Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism

ARTICLE Received 16 Sep 2015 | Accepted 12 May 2016 | Published 23 Jun 2016 DOI: 10.1038/ncomms11919 OPEN Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism Catherine Marquer1, Huasong Tian1,w, Julie Yi1, Jayson Bastien1,w, Claudia Dall’Armi1,w, YoungJoo Yang-Klingler1, Bowen Zhou1, Robin Barry Chan1 & Gilbert Di Paolo1,w Small GTPases play a critical role in membrane traffic. Among them, Arf6 mediates transport to and from the plasma membrane, as well as phosphoinositide signalling and cholesterol homeostasis. Here we delineate the molecular basis for the link between Arf6 and cholesterol homeostasis using an inducible knockout (KO) model of mouse embryonic fibroblasts (MEFs). We find that accumulation of free cholesterol in the late endosomes/lysosomes of Arf6 KO MEFs results from mistrafficking of Niemann–Pick type C protein NPC2, a cargo of the cation-independent mannose-6-phosphate receptor (CI-M6PR). This is caused by a selective increase in an endosomal pool of phosphatidylinositol-4-phosphate (PI4P) and a perturbation of retromer, which controls the retrograde transport of CI-M6PR via sorting nexins, including the PI4P effector SNX6. Finally, reducing PI4P levels in KO MEFs through independent mechanisms rescues aberrant retromer tubulation and cholesterol mistrafficking. Our study highlights a phosphoinositide-based mechanism for control of cholesterol distribution via retromer. 1 Department of Pathology and Cell Biology, Columbia University Medical Center, New York City, New York 10032, USA. w Present addresses: Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York City, New York 10021, USA (H.T.); Laura & Isaac Perlmutter Cancer Center, NYU Langone Medical Center, New York City, New York 10016, USA (J.B.); IRBM Science Park S.P.A, 00071 Pomezia, Italy (C.D.); Denali Therapeutics, South San Francisco, California 94080, USA (G.D.P.). Correspondence and requests for materials should be addressed to C.M. (e-mail: ) or to G.D.P. (e-mail: ). NATURE COMMUNICATIONS | 7:11919 | DOI: 10.1038/ncomms11919 | www.nature.com/naturecommunications 1 ARTICLE I NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11919 ntracellular transport routes are under strict regulatory control in eukaryotic cells to ensure proper sorting of cargoes, maintain organelle identity and ultimately guarantee cell homeostasis. Among the key regulators of intracellular trafficking pathways, small GTPases, such as ADP ribosylation factor (Arf) family members, play a fundamental role in a compartmentspecific manner. Similar to other GTPases, Arf proteins cycle between an inactive GDP-bound form and an active GTP-bound form1. Unlike the other Arf family members (that is, Arf1-5), Arf6 is uniquely localized to the plasma membrane and to endosomes2,3, where it influences membrane trafficking. The role of Arf6 in various clathrin-dependent and -independent endocytic pathways as well as in recycling to the plasma membrane has been extensively studied4,5. A role for Arf6 in multivesicular body formation has also been recently described6. In addition, Arf6 regulates actin remodeling in such contexts as cell spreading, migration, cytokinesis, phagocytosis and neurite outgrowth5,7. In vivo, Arf6 ablation is embryonically lethal in the mouse8 but a conditional knockout (KO) model revealed a non-cell autonomous role for neuronal Arf6 in oligodendrocyte precursor cell migration and myelination9. One of the major mechanisms of action of Arf6 occurs through the control of lipid metabolism. Indeed, Arf6 binds and activates phosphatidylinositol-4-phosphate 5-kinases (PI4P5Ks), also known as type I PIPKs (PIPKIs), which phosphorylate PI4P into PI(4,5)P2 (ref. 10,11). In addition, Arf6 can activate phospholipase D (PLD)12, whose product phosphatidic acid can in turn activate PIPKIs (ref. 13). Overexpressing a constitutively active mutant of Arf6 (Arf6 Q67L) also results in enlarged endosomes that contain high levels of PI(4,5)P2 (ref. 14). More recently, Arf6 has been implicated in the regulation of cellular cholesterol distribution. In cultured cells, most cholesterol is derived from cholesteryl ester-rich LDL particles present in the media. LDL-particles are internalized by the LDL receptor (LDLR) and trafficked to the lumen of late endosomes/ lysosomes (LE/LYS). Cholesteryl esters are first hydrolysed by lysosomal acid lipase to free cholesterol, which is believed to be transferred by NPC2, a small soluble protein of the LE/LYS lumen, to the polytopic membrane protein NPC1. The latter mediates the egress of cholesterol from the endolysosomal system, allowing for its distribution to other cellular compartments and inhibition of cholesterol biosynthesis via the cholesterol-sensing machinery operating in the endoplasmic reticulum15,16. Perturbation of cholesterol traffic through mutations of NPC1 or NPC2 genes causes Niemann–Pick Type C (NPC) disease, a fatal neurodegenerative disorder associated with accumulation of free cholesterol and other lipids in the endolysosomal system17. Several studies provided hints that Arf6 is involved in the control of cholesterol homeostasis. First, Arf6 Q67L enlarged endosomes contain high levels of free cholesterol, labelled with filipin18. Second, silencing Arf6 was found to increase cellular cholesterol content in HeLa cells, reminiscent of an NPC disease phenotype19. In addition, Arf6 and components of its interactome were identified as ‘hits’ in a transcriptomic screen performed after cholesterol levels of cultured rat neurons were acutely increased20. More recently, Arf6 was a hit in a genomewide RNA-interference screen designed to identify genes required for intracellular transport of LDL-derived cholesterol, although this link was not explored further21. While these studies point to a link between Arf6 and cholesterol metabolism, the molecular basis underlying this relationship is unknown. To address this fundamental question, we developed a novel model of Arf6 conditional KO in immortalized mouse embryonic fibroblasts (MEFs). We found that Arf6 depletion leads to cholesterol redistribution and accumulation in LE/LYS, a phenotype due to the mistargeting of NPC2 away from 2 lysosomes. Because NPC2 is a cargo of the mannose-6-phosphate receptor (M6PR)22, we examined the function of retromer, which regulates the retrograde transport of the M6PR. We found that retromer function is impaired in Arf6 KO cells, thus leading to mislocalization of the cation-independent M6PR (CI)-M6PR away from the trans-Golgi network (TGN). We also found that PI4P levels were increased in Arf6 KO MEFs and that retromerpositive aberrant tubules emerged in part from PI4P-containing endosomes. Finally, we demonstrated that restoring PI4P levels rescues aberrant retromer tubules and cholesterol accumulation. This work therefore highlights a novel role for Arf6 in the regulation of retromer with critical implications for (...truncated)