Date Published: September 20, 2018
Publisher: Public Library of Science
Author(s): Megan T. Harris, Syed Saad Hussain, Candice M. Inouye, Anna M. Castle, J. David Castle, Xuewei Zhu.
The ABC transporter ABCG1 contributes to the regulation of cholesterol efflux from cells and to the distribution of cholesterol within cells. We showed previously that ABCG1 deficiency inhibits insulin secretion by pancreatic beta cells and, based on its immunolocalization to insulin granules, proposed its essential role in forming granule membranes that are enriched in cholesterol. While we confirm elsewhere that ABCG1, alongside ABCA1 and oxysterol binding protein OSBP, supports insulin granule formation, the aim here is to clarify the localization of ABCG1 within insulin-secreting cells and to provide added insight regarding ABCG1’s trafficking and sites of function. We show that stably expressed GFP-tagged ABCG1 closely mimics the distribution of endogenous ABCG1 in pancreatic INS1 cells and accumulates in the trans-Golgi network (TGN), endosomal recycling compartment (ERC) and on the cell surface but not on insulin granules, early or late endosomes. Notably, ABCG1 is short-lived, and proteasomal and lysosomal inhibitors both decrease its degradation. Following blockade of protein synthesis, GFP-tagged ABCG1 first disappears from the ER and TGN and later from the ERC and plasma membrane. In addition to aiding granule formation, our findings raise the prospect that ABCG1 may act beyond the TGN to regulate activities involving the endocytic pathway, especially as the amount of transferrin receptor is increased in ABCG1-deficient cells. Thus, ABCG1 may function at multiple intracellular sites and the plasma membrane as a roving sensor and modulator of cholesterol distribution, membrane trafficking and cholesterol efflux.
In eukaryotic cells, the ATP Binding Cassette (ABC) transporters ABCA1 and ABCG1 are known to promote cholesterol export from cells and have been of substantial interest due to their complementary roles alongside cholesterol uptake, biosynthesis and storage in maintaining intracellular cholesterol homeostasis [1,2]. While these transporters and their homologs among mammals and lower organisms are broadly expressed , their levels are amplified in cells, e.g., macrophages and type-2 pneumocytes that are specialized for processing and exporting lipids including cholesterol physiologically [4–6]. A major focus in studying their actions has been on the mechanisms and pathways they use to transfer cholesterol to plasma lipoproteins (reviewed in ). Several studies have also highlighted the ability of ABCA1 and ABCG1 to promote cholesterol esterification and storage under conditions that preclude cholesterol export [8–10] and to regulate the degree of lipid ordering in membranes and membrane content of cholesterol. The latter actions of the transporters may couple cholesterol export or redistribution to various processes including modulation of cell-to-cell versus cell-to-extracellular matrix interactions and inflammatory responses (ABCA1: [11,12]), proliferation of immune and hematopoietic cells (ABCG1: [13–15]), and insulin secretion (ABCs A1 and G1: [16–19]). ABCG1 has not been studied as extensively as ABCA1, which gained early and enduring attention due to the link between its deficiency and Tangier disease, where intracellullar cholesterol levels are increased and plasma levels of high density lipoprotein are dramatically decreased . ABCG1 is able to contribute to the export of cholesterol , especially when experimentally induced or overexpressed or under conditions of increased cellular cholesterol load [2,4,9,21–25], and its deficiency leads to profound intracellular cholesterol accumulation in certain cell types . While these actions implicate its presence on the plasma membrane, other findings argue that ABCG1 is mainly concentrated intracellularly rather than at the cell surface [5,17,27]. Further, its deficiency in some cells has either a modest or no effect on cellular cholesterol levels [4,5,14,17]. Thus, increasing attention has been paid to ABCG1’s possible intracellular roles that might complement its function in cholesterol export .
In this study, our main mission has been to reevaluate and clarify the localization, trafficking, and sites of function of ABCG1 in pancreatic beta cells. Because our cell fractionation studies (Fig 1) showed that neither ABCG1 nor GFP-G1 distributed appreciably with markers of insulin granules, we felt compelled to modify our previous view that ABCG1 is extensively associated with these granules  and to identify the technical limitations that were probably responsible for the earlier interpretation. Indeed, our current observation showing that little ABCG1 is associated with insulin granules, which are generally long-lived, is consistent with the short half-life of the transporter that we now report (Fig 4). However, we note that ABCG1’s presence at low levels in granule membranes is not fully ruled out, especially in islet beta cells that are more differentiated than INS1 and MIN6 cells and maintain a much larger granule storage pool. Indeed, our earlier fractionation of pancreatic islets indicated presence of ABCG1 in lower density granule fractions . Consequently, ABCG1 may maintain an association with a subset of insulin granules, particularly during their formation and be progressively removed as the granules mature. This possibility is consistent with our recent demonstration that ABCG1 supports the process of insulin granule formation . Nevertheless, based on our present results, the majority of ABCG1 is clearly not granule-associated.