Department of Biology, East Carolina University, Greenville, NC, USA.
Department of Biological Sciences, University of North Texas, Denton, TX, USA.
Methods Mol Biol. 2021;2295:295-320. doi: 10.1007/978-1-0716-1362-7_16.
Cytosolic lipid droplets (LDs) are organelles which emulsify a variety of hydrophobic molecules in the aqueous cytoplasm of essentially all plant cells. Most familiar are the LDs from oilseeds or oleaginous fruits that primarily store triacylglycerols and serve a storage function. However, similar hydrophobic particles are found in cells of plant tissues that package terpenoids, sterol esters, wax esters, or other types of nonpolar lipids. The various hydrophobic lipids inside LDs are coated with a phospholipid monolayer, mostly derived from membrane phospholipids during their ontogeny. Various proteins have been identified to be associated with LDs, and these may be cell-type, tissue-type, or even species specific. While major LD proteins like oleosins have been known for decades, more recently a growing list of LD proteins has been identified, primarily by proteomics analyses of isolated LDs and confirmation of their localization by confocal microscopy. LDs, unlike other organelles, have a density less than that of water, and consequently can be isolated and enriched in cellular fractions by flotation centrifugation for composition studies. However, due to its deep coverage, modern proteomics approaches are also prone to identify contaminants, making control experiments necessary. Here, procedures for the isolation of LDs, and analysis of LD components are provided as well as methods to validate the LD localization of proteins.
细胞质脂滴（LDs）是一种细胞器，可将各种疏水分子乳化在基本所有植物细胞的水细胞质中。最常见的是来自油籽或含油果实的 LDs，它们主要储存三酰基甘油并起到储存作用。然而，在植物组织的细胞中也发现了类似的疏水性颗粒，它们用于包装萜类、固醇酯、蜡酯或其他类型的非极性脂质。LD 内的各种疏水性脂质都被一层磷脂单层包裹，这些磷脂主要来源于它们在个体发育过程中的膜磷脂。已经鉴定出多种与 LDs 相关的蛋白质，这些蛋白质可能是细胞类型、组织类型，甚至是物种特异性的。虽然几十年来人们一直知道主要的 LD 蛋白，如油体蛋白，但最近越来越多的 LD 蛋白被鉴定出来，主要是通过对分离的 LDs 的蛋白质组学分析，并通过共聚焦显微镜确认其定位。与其他细胞器不同，LDs 的密度小于水，因此可以通过漂浮离心法在细胞组分中分离和富集，用于组成研究。然而，由于其深度覆盖，现代蛋白质组学方法也容易识别污染物，因此需要进行对照实验。本文提供了 LDs 的分离和 LD 成分分析的程序，以及验证蛋白质 LD 定位的方法。
