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. 2012 May 4;287(19):15523-32.
doi: 10.1074/jbc.M112.343038. Epub 2012 Mar 15.

Reconstitution of glucosylceramide flip-flop across endoplasmic reticulum: implications for mechanism of glycosphingolipid biosynthesis

Madhavan Chalat  1 , Indu Menon, Zeynep Turan, Anant K Menon
Affiliations

Reconstitution of glucosylceramide flip-flop across endoplasmic reticulum: implications for mechanism of glycosphingolipid biosynthesis

Madhavan Chalat et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Intracellular transport of glucosylceramide and structure of fluorescent lipids. A, trafficking of glucosylceramide. GlcCer is synthesized on the cytoplasmic face of the Golgi complex (indicated by the asterisk) (1). The majority of GlcCer is moved to the cytoplasmic face of the ER by FAPP2, a cytoplasmic lipid transfer protein. This is probably a bidirectional process (2). GlcCer is flipped across the ER membrane by an ATP-independent flippase (3). Lumenal GlcCer enters ER-derived transport vesicles and thus enters the lumen of the Golgi complex on vesicle fusion (4). In the Golgi lumen, GlcCer is galactosylated to form lactosylceramide, which is then further elaborated to generate higher order GSLs (5). A minor fraction of GlcCer moves to the PM by a non-vesicular route and is flipped across the plasma membrane. The model is based the work of Halter et al. (13). B, NBD-PE and NBD-GlcCer.
FIGURE 2.
FIGURE 2.
Spontaneous flipping of NBD-GlcCer and NBD-PE. Asymmetric liposomes with the NBD-labeled lipid confined to the inner leaflet at time zero were incubated at 23 °C. Transbilayer movement of the labeled lipid from the inner to the outer leaflet was monitored over time by determining the fraction of sample fluorescence that could be eliminated by dithionite, a membrane-impermeant reductant. The data were fit to a monoexponential equation of the form, % flipped = 50(1 − exp(−kt)). The half-time of flipping (equal to 0.69/k) was ∼80 h for NBD-PE and ∼9 h for NBD-GlcCer.
FIGURE 3.
FIGURE 3.
Reconstitution of NBD-PE and NBD-GlcCer flipping in proteoliposomes. Large unilamellar vesicles were prepared from Triton X-100-solubilized egg phosphatidylcholine, egg phosphatidylglycerol, and either NBD-PE or NBD-GlcCer. Different amounts of Triton X-100-solubilized rat liver ER membrane proteins were included in the reconstitution mixture to generate vesicles with different PPR values (mg of protein/mmol of phospholipid). A, rat liver homogenates were fractionated to yield smooth microsomes (SM) and RER. Smooth microsomes and RER samples were analyzed (equal protein loading) by SDS-PAGE and immunoblotting using antibodies to ribophorin I (∼65 kDa), α2,6-sialyltransferase (α2,6 ST; ∼50 kDa) and dipeptidylpeptidase IV (DPPIV; ∼110 kDa). B, thin layer chromatograms of organic solvent extracts of liposomes and proteoliposomes reconstituted with NBD-PE or NBD-GlcCer. The migration position of NBD-lipid standards is indicated; o, origin; f, solvent front. C, liposomes (L) and proteoliposomes (P1 and P2, with PPR of 3.7 and 37 mg/mmol) were reconstituted with NBD-PE. Dithionite was added at time 0, and the reduction in fluorescence was monitored over time at ambient temperature. D, as in C, except that vesicles were reconstituted with NBD-GlcCer. E, raw data obtained from experiments, such as those shown in C and D, indicate the percentage (y) of NBD-PE or NBD-GlcCer that can be reduced by dithionite as a function of the PPR of the reconstituted sample. The transformation f(y) = (y − y0)/(ymax − y0), where y0 is the percentage reduction obtained with liposomes and ymax is the maximum percentage reduction observed, yields a monoexponential graph of p(≥1) versus PPR, where p(≥1) is the probability that a particular vesicle in the sample has ≥1 flippase. The monoexponential fit constant for this graph is the PPR value at which the average number of flippases per vesicle is 1, and ∼63% of the vesicles in the population possess ≥1 flippase (17).
FIGURE 4.
FIGURE 4.
Effect of protein modification reagents on translocation of NBD-GlcCer and NBD-PE. Triton X-100-solubilized rat liver ER membrane proteins were treated with NEM (40 mm), DEPC (40 mm), or both for 45 min at ambient temperature before being taken for reconstitution. Mock-treated samples were used as controls. A, fluorescence traces generated on adding dithionite to NBD-GlcCer vesicles prepared without protein (liposome), with rat liver ER membrane proteins (proteoliposome), or with protein samples treated with NEM, DEPC, or NEM + DEPC as indicated. The PPR of all of the protein-containing samples was ∼3.7 mg/mmol. B, combined data (means ± S.E. (error bars) from at least three independent experiments) showing the effect of protein modification reagents on the translocation of NBD-GlcCer and NBD-PE in vesicles reconstituted at a PPR of ∼3.7 mg/mmol.
