**6. Post-Transcriptional Regulation**

Less is known about the post-transcriptional regulation of the G5G8. The half transporters are retained within the endoplasmic reticulum (ER) unless co-expressed [124,125]. Formation of the complex appears to be relatively inefficient in cultured cells, is dependent upon the presence of N-linked glycans that reside in the third extracellular loop of each protein, and can be enhanced by the expression of the lectin chaperones, Calnexin and Calreticulin [124–127]. Using chimeric approaches, the ER-retention motif was localized to the N-terminal, cytosolic domain, but has yet to be defined [128]. Failure to form complexes within the ER results in rapid degradation of each half transporter [127,129]. At the cell surface, the mature G5G8 complex resides within apical membranes of both hepatocytes and enterocytes [124]. There is also evidence of an intracellular, recruitable pool of G5G8 that translocates to the canalicular surface in response to cAMP and in response to diets containing cholate and cholesterol [114,130]. However, the stimuli and signaling pathways involved in intracellular trafficking of G5G8 have yet to be elucidated.

A number of approaches have been utilized to investigate the activity of G5G8. Heterologous expression in HEK293 and dog gall bladder epithelial cells demonstrated G5G8-dependent cholesterol efflux to bile acid micelles, but not HDL or apolipoprotein A1 [131,132]. Native mouse and recombinant human and mouse G5G8 have been purified to varying degrees from liver, rat hepatocytes, Sf9 insect cells, and *Pichia pastoris* [133–136]. These studies demonstrated ATP- and magnesium-dependent, vanadate-sensitive ATPase, and sterol transport activity. Various bile acids stimulate ATP hydrolysis. Among the species tested, G5G8 activity was most sensitive to cholate [133]. Perhaps surprisingly, neither cholesterol nor phytosterols stimulated ATPase or sterol transport activity in preparations from Sf9 cells [135,136]. Using inside-out vesicles in this same system, Wang et al. showed that other nucleotides could support sterol transfer, albeit less efficiently [135]

The nucleotide binding sites of G5G8 were proposed, and later confirmed by crystallography, to be comprised of Walker A and B domains of one partner and the signature motif of the other [137,138]. The Walker A and B domains of G8 juxtaposed to the signature motif of G5 were designated nucleotide binding site (NBS) 1. While both NBSs bind 8-Azido ATP, mutations in highly conserved residues within the Walker A and B domain of G5 (NBS2), but not G8 (NBS1), abolished ATP binding and hydrolysis. These findings were confirmed for G5G8-mediated biliary cholesterol secretion by expressing the mutants in G5G8-defecient mice. Domain swapping experiments between G5 and G8 confirmed that ATP hydrolysis in NBS2 is indispensable for activity [139]. G5G8 was crystallized as a heterodimer in lipid bilayers (bicelles) in the presence of cholesterol in the nucleotide free state to a resolution of four angstroms [138]. G5G8 was designated as a Type II Exporter. Key molecular interactions inferred from this structure were validated as essential for cholesterol transport in vivo by expressing recombinant mutants in G5G8-deficient mice. Naturally occurring missense variants and mutants can provide mechanistic insight to protein structure–function. The potential impact of mutations and polymorphisms on G5G8 structure function inferred from the available crystal structure were recently reviewed [140]. However, formal investigations into the impact of missense variants of any type on G5G8 trafficking, stability, and activity have been limited to only a few.
