In this study we have examined the cellular itinerary and fate of recombinant, epitope-tagged ABCG5 and ABCG8 in three cell types: CHO-K1 cells, nonpolarized cultured rat hepatocytes (CRL-1601 cells), and polarized hepatocytes (WIF-B cells). We provide evidence that ABCG5 and ABCG8 are physically associated in these cells and that they chaperone each other out of the ER en route to the plasma membrane. The transporters colocalized with each other in the ER and plasma membranes of stably expressing cultured hepatocytes and were present on the apical (canalicular) membranes of WIF-B cells. Collectively, these data indicate that an ABCG5/ABCG8 complex is assembled in the ER, moves through the Golgi complex, and is targeted to the apical surface of cultured cells, suggesting that ABCG5 and ABCG8 may function as a heterodimer on the apical surfaces of cells to export neutral sterols. To our knowledge this is the first direct demonstration that trafficking of an ABC half-transporter to the cell surface requires the presence of its dimerization partner.
All experiments in this study were performed using epitope-tagged versions of ABCG5 and ABCG8, since antibodies that recognize the native proteins are not available. Although we cannot exclude the possibility that epitope tagging of the proteins alters their behavior in cells, experiments using tagged and untagged versions of ABCG8 indicate that this possibility is unlikely. The exit of ABCG5 from the ER requires coexpression of ABCG8. Both tagged and nontagged forms of ABCG8 allow transport of ABCG5 to the plasma membrane. These results indicate that the formation of ABCG5/ABCG8 complex and its trafficking to the cell surface are not influenced by the presence of the epitope tag. Similarly, the substitution of a proline residue for arginine at residue 220 of ABCG8 had no effect on the half-life, processing, or formation of heterodimers in CHO-K1 cells.
Both ABCG5 and ABCG8 are N-glycosylated. When each transporter is expressed individually in cells, the added sugars do not undergo maturation and the proteins are more rapidly degraded. When the two proteins are coexpressed, higher–molecular weight, Endo H–resistant, neuraminidase-sensitive forms of both proteins appear. The higher–molecular weight forms of the proteins have significantly longer half-lives, suggesting that formation of the complex between the two half-transporters in the ER promotes their transit from the ER to the trans-Golgi complex and thus diverts them from a degradative pathway. Cell fractionation and immunolocalization studies in cultured hepatocytes confirm that ABCG5 is retained in the ER unless ABCG8 is expressed in the same cells. These data indicate that the cellular localizations of the proteins are contingent on their oligomeric structure.
The observation that mutations in either ABCG5
cause an identical phenotype (1
) is also consistent with ABCG5
forming a complex. Since ABC half-transporters contain only a single nucleotide-binding fold and six putative transmembrane segments, they must form homodimers or heterodimers to be active (14
). Some half-transporters, such as TAP1 and TAP2, function as heterodimers; mutations in either gene result in the loss of peptide transport activity (16
). The four peroxisomal half-transporters (ABCD1–ABCD4) form homodimers and heterodimers in vitro (17
), but the composition of the functional complexes of these proteins is not known. The ABCG subfamily members scarlet, brown, and white also function as heterodimers (38
), whereas ABCG2 transports substrates when expressed alone in insect cells, suggesting that it functions as a homodimer (39
). The finding that ABCG5 and ABCG8 coimmunoprecipitate indicates that the two proteins are associated in cells and suggests that they heterodimerize. The interaction between these two proteins is specific and is not a post–cell lysis artifact, since the half-transporters do not coimmunoprecipitate if lysates from cells expressing each half-transporter alone are mixed. Coimmunoprecipitation of the high–molecular weight, Endo H–resistant forms of ABCG5-myc and ABCG8-HA is almost quantitative, further indicating that ABCG5/ABCG8 complex formation is required for ER exit.
A small fraction of expressed ABCG5 and ABCG8 also immunoprecipitates as homo-oligomeric complexes. For both proteins, only the lower–molecular weight forms are present in these complexes, indicating that if ABCG5 or ABCG8 form homodimers or other homo-oligomers, these complexes fail to exit the ER. Consistent with this interpretation, ABCG5-myc is not transported to the apical membrane in WIF-B cells if expressed alone. In contrast, ABCG2 appears to function as a homodimer and has been observed on the bile canalicular membrane (19
). Further studies will be required to determine whether the ER-associated forms of ABCG5 and ABCG8 function as homodimers, represent nonspecific protein aggregates, or are mis-paired half-transporters that fail to achieve the appropriate conformation to escape the ER.
The observation that ABCG5-myc and ABCG8-HA reach the trans
–Golgi complex only when the two proteins are coexpressed indicates that monomeric and multimeric homodimers of these proteins are specifically retained in the ER. The mechanism for ER retention of ABCG5 and ABCG8 is not known. Other oligomeric membrane proteins contain ER retention motifs that are masked when the proteins complex. For example, heterodimerization is required for the T cell antigen receptor (TCR), the H2a subunit of the asialoglycoprotein receptor (ASGPR), and the GABAB receptor to exit the ER (40
). Each subunit of these proteins contains a different ER retention motif (GLRILLLKV, EGHRG, and RXR for TCR, ASGPR, and GABAB, respectively) that is masked by heterodimerization, allowing the protein complex to exit the ER. Only the RXR motif is present in mouse ABCG5 and ABCG8. One copy of this motif is conserved between the mouse and human proteins (residues 242–244 in ABCG5 and residues 196–198 in ABCG8), but the role of these residues in ER retention remains to be examined. Other mechanisms for the retention of improperly complexed oligomers include binding of proteins to ER chaperones that contain KDEL motifs, and thiol retention whereby the masking of cysteine residues by disulfide bonding allows proteins to exit the ER (43
). ABCG5 and ABCG8 contain numerous cysteine residues, but whether intramolecular or intermolecular disulfide bonds exist in the ABCG5/ABCG8 complex has not been examined. Additional studies will be required to determine the mechanisms by which ABCG5 and ABCG8 monomers, and perhaps oligomers, are recognized by the ER quality control system.
A potentially interesting observation is the finding of higher–molecular weight forms of ABCG5 and ABCG8 in the ER, as well as in the non-ER, fractions of cultured hepatocytes expressing both half-transporters. The presence of the mature forms in the ER fractions could represent contamination of these fractions with non-ER components of the cell, although none of the non-ER marker proteins are present in the ER fractions (Figure b). The fully mature, glycosylated forms of the proteins may have recycled from the Golgi complex compartment back to the ER, as has been described for SREBP cleavage–activating protein (SCAP) (45
). More experiments will be required to determine the source of the mature forms of the half-transporters in the ER.
ABCG5 and ABCG8 colocalize at the cell surface in cultured hepatocytes, and immunoelectron microscopy confirms that the proteins are associated with the plasma membrane in these cells. The proteins are located predominantly on the apical plasma membrane, suggesting that ABCG5 and ABCG8 may participate directly in the transport of neutral sterols across the apical membrane into bile. It is not known whether neutral sterols are the primary transport substrate of ABCG5 and ABCG8 or whether another molecule is actively transported and sterol efflux is secondary. Phospholipids are the primary transport substrate for a number of other ABC lipid transporters, and phospholipid transport and cholesterol transport into bile are tightly interrelated. For example, biliary cholesterol excretion is markedly reduced in mice lacking the phosphatidylcholine transporter ABCB4 (MDR2) (46
). Sitosterolemic patients have a marked reduction in fractional excretion of biliary sterols but have normal biliary excretion of phospholipids (7
). Conversely, overexpression of ABCG5 and ABCG8 in mice causes a selective increase in cholesterol excretion into the bile (47
). These findings suggest that neutral sterols or a neutral sterol carrier protein, rather than phospholipids, are the primary transport substrate of ABCG5 and ABCG8.
The observation that ABCG5 and ABCG8 form a heterodimer does not exclude the possibility that either protein may form functional complexes with other ABC half-transporters. In Drosophila
, white forms functional complexes with both scarlet and brown to transport different substrates (38
). The mammalian ABCG transporter subfamily includes six known members. Although the tissue expression patterns of ABCG5 and ABCG8 differ from those of the other ABCG subfamily members, both ABCG1 and ABCG2 are expressed in the liver and intestine (19
). ABCG3 and ABCG4 are expressed at only low levels in the liver and intestine of mice (data not shown); the tissue distribution of expression of these proteins in humans is not known. Thus, it remains possible that ABCG5 and ABCG8 are not monogamous and that the half-transporters associate with other subfamily members to transport different substrates. The formation of dimer pairs between various members of this subfamily of proteins may permit transport of a more diverse array of substrates, or provide tissue- and/or substrate-specific control of solute transport.