FIGURE 5.
FIGURE 5.
Velocity gradient sedimentation analysis and hydroxyapatite chromatography of NBD-PE and NBD-GlcCer flippase activities. A–C, velocity gradient sedimentation analysis of TE; D, hydroxyapatite chromatography of TE. A, refractive index of individual fractions and protein content of fractions pooled pairwise, starting with fraction 2. The gray bar running through all three panels indicates the peak of the protein recovery profile. B, sedimentation behavior of ovalbumin (3.6 S), bovine serum albumin (4.2 S), and catalase (11 S). Fractions were analyzed by SDS-PAGE, and the relative amount of a particular standard in each fraction was determined by densitometry of the Coomassie-stained gel. C, fractions were pooled pairwise, starting with fraction 2, subjected to buffer exchange, and then reconstituted at the same PPR value (∼1 mg/mmol) to determine NBD-PE and NBD-GlcCer translocation activity. Unfractionated TE was reconstituted in parallel. A specific activity (SA) measure was obtained by determining the extent of fluorescence reduction (yfrac and yTE), subtracting the extent of reduction obtained with protein-free liposomes (yo), and normalizing to the protein amount (p) used for reconstitution. Thus, SAfrac = (yfrac − yo)/p. Total flippase activity was calculated as the product of specific activity and the total protein amount in the sample. The graph shows the total activity in each pooled fraction normalized to that of the load (TE). D, TE was fractionated on hydroxyapatite to yield a flow-through fraction as well as fractions eluted from the resin with 1 m NaCl and 0.3 m phosphate. The chart shows GlcCer and PL flippase activity (calculated as described in the legend to C) recovered in each fraction (mean ± range (error bars) of two independent experiments).
FIGURE 6.
FIGURE 6.
NBD-GlcCer translocation in proteoliposomes containing yeast ER proteins. A, liposomes (L) and proteoliposomes containing yeast ER membrane proteins (P1 and P2, with PPR of 3.2 and 16 mg/mmol) were reconstituted with NBD-GlcCer. Dithionite was added at time 0, and the reduction in fluorescence was monitored over time at ambient temperature. B, raw data for NBD-GlcCer and NBD-PE flipping were obtained from experiments such as the one shown in A, using proteoliposomes prepared at a range of PPR values. The data were processed as described in the legend to Fig. 3C to generate a graph of p(≥1) versus PPR. The lines through the two data sets are monoexponential fits with a fit constant (in each case) of ∼7.5 mg/mmol.
FIGURE 7.
FIGURE 7.
A role for reticulons and Yop1 in lipid flipping? A, proteoliposomes prepared at different protein/phospholipid ratio using TE from BY4741 wild-type cells as well as TE from NDY257 cells lacking Rtn1, Rtn2, and Yop1 were assayed for their ability to translocate NBD-PE and NBD-GlcCer. The line through the points is a monoexponential fit encompassing all four data sets. B, Yop1-HA was expressed in NDY257 cells and affinity-purified after preparing TE. The purified sample was analyzed by SDS-PAGE and immunoblotting using anti-HA antibodies or by silver-staining the gel. C, purified Yop1-HA was reconstituted into proteoliposomes. Membrane association of Yop1-HA was demonstrated by flotation in a discontinuous sucrose gradient, as described previously (21), using steps of 750, 750, 1000, 1000, and 750 μl of 0, 5, 10, 20, and 30% (w/v) sucrose, respectively. The vesicles floated as a sharp band at the interface between the 10 and 20% sucrose layers. Fractions (600, 600, 600, 400, 800, 600, and 650 μl, harvested from the top) were analyzed for phospholipid content and taken for SDS-PAGE immunoblotting to quantitate Yop1-HA. Fraction 5 includes the interface between the 10 and 20% sucrose layers. D, proteoliposomes containing purified Yop1-HA were tested for their ability to flip NBD-PE and NBD-GlcCer. Vesicles reconstituted with TE from Yop1-HA-expressing NDY257 cells were used for comparison. The concentration of Yop1-HA was adjusted to be the same as that of Yop1-HA in TE; this was verified by immunoblotting. We determined the extