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Flippase activity in proteoliposomes reconstituted with Spinacea oleracea endoplasmic reticulum membrane proteins: Evidence of biogenic membrane flippase in plants
Published in
2008
PMID: 18767811
Volume: 47
   
Issue: 39
Pages: 10481 - 10490
Abstract
Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca2+-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals. © 2008 American Chemical Society.
About the journal
JournalBiochemistry
ISSN00062960
Open AccessNo
Concepts (59)
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    Calcium
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    Cell membranes
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    Flip flop circuits
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    LIFE SUPPORT SYSTEMS (SPACECRAFT)
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    Phospholipids
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    Proteins
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    CYTOPLASMIC MEMBRANES
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    Endoplasmic reticulum
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    ENDOPLASMIC RETICULUM MEMBRANES
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    EUKARYOTIC CELLS
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    FLIPPASE
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    IN PLANTS
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    MEMBRANE BIOGENESIS
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    METABOLIC ENERGY
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    MODEL SYSTEMS
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    Phosphatidyl choline
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    PHOSPHATIDYL ETHANOLAMINE
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    PROTEOLIPOSOMES
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    RAT LIVERS
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    TRANSLOCATOR
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    Triton x-100
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    Membranes
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    Adenosine triphosphate
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    Carrier protein
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    FLIPPASE
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    Fluorescent dye
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    MEMBRANE ENZYME
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    Membrane protein
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    Phosphatidylcholine
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    Phosphatidylethanolamine
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    Phospholipid
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    Proteinase
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    Reagent
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    SCRAMBLASE
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    Triton x 100
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    Unclassified drug
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    Article
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    Biogenesis
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    Cell membrane
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    Energy
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    Enzyme activity
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    Enzyme assay
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    EUKARYOTIC CELL
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    Membrane vesicle
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    Nonhuman
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    Priority journal
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    PROTEOLIPOSOME
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    Spinach
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    CASP8 AND FADD-LIKE APOPTOSIS REGULATING PROTEIN
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    Liposomes
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    Membrane proteins
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    PLANT LEAVES
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    PLANT PROTEINS
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    PROTEOLIPIDS
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    Spinacia oleracea
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    Animalia
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    Bacteria (microorganisms)
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    Eukaryota
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    Rattus