Identification and characterization of nonsedimentable lipid-protein microvesicles.

Abstract

Previously uncharacterized lipid-protein microvesicles have been isolated from young and senescing bean cotyledon tissue. The microvesicles are nonsedimentable and enriched in phospholipid degradation products (free fatty acids, long-chain aldehydes, and long-chain hydrocarbons). They range from 70 to 170 nm (radius) with a mean radius of 132 nm, and it is clear from freeze-fracture electron micrographs that they are bilayered in nature. Nonsedimentable lipid-protein microvesicles containing the same products of phospholipid degradation but smaller were also formed in vitro when smooth microsomal membranes from young cotyledon tissue were treated with Ca2' to stimulate enzymatic degradation of phospholipids. The data suggest that these microvesices comprise an intermediate stage of membrane lipid deterioration. They appear to serve as a vehicle for moving phospholipid degradation products out of membranes into the cytosol during senescence and perhaps also during normal membrane lipid turnover.Membrane lipid deterioration is an inherent feature of plant senescence and of capitulation of plant tissues to certain types of stress including drought and freezing (1)(2)(3)(4). One of the clearest manifestations of this is a progressive decline in phospholipid phosphate and fatty acids resulting in an increase in the sterol/esterified fatty acid ratio in membranes and a corresponding decrease in membrane bulk lipid fluidity (1). There is also an accumulation of free fatty acids and peroxidized lipids in senescing membranes that alter the phase properties of membrane lipids and introduce packing perturbations in the bilayer that are thought to facilitate enzymatic degradation of lipids (1,(5)(6)(7). In addition, membrane phospholipids have been shown to have relatively rapid turnover rates ranging in half-life from 1 to 10 hr (8).Several lipolytic enzymes that could participate in membrane lipid turnover and in the net degradation of membrane lipids that accompanies senescence and capitulation to stress have been found associated with plant membranes. Phospholipase D activity has been detected in isolated fractions of endoplasmic reticulum, Golgi membranes, tonoplast, and plasmalemma (9-12), and it has been proposed that the membrane-associated form ofthe enzyme participates in lipid turnover (13). Phosphatidic acid phosphatase has been found on endoplasmic reticulum and in association with other isolated membrane fractions (14-16), and there are reports of lipolytic acyl hydrolase being associated with microsomal membranes (17,18). Moreover, a recent study has indicated that phospholipase D, phosphatidic acid phosphatase, and lipolytic acyl hydrolase are all enriched in the sedimentable material obtained after partial solubilization ofmicrosomes in Triton X-100, suggesting that they are tightly associated with the membranes (19).The precise mechanism by which phospholipid degradation products are removed from membranes has not been established, although there have been some reports of microvesiculation from mem...

Disruption of Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Gene Altered Cuticular Lipid Composition, Increased Plastoglobules, and Enhanced Susceptibility to Infection by the Fungal Pathogen Alternaria brassicicola

“…However, these phenomena were not observed in the Arabidopsis cer5 and wbc11 mutants, which were deficient in cuticular lipid export. Plastoglobules are lipid bodies formed as a result of a "blistering" of the stroma-side leaflet of the thylakoid membrane (Yao et al, 1991;Austin et al, 2006). The size and number of plastoglobules change during plastid development and differentiation as well as under stress conditions.…”

A Role for Diacylglycerol Acyltransferase during Leaf Senescence

“…Internal standards (diheptadecanoyl l-␣-phosphatidylcholine, diarachidin, heptadecanoic acid, triheptadecanoic acid, and cholesteryl arachidate) were added before extraction. Lipid extracts were fractionated by thin layer chromatography (Yao et al, 1991), and the separated lipids were visualized with iodine vapor and identified using authentic standards. Fatty acids of the separated lipid fractions were transmethylated (Morrison and Smith, 1964), and the resultant fatty acid methyl esters were quantified by gas chromatographymass spectrometry (HP-5890 series II gas chromatograph equipped with a DB Wax column; 30-ϫ 0.25-mm i.d., 0.25-m film, J&W Scientific, Folsom, CA) and a mass detector (HP-5970) with a scan range of m/z 35-150 operating at 0.16 s/scan.…”

Subcellular Localization of Secondary Lipid Metabolites Including Fragrance Volatiles in Carnation Petals

“…The cytosolic lipid-protein particles also contain a lower-molecular-weight protein (approximately 17 kD) that may be analogous to oil body oleosin (Hudak and Thompson, 1996). Lipid-protein particles similar to those in carnation petals have also been isolated from the cytosol of bean cotyledon tissue (Yao et al, 1991a;McKegney et al, 1995) and from the stroma of chloroplasts (Ghosh et al, 1994). Of particular interest is the finding that the cytosolic and organellar lipid-protein particles are enriched in membrane lipid and protein metabolites (Yao et al, 1991a;Ghosh et al, 1994;Hudak et al, 1995).…”

“…Lipid-protein particles similar to those in carnation petals have also been isolated from the cytosol of bean cotyledon tissue (Yao et al, 1991a;McKegney et al, 1995) and from the stroma of chloroplasts (Ghosh et al, 1994). Of particular interest is the finding that the cytosolic and organellar lipid-protein particles are enriched in membrane lipid and protein metabolites (Yao et al, 1991a;Ghosh et al, 1994;Hudak et al, 1995). This has prompted the proposal that their formation may be an integral feature of membrane turnover, allowing removal of metabolites that would otherwise destabilize the structure of the membrane bilayer (Yao et al, 1991a;Hudak et al, 1995).…”