Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains - eScholarship

JCB: Article Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains Adam Kassan, 1 Albert Herms, 1 Andrea Fernandez-Vidal, 1 Marta Bosch, 1 Nicole L. Schieber, 2,3 Babu J.N. Reddy, 4 Alba Fajardo, 1 Mariona Gelabert-Baldrich, 1 Francesc Tebar, 1,5 Carlos Enrich, 1,5 Steven P. Gross, 4 Robert G. Parton, 2,3 and Albert Pol 1,5,6 Equip de Senyalitzacio i Proliferacio Cellular, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain The Institute for Molecular Bioscience and 3 Centre for Microscopy and Microanalysis, The University of Queensland, Brisbane, Queensland 4072, Australia Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697 Departament de Biologia Cellular, Immunologia i Neurociencies, Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain Institucio Catalana de Recerca i Estudis Avancats (ICREA), 08010 Barcelona, Spain THE JOURNAL OF CELL BIOLOGY C ontrol of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, pro- cesses. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of “emerging” LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demon- strates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storage. Introduction Lipid droplets (LDs) are ubiquitous organelles that collect, store, and supply lipids (Walther and Farese, 2012). Nonethe- less, excessive or reduced accumulations of LDs are hallmarks of prevalent human diseases including steatohepatitis, obesity, diabetes, myopathies, arteriosclerosis, or lipodystrophies. How- ever, relatively little is known about the molecular processes and sites that control LD formation. In eukaryotes, LDs likely form de novo by accumulation of neutral lipids in the ER. Con- sistent with this, the ER harbors enzymes required for neutral lipid synthesis (Buhman et al., 2001), many ER proteins are re- quired for LD formation and expansion (Brasaemle and Wolins, 2012), LDs can be generated in vitro with ER microsomes (Lacey et al., 1999; Marchesan et al., 2003), and there exists an active partitioning of proteins between the ER and LDs (Jacquier et al., 2011). However, although it has been possible A. Kassan, A. Herms, and A. Fernandez-Vidal contributed equally to this paper. Correspondence to Robert G. Parton: r.parton@imb.uq.edu.au; or Albert Pol: apols@ub.edu Abbreviations used in this paper: ACSL, acyl-CoA synthetase long-chain family; LD, lipid droplet; OA, oleic acid; OFP, orange fluorescent protein; pre-LD, pre- existing LD; qRT-PCR, quantitative RT-PCR. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 203 No. 6 985–1001 www.jcb.org/cgi/doi/10.1083/jcb.201305142 to detect early LDs in the proximity of the ER (Pol et al., 2004; Wolins et al., 2005; Turro et al., 2006; Kuerschner et al., 2008; Skinner et al., 2009; Poppelreuther et al., 2012), whether these are indeed nascent LDs, and whether specialized microdomains existed before the recruitment of these proteins was unknown. Indeed, it was commonly assumed that direct imaging of newly forming LDs was impossible with the current methodology (Salo et al., 2011; Suzuki et al., 2011). The generally accepted model is that triglycerides are deposited as a lens within the ER bilayer. This process is presumably regulated by proteins, as LD formation is not spontaneously triggered by accumulation of neutral lipids in the ER (Gubern et al., 2008; Adeyo et al., 2011). Thus, ER proteins that can extend hydrophobic domains into the bi- layer are attractive candidates to recognize and organize the sites of nucleation. We previously identified a localization signal for sorting proteins with a hydrophobic domain within © 2013 Kassan et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- lication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). JCB