Phospholipids in tissues of the eye I. Isolation, characterization and quantitative analysis by two-dimensional thin-layer chromatography of diacyl and vinyl-ether phospholipids

Broekhuyse, R.M.

doi:10.1016/0005-2760(68)90038-6

Cited by 322 publications

(88 citation statements)

References 19 publications

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“…Two-dimensional thin layer chromatography was used to separate the individual phospholipid classes (94). Finally, phospholipids were stained with iodine vapor, scraped off, and quantified (95).…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Schuler¹,

Bartolomeo

Mårtensson

et al. 2016

Journal of Biological Chemistry

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Two protein translocases transport precursor proteins into or across the inner mitochondrial membrane. The presequence translocase (TIM23 complex) sorts precursor proteins with a cleavable presequence either into the matrix or into the inner membrane. The carrier translocase (TIM22 complex) inserts multispanning proteins into the inner membrane. Both protein import pathways depend on the presence of a membrane potential, which is generated by the activity of the respiratory chain. The non-bilayer-forming phospholipids cardiolipin and phosphatidylethanolamine are required for the activity of the respiratory chain and therefore to maintain the membrane potential for protein import. Depletion of cardiolipin further affects the stability of the TIM23 complex. The role of bilayer-forming phospholipids like phosphatidylcholine (PC) in protein transport into the inner membrane and the matrix is unknown. Here, we report that import of presequence-containing precursors and carrier proteins is impaired in PC-deficient mitochondria. Surprisingly, depletion of PC does not affect stability and activity of respiratory supercomplexes, and the membrane potential is maintained. Instead, the dynamic TIM23 complex is destabilized when the PC levels are reduced, whereas the TIM22 complex remains intact. Our analysis further revealed that initial precursor binding to the TIM23 complex is impaired in PC-deficient mitochondria. We conclude that reduced PC levels differentially affect the TIM22 and TIM23 complexes in mitochondrial protein transport.Mitochondria fulfill essential functions for the survival of the cell like energy conversion to produce ATP, synthesis of amino acids, lipids, and heme, as well as the generation of iron-sulfur clusters. They contain about 1000 proteins in yeast and 1500 proteins in humans (1, 2). More than 99% of the mitochondrial proteins are synthesized as precursors on cytosolic ribosomes. Mitochondria contain a sophisticated system of protein translocases to import precursor proteins (3-9). The translocase of the outer membrane (TOM 3 complex) forms the general entry gate for most precursor proteins. After passage of the TOM channel, distinct protein translocases sort the preproteins into the different subcompartments: the outer and inner membrane as well as the two aqueous compartments, the matrix and intermembrane space.The majority of mitochondrial proteins are sorted into the inner membrane and the matrix. Two inner membrane-bound protein complexes mediate protein import. The presequence translocase (also termed TIM23 complex) transports precursor proteins with a cleavable presequence into the inner membrane and the matrix, whereas the carrier translocase (also termed TIM22 complex) inserts proteins with multiple transmembrane segments into the inner membrane (3-9). The membrane potential across the inner membrane provides the driving force for both protein import pathways and is generated by the activity of the respiratory chain. Presequence-containing preproteins are directly transferred from the ...

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Section: Methodsmentioning

confidence: 99%

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Schuler¹,

Bartolomeo

Mårtensson

et al. 2016

Journal of Biological Chemistry

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“…For quantification of total phospholipids, a lipid extract of total cell extracts (800 g of protein) was analyzed by the method of Broekhuyse (34). Individual phospholipids were analyzed from total cell lipid extracts (2 mg of protein) by twodimensional TLC using chloroform/methanol/25% ammonia (65:35:5 per volume) as solvent system for the first dimension, and chloroform/acetone/methanol/acetic acid/water (50:20: 10:10:5 per volume) for the second dimension.…”

Section: Methodsmentioning

confidence: 99%

“…Individual phospholipids were analyzed from total cell lipid extracts (2 mg of protein) by twodimensional TLC using chloroform/methanol/25% ammonia (65:35:5 per volume) as solvent system for the first dimension, and chloroform/acetone/methanol/acetic acid/water (50:20: 10:10:5 per volume) for the second dimension. Spots were visualized by staining with iodine vapor, scraped off, and quantified by the method of Broekhuyse (34).…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Yeast Triacylglycerol Lipase Tgl3p by Formation of Nonpolar Lipids

Schmidt

Athenstaedt

Koch

et al. 2013

Journal of Biological Chemistry

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Background: Tgl3p from yeast serves as the major triacylglycerol lipase but also as lysophospholipid acyltransferase. Results: Formation of nonpolar lipids strongly affects subcellular localization and function of Tgl3p. Conclusion: Tgl3p activity is mainly regulated by the presence/absence of lipid droplets. Significance: The yeast lipase/acyltransferase Tgl3p is an important player in lipid homeostasis.

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“…Phospholipids were separated by two-dimensional thin-layer chromatography on silica gel 60 plates (Merck, Darmstadt, Germany) using chloroform/methanol/25% NH 3 (65:35:5; per volume) as the first solvent and chloroform/acetone/ methanol/acetic acid/water (50:20:10:10:5; per volume) as the second solvent. Phospholipids were visualized on TLC plates by staining with iodine vapor, scraped off, and quantified (Broekhuyse, 1968).…”

Section: Methodsmentioning

confidence: 99%

Loss of Swiss Cheese/Neuropathy Target Esterase Activity Causes Disruption of Phosphatidylcholine Homeostasis and Neuronal and Glial Death in AdultDrosophila

Mühlig-Versen¹,

Cruz²,

Tschäpe³

et al. 2005

J. Neurosci.

133

140

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The Drosophila Swiss cheese (sws) mutant is characterized by progressive degeneration of the adult nervous system, glial hyperwrapping, and neuronal apoptosis. The Swiss cheese protein (SWS) shares 39% sequence identity with human neuropathy target esterase (NTE), and a brain-specific deletion of SWS/NTE in mice causes a similar pattern of progressive neuronal degeneration. NTE reacts with organophosphate compounds that cause a paralyzing axonal degeneration in humans and has been shown to degrade endoplasmic reticulum-associated phosphatidylcholine (PtdCho) in cultured mammalian cells. However, its function within the nervous system has remained unknown. Here, we show that both the fly and mouse SWS proteins can rescue the defects that arise in sws mutant flies, whereas a point mutation in the proposed active site cannot restore SWS function. Overexpression of catalytically active SWS caused formation of abnormal intracellular membraneous structures and cell death. Cell-specific expression revealed that not only neurons but also glia depend autonomously on SWS. In wild-type flies, endogenous SWS was detected by immmunohistochemistry in the endoplasmic reticulum (the primary site of PtdCho processing) of neurons and in some glia. sws mutant flies lacked NTE-like esterase activity and had increased levels of PtdCho. Conversely, overexpression of SWS resulted in increased esterase activity and reduced PtdCho. We conclude that SWS is essential for membrane lipid homeostasis and cell survival in both neurons and glia of the adult Drosophila brain and that NTE may play an analogous role in vertebrates.

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Phospholipids in tissues of the eye I. Isolation, characterization and quantitative analysis by two-dimensional thin-layer chromatography of diacyl and vinyl-ether phospholipids

Cited by 322 publications

References 19 publications

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Regulation of the Yeast Triacylglycerol Lipase Tgl3p by Formation of Nonpolar Lipids

Loss of Swiss Cheese/Neuropathy Target Esterase Activity Causes Disruption of Phosphatidylcholine Homeostasis and Neuronal and Glial Death in AdultDrosophila

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