Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1.
We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C(16) species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.
Klappe
Karin
K
Department of Cell Biology, University Medical Center Groningen, The Netherlands.
Dijkhuis
Anne-Jan
AJ
Hummel
Ina
I
van Dam
Annie
A
Ivanova
Pavlina T
PT
Milne
Stephen B
SB
Myers
David S
DS
Brown
H Alex
HA
Permentier
Hjalmar
H
Kok
Jan W
JW
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D020404:Glycerophospholipids / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008055:Lipids / Q000032:analysis / Q000737:chemistry; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013107:Sphingolipids / Q000737:chemistry / Q000378:metabolism*
D002784:Cholesterol; D005227:Fatty Acids; D005229:Fatty Acids, Monounsaturated; D005452:Fluoresceins; D020404:Glycerophospholipids; D008055:Lipids; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011092:Polyethylene Glycols; D013107:Sphingolipids; C024098:6-carboxyfluorescein; C005092:lubrol; C421109:multidrug resistance-associated protein 1; C001996:thermozymocidin