. (B) Site-directed mutagenesis was used to introduce premature stop codons into the C-terminal domains of G5 and G8 at the locations indicated. (C) WIF-B cells were grown on glass coverslips, infected with the indicated recombinant adenoviruses, and processed for immunofluorescence microscopy. Fixed and permeabilized WIF-B cells were incubated with anti-G5 pAb (4591) and anti-G8 mAb (1B10A5), followed by goat anti-rabbit Alexa 488 and anti-mouse Alexa 568, respectively. Cells expressing G5 and G8, G5-T and G8, G5 plus either G8-T1, G8-T2, or G8-T3 are shown. Colocalization is shown as a merged image.

Solubilization of native G5G8 and separation of mature G5G8 from the immature forms of the half-transporters. Postnuclear liver membranes from wild-type mice were prepared and solubilized as described in the Experimental Procedures. (A) Immature (ER) and mature forms of G5 and G8 were separated by sequential Blue 4 and Blue 2 chromatography, as described in the Experimental Procedures. The crude detergent extract (25 µg), the proteins eluted from the Blue 4 column by 1.5 M NaCl and further purified by glycerol-gradient centrifugation (10 µg), and the Blue 2 column eluate (2 µg) were subjected with SDS–PAGE and immunoblotted with an anti-G5 Ab. (B) Oligomeric structure of G5 and G8 in the crude detergent extract and the Blue 2 column fraction from wild-type mouse livers. Crude detergent extract of wild-type mouse liver membranes (60 µg) and the Blue 2 column eluate (5 µg) were resolved by BN–PAGE and analyzed by immunoblotting using antibodies to G5 and G8. (C) Lanes from the gel shown in B containing the crude membrane extract and the eluate from the Blue 2 column were cut into strips and placed horizontally on a SDS–PAGE gel (10%). Also included in the experiment was an ion-exchange column (Mono Q) fraction of the crude extract. The SDS–PAGE was run as the second dimension and analyzed by Western blotting using anti-G5 and anti-G8 antibodies.

Purification of native mouse G5G8 by G5 Ab-affinity chromatography. Native G5G8 was affinity-purified using G5 Ab-conjugated beads as described in the Experimental Procedures. An aliquot of the purified protein (25 ng) was treated with peptide N-glycosidase F (PNGase), and the proteins were size-fractionated on a 12.5% SDS–PAGE before the gel was stained with silver (top) or analyzed by Western blotting (0.5 ng of protein) using anti-G5 and anti-G8 antibodies (bottom).

Protein concentration dependence and time course of cholesterol-transfer activity by purified native mouse G5G8. (A) Purified native G5G8 prepared as described in the Experimental Procedures was concentrated using a Microcon filter (Amicon) and reconstituted into proteoliposomes. In this assay, the concentration of G5G8 was such that the same volume of proteoliposomes was used for each cholesterol uptake assay. ATP-dependent cholesterol transfer was measured as described in Figure 5. (B) Time course of ATP-dependent cholesterol uptake was determined by measuring ATP-dependent [³H]-cholesterol transfer from the donor liposomes to the proteoliposomes containing native G5G8 (20 ng) at the designated time intervals.

Functional analysis of the palmitoylation of G5. G5G8^−/− mice were infected with adenoviruses encoding wild-type or mutant G5 (C61A) and wild-type G8 (8 mice/group). (A) Immunoblot analysis of liver membrane fractions from a representative mouse from each group was performed using antibodies to mouse G5 (4591) and G8 (1B10A5). (B) Gallbladder bile was collected following a 4 h fast, and biliary cholesterol was measured using GC–MS, as described in the Experimental Procedures.

Effects of C-terminal deletion of G5 or G8 on biliary sterol secretion in vivo. G5G8^−/− mice were infected with adenoviruses encoding wild-type or truncated G5 and G8 (3 mice/group). (A) A total of 3 days after infection, liver samples from these mice were pooled and subjected to immunoblotting as described in the Experimental Procedures. (B) Gallbladder bile was collected from these mice following a 4 h fast, and biliary cholesterol was measured using GC–MS. This experiment was repeated once, and similar results were obtained.