Role of STARD 4 in sterol transport between the endocytic recycling compartment and the plasma membrane

Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and nonvesicular sterol transport processes. Using the fluorescent cholesterol analogue dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a t1/2 =12–15 min. Approximately 70% of sterol transport is ATP independent and therefore is nonvesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. A soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of nonvesicular sterol transport between the plasma membrane and ERC. This study shows that nonvesicular sterol transport mechanisms and STARD4 in particular account for a large fraction of sterol transport between the plasma membrane and the ERC. INTRODUCTION Cholesterol is an essential constituent of biological membranes. Structurally, cholesterol has a single hydroxyl as the sole polar component, four rings, and a short alkyl chain, whereas most other lipids, such as glycerophospholipids and sphingolipids, are composed of large polar head groups and two long hydrocarbon chains (van Meer, 2005; Iaea and Maxfield, 2015). The unique chemical structure of cholesterol allows it to be absorbed into and extracted from membranes easily (Phillips et al., 1987). Cholesterol substantially affects the biophysical properties of membranes because it is able to pack against the acyl chains of phospholipids, reduce membrane permeability, and facilitate the formation of liquid-ordered domains in membranes (Huang and Feigenson, 1999; Pani et al., 2008; Galan et al., 2010). The abundance and distribution of cholesterol within membranes are tightly controlled by complex homeostatic mechanisms (Mesmin and Maxfield, 2009; Iaea et al., 2014; Iaea and Maxfield, 2015). Understanding the intracellular dynamics of cholesterol is critical because the proper distribution among the plasma membrane and cellular organelle membranes is important for cellular function (van Meer and Sprong, 2004; Maxfield and Menon, 2006; Simons and Gerl, 2010). Cholesterol distribution among cellular compartments and within the membrane is heterogeneous (Iaea and Maxfield, 2015). The plasma membrane is the largest cholesterol pool in the cell and contains ∼60% of total cellular cholesterol in cells such as fibroblasts (Liscum and Munn, 1999). Cholesterol can flip rapidly between two membrane leaflets in a bilayer, and it has been shown that the half-time is <1 s in erythrocytes (Lange et al., 1981; Steck et al., 2002). Cholesterol is also highly enriched in the endocytic Monitoring Editor Howard Riezman University of Geneva Received: Aug 24, 2016 Revised: Feb 3, 2017 Accepted: Feb 8, 2017 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E16-07-0499) on February 16, 2017. Present addresses: †Genentech, 1 DNA Way, South San Francisco, CA 94080; ‡Regeneron Pharmaceuticals, 777 Old Saw Mill River Rd., Tarrytown, NY 10591. *Address correspondence to: Frederick R. Maxfield (frmaxfie@med.cornell.edu). © 2017 Iaea et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. Abbreviations used: ACAT, Acyl-CoA:cholesterol acyltransferase; ATP, adenosine triphosphate; CRISPR, clustered regularly interspaced short palindromic repeats; DHE, dehydroergosterol; ER, endoplasmic reticulum; ERC, endocytic recycling compartment; FRAP, fluorescence recovery after photobleaching; HPCD, hydroxypropyl cyclodextrin; LPDS, lipoprotein-depleted serum; MCD, methyl-βcyclodextrin; SRA, scavenger receptor A; SREBP-2, sterol-responsive element– binding protein-2; StAR, steroidogenic acute regulatory protein; STARD, START domain; START, StAR–related lipid-transfer; STPs, sterol transfer proteins; Tf, transferrin; U2OS, human osteosarcoma. David B. Iaeaa,b,†, Shu Maoa,‡, Frederik W. Lunda, and Frederick R. Maxfielda,b,* aDepartment of Biochemistry, Weill Cornell Medical College, New York, NY 10065; bWeill Cornell Medical College, Rockefeller University, and Memorial Sloan-Kettering Cancer Center Tri-Institutional Chemical Biology Program, New York, NY 10065