In this paper, we describe the first purification and functional reconstitution of native G5G8. The native proteins were co-purified as a heterodimer, and we found no evidence for higher order oligomerization. Previously, we showed that recombinant, epitope-tagged forms of G5G8 expressed in insect cells promote the transfer of sterols from donor vesicles to proteoliposomes. Here, we demonstrated that sterols are the direct transport substrates of native G5G8. Native G5G8 was significantly more stable than the recombinant transporter, which allowed for more detailed biochemical characterization of the transporter.
The stoichiometric amount of ATP hydrolyzed by ABC transporters per allocrite translocation event is a major unresolved question. Models proposing hydrolysis of one or two ATP molecules per transport cycle have been put forward (26
), but measured ratios of ATPase activity to allocrite transport vary widely, even for the same transporter (27
). In our previous study using purified recombinant G5G8, sterol-transfer activity was not sufficiently stable over time to allow for a quantitative estimate of the allocrite transfer rate. In the present study, the native purified complex maintained linear cholesterol-transfer activity of 0.1% of labeled cholesterol per minute for over 90 min (). Assuming that the labeled cholesterol traces all of the cholesterol in the donor particle, this activity represents a transfer rate of 0.6 µmol of cholesterol (mg of G5G8)−1
. The ATPase activity of the native complex was 0.39 µmol mg−1
, suggesting a coupling ratio of 1 ATP molecule hydrolyzed per sterol molecule translocated. This ratio cannot be considered definitive, because both the sterol transfer rate and the ATPase activity of the reconstituted transporter are subject to measuring errors. Nonetheless, a coupling ratio of 1:1 is consistent with our earlier observation that only one of the nucleotide-binding domains of the G5G8 complex hydrolyzes ATP (12
Our current knowledge concerning the mechanisms of action of ABC transporters has been inferred almost entirely from recombinant proteins expressed in heterologous cells or membranes. Few studies have examined in detail the function of native ABC transporters. An exception is ABCR (ABCA4), which was purified from bovine rod outer segments (27
). Although the putative substrate, all-trans
retinal, increased ATPase activity of the transporter, Sun et al. did not report retinal transport by the purified protein (27
). Mao et al. (28
) purified native MRP1 (ABCC1) from doxorubicin-selected cultured lung tumor cells and reconstituted active transport of cysteinyl leukotriene C, an endogenous substrate of the transporter. Thus, the present study describes the first purification and reconstitution of substrate transport by a mammalian ABC transporter purified from the tissues in which it is expressed.
Although the use of recombinant proteins has many experimental advantages, the fidelity with which they reflect the properties of the native protein is uncertain. Differences in post-translational modifications, protein folding, and complex assembly may affect the properties of the recombinant protein. Although the results that we obtained using the native protein purified from mouse liver were qualitatively similar to those obtained using the recombinant protein produced in Sf9 cells, use of the native complex had several advantages. A major advantage of using the native protein was its stability. Previously, we have used membrane vesicles from recombinant Sf9 cells for our functional studies because the purified recombinant proteins produced in these cells is highly unstable. The purified insect-G5G8 is freeze-labile and loses activity within 24 h, even after it is reconstituted into proteoliposomes. In contrast, reconstituted native G5G8 retains maximal activity for several days when maintained at 4 °C (data not shown). The G5G8 purified from the liver can be stored at −80 °C and undergoes freeze–thawing up to 3 times without any reduction in sterol-transfer activity (data not shown). In addition, the purified complex can be concentrated 10-fold using Microcon filters (Millipore) without inducing aggregation.
Another difference between the insect-derived and native G5G8 was in the selectivity of lipid transport. Recombinant G5G8 synthesized in insect cells consistently transferred small amounts of cholesteryl esters and phospholipids, whereas essentially no ATP-dependent transfer of these molecules was observed when the native protein was used in the same assays. The results of these experiments demonstrate the strict substrate selectivity of G5G8. Furthermore, the present results confirm that G5G8-mediated transfer of cholesterol is exquisitely stereoselective; we observed no ATP-dependent transfer of the enantiomer of cholesterol by native G5G8. We do not know if the observed differences between the recombinant and native proteins are attributable to differences in glycosylation of the proteins expressed in insect versus mammalian cells, the presence of epitope tags at the C termini of the recombinant proteins, or to other yet-to-be-identified factors.
Recombinant G5 and G8 were co-purified as a heterodimer from Sf9
cells, consistent with current structural models of ABC transporters (29
). Higher order oligomeric structures have recently been reported for ABCG2 (30
) and ABCA1 (31
). Interestingly, ABCA1 appeared to transition from a dimer to a higher order oligomer during the catalytic cycle (31
). We used native gel electrophoresis, density-gradient ultracentrifugation, and chemical cross-linking studies to examine the oligomeric structure of native G5G8. All three methods indicated that the functional complex is a heterodimer, and no higher order oligomeric forms were observed at any stage in the catalytic cycle.
ATPase activity is stimulated by the addition of the transport substrate for most ABC transporters (36
). In our previous study using recombinant G5G8 expressed in insect cells, cholesterol appeared to cause a slight reduction in ATPase activity but the functional instability and low activity of the purified recombinant protein made it difficult to obtain consistent ATPase measurements (11
). The stable activity of purified native G5G8 provided us with an opportunity to address this issue more definitively. Here, we have confirmed a mild but consistent inhibition of G5G8 ATPase activity by both cholesterol and sitosterol. A similar phenomenon was reported for ABCA1; the ATPase activity of this cholesterol transporter is also slightly inhibited by the addition of sterol (24
). It is possible that the addition of cholesterol to the membrane in the absence of an acceptor affects ATPase activity by altering the conformation of the protein. In vivo
, mixed micelles of PC/bile acids serve as the natural acceptor for cholesterol transport to the bile. Further studies will be required to determine the effect of cholesterol-binding on the conformation of the NBDs in the G5G8 transporter.
We also demonstrated that G5 but not G8 is palmitoylated through a thioester linkage at C61 (). Only the mature form of G5 is palmitoylated, indicating that the modification occurs after the protein leaves the ER, presumably in the Golgi. Studies in yeast have shown that protein palmitoylation is catalyzed by a family of cysteine-rich Asp-His-His-Cys (DHHC) domain-containing proteins (DHHC proteins) (32
). Of 23 predicted DHHC proteins in the human genome (33
), Fernández-Hernando et al. reported that DHHC-2, -3, -7, -8, and -21 were co-localized with the Golgi matrix protein GM-130 in COS-7 cells (34
). Therefore, it is possible that some of these DHHC proteins are involved in the palmitoylation of G5.
This is the first ABC transporter demonstrated to be palmityolated. In other proteins, palmitoylation has been shown to modulate protein–protein interactions and enzymatic activity as well as promote subcellular trafficking and interaction with the cell membrane (35
). Although the functional role of palmitoylation of G5 is not clear, we have shown that the modification is not required for heterodimerization, trafficking of the protein to the apical membrane, or function of the complex.
The architecture of ABC transporters includes two NBDs that catalyze the hydrolysis of ATP and two transmembrane domains that are believed to mediate substrate binding and transport. Much less is known about the role of the short C-terminal, cytoplasmic regions of the proteins. The cytoplasmic tails of some ABC transporters may be important for their subcellular localization. For example, the C-terminal regions of CFTR and MRP2 contain PDZ-interacting motifs that may play a role in sorting to the apical membrane. It has also been demonstrated that the removal of only two or three amino acids from the C terminus of ABCG2 was enough to cause impaired trafficking of the transporter (36
G5 and G8 are transported efficiently and specifically to the apical membrane of both hepatoctyes and enterocytes, yet the signals that target the heterodimer to the apical membrane are not known. In the current study, we examined whether the C-terminal regions of G5 and G8 contribute to localization of the transporter. Both of the C-terminal cytoplasmic regions of G5 and G8 are highly conserved across species (), although neither half-transporter contains a consensus sequence for established membrane targeting motifs, such as a PDZ-binding domain or a tyrosine motif. Surprisingly, despite strong conservation of the terminal 4 amino acids of G5 and the 7 amino acids of G8, these sequences were not essential for heterodimerization, apical trafficking, or sterol-mediated transport of G5G8. We cannot rule out that these sequences have more subtle effects on protein trafficking or function that were not detected using our methods.
We have shown that G5G8 can promote the transfer of cholesterol between membranes, but it is not known if the complex directly mediates movement of cholesterol from an intracellular donor to the apical membrane, from the inner to the outer leaflet, or across the bilayer to an acceptor (37
). To determine if G5G8 can directly mediate cholesterol transport across the membrane bilayer, we attempted to trap cholesterol in the interior of proteoliposomes using methyl-β-cyclodextrin (MCD). We were unable to demonstrate MCD-dependent sequestration of cholesterol in this assay. It is possible that the MCD-binding sites were already saturated with cholesterol before the assays were performed. Cholesterol from liposomes trapped with MCD during the formation of the proteoliposomes or cholesterol extracted from the membrane lipids, which have a high liposomes/protein ratio, could saturate the MCD-binding sites.
Our failure to demonstrate G5G8-mediated trans-bilayer transport may reflect inadequacies in our assay conditions. Alternatively, it may indicate that the true function of G5G8 is to transfer cholesterol to the membrane or between leaflets of the membrane rather than to transport cholesterol across the membrane. Further studies will be required to elucidate the molecular mechanism by which G5G8 promotes cholesterol secretion. The purification of a stable, functional G5G8 complex provides a valuable reagent with which to pursue these studies.