title: New fluorescent probes reveal that flippase-mediated flip-flop of phosphatidylinositol across the endoplasmic reticulum membrane does not depend on the stereochemistry of the lipid.
abstract: Glycerophospholipid flip-flop across biogenic membranes such as the endoplasmic reticulum (ER) is a fundamental feature of membrane biogenesis. Flip-flop requires the activity of specific membrane proteins called flippases. These proteins have yet to be identified in biogenic membranes and the molecular basis of their action is unknown. It is generally believed that flippase-facilitated glycerophospholipid flip-flop across the ER is governed by the stereochemistry of the glycerolipid, but this important issue has not been resolved. Here we investigate whether the ER flippase stereochemically recognizes the glycerophospholipids that it transports. To address this question we selected phosphatidylinositol (PI), a biologically important molecule with chiral centres in both its myo-inositol headgroup and its glycerol-lipid tail. The flip-flop of PI across the ER has not been previously reported. We synthesized fluorescence-labeled forms of all four diastereoisomers of PI and evaluated their flipping in rat liver ER vesicles, as well as in flippase-containing proteoliposomes reconstituted from a detergent extract of ER. Our results show that the flippase is able to translocate all four PI isomers and that both glycerol isomers of PI flip-flop across the ER membrane at rates similar to that measured for fluorescence-labeled phosphatidylcholine. Our data have important implications for recent hypotheses concerning the evolution of distinct homochiral glycerophospholipid membranes during the speciation of archaea and bacteria/eukarya from a common cellular ancestor.
authors.affiliation: Bio-Organic Chemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India. ram@nii.res.in
authors.forename: Ram A
authors.initials: RA
authors.lastname: Vishwakarma
authors.forename: Stefanie
authors.initials: S
authors.lastname: Vehring
authors.forename: Anuradha
authors.initials: A
authors.lastname: Mehta
authors.forename: Archana
authors.initials: A
authors.lastname: Sinha
authors.forename: Thomas
authors.initials: T
authors.lastname: Pomorski
authors.forename: Andreas
authors.initials: A
authors.lastname: Herrmann
authors.forename: Anant K
authors.initials: AK
authors.lastname: Menon
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry; D000818:Animals; D004227:Dithionite / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D010716:Phosphatidylinositols / Q000138:chemical synthesis / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D005456:Fluorescent Dyes; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D004227:Dithionite; D009327:4-Chloro-7-nitrobenzofurazan