No abstract

The lipid droplet (LD) is a cell organelle that has been linked to human metabolic syndromes and that can be exploited for the development of biofuels. The isolation of LDs is crucial for carrying out morphological and biochemical studies of this organelle. In the past two decades, LDs have been isolated from several organisms and investigated by microscopy, proteomics and lipidomics. However, these studies need to be extended to more model organisms, as well as to more animal tissues. Thus, a standard method that can be easily applied to these new samples with the need for minimal optimization is essential. Here we provide an LD isolation protocol that is relatively simple and suitable for a wide range of tissues and organisms. On the basis of previous studies, this 7-h protocol can yield 15-100 μg of protein-equivalent high-quality LDs that satisfy the requirements for current LD research in most organisms.

Lipid droplets (LDs) are intracellular organelles with neutral lipid cores surrounded by a phospholipid monolayer and coated with various proteins ( 1-3 ). LDs have been found in almost all eukaryotic organisms from yeast to mammals ( 4 ). They interact with other cellular organelles ( 5-8 ), and their dynamics is closely related to the progression of metabolic diseases, such as obesity, fatty liver, type 2 diabetes mellitus, and atherosclerosis ( 9 ). Recent studies have also shown that LDs are involved in the reproduction of infectious hepatitis C virus particles ( 10 ) and in protecting cells from damage ( 11 ). The identifi cation of perilipin, ADRP, and Tip47 (PAT) family proteins has provided useful marker proteins to facilitate the purifi cation of LDs. Recent proteomic studies suggesting that LDs are not simply inert cellular inclusions for the storage of neutral lipids, but rather functional cellular organelles, has established a new era in LD research ( 3,(12)(13)(14)(15)(16)(17)(18).Although LDs are highly dynamic organelles involved in many cellular activities, especially lipid metabolism, the molecular mechanisms that govern LD formation remain largely unknown. The current model of LD biogenesis speculates that LDs are derived from the endoplasmic reticulum (ER) by a process that begins with the accumulation of neutral lipids between the leafl ets of phospholipid bilayers ( 3,19 ). Many studies have attempted to unravel how LDs form and grow, but this hypothesis still lacks direct evidence and the molecular mechanism Abstract Storage of cellular triacylglycerols (TAGs) in lipid droplets (LDs) has been linked to the progression of many metabolic diseases in humans, and to the development of biofuels from plants and microorganisms. However, the biogenesis and dynamics of LDs are poorly understood. Compared with other organisms, bacteria seem to be a better model system for studying LD biology, because they are relatively simple and are highly effi cient in converting biomass to TAG. We obtained highly purifi ed LDs from Rhodococcus sp. RHA1, a bacterium that can produce TAG from many carbon sources, and then comprehensively characterized the LD proteome. Of the 228 LD-associated proteins identifi ed, two major proteins, ro02104 and PspA, constituted about 15% of the total LD protein. (Grant 2009CB919000, Grant 2010CB833703; Grant 2011CBA00900), and the National Natural Science Foundation of China (Grant 30871229, Grant 30971431, and Grant 31000365 Abbreviations: ER, endoplasmic reticulum; LD, lipid droplet; MLDS, microorganism lipid droplet small; MSM, mineral salt medium; NB, nutrient broth; PAT, perilipin, ADRP, and Tip47; PspA, phage shock protein A; TAG, triacylglycerol; TEM, transmission electron microscopy.