Reconstitution of glucosylceramide flip-flop across endoplasmic reticulum: implications for mechanism of glycosphingolipid biosynthesis. Academic Article uri icon

Overview

MeSH

  • Animals
  • Biological Transport
  • Intracellular Membranes
  • Lipid Bilayers
  • Liposomes
  • Membrane Proteins
  • Membrane Transport Proteins
  • Microsomes, Liver
  • Models, Biological
  • Octoxynol
  • Phospholipid Transfer Proteins
  • Proteolipids
  • Rats
  • Saccharomyces cerevisiae Proteins
  • Transport Vesicles

MeSH Major

  • Endoplasmic Reticulum
  • Glucosylceramides
  • Glycosphingolipids
  • Golgi Apparatus

abstract

  • Most glycosphingolipids are synthesized by the sequential addition of monosaccharides to glucosylceramide (GlcCer) in the lumen of the Golgi apparatus. Because GlcCer is synthesized on the cytoplasmic face of Golgi membranes, it must be flipped to the non-cytoplasmic face by a lipid flippase in order to nucleate glycosphingolipid synthesis. Halter et al. (Halter, D., Neumann, S., van Dijk, S. M., Wolthoorn, J., de Mazière, A. M., Vieira, O. V., Mattjus, P., Klumperman, J., van Meer, G., and Sprong, H. (2007) Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol. 179, 101-115) proposed that this essential flipping step is accomplished via a complex trafficking itinerary; GlcCer is moved from the cytoplasmic face of the Golgi to the endoplasmic reticulum (ER) by FAPP2, a cytoplasmic lipid transfer protein, flipped across the ER membrane, then delivered to the lumen of the Golgi complex by vesicular transport. We now report biochemical reconstitution studies to analyze GlcCer flipping at the ER. Using proteoliposomes reconstituted from Triton X-100-solubilized rat liver ER membrane proteins, we demonstrate rapid (t(½) < 20 s), ATP-independent flip-flop of N-(6-((7-nitro-2-1,3-benzoxadiazol-4-yl)amino)hexanoyl)-D-glucosyl-β1-1'-sphingosine, a fluorescent GlcCer analog. Further studies involving protein modification, biochemical fractionation, and analyses of flip-flop in proteoliposomes reconstituted with ER membrane proteins from yeast indicate that GlcCer translocation is facilitated by well characterized ER phospholipid flippases that remain to be identified at the molecular level. By reason of their abundance and membrane bending activity, we considered that the ER reticulons and the related Yop1 protein could function as phospholipid-GlcCer flippases. Direct tests showed that these proteins have no flippase activity.

publication date

  • May 4, 2012

has subject area

  • Animals
  • Biological Transport
  • Endoplasmic Reticulum
  • Glucosylceramides
  • Glycosphingolipids
  • Golgi Apparatus
  • Intracellular Membranes
  • Lipid Bilayers
  • Liposomes
  • Membrane Proteins
  • Membrane Transport Proteins
  • Microsomes, Liver
  • Models, Biological
  • Octoxynol
  • Phospholipid Transfer Proteins
  • Proteolipids
  • Rats
  • Saccharomyces cerevisiae Proteins
  • Transport Vesicles

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC3346150

Digital Object Identifier (DOI)

  • 10.1074/jbc.M112.343038

PubMed ID

  • 22427661

Additional Document Info

start page

  • 15523

end page

  • 15532

volume

  • 287

number

  • 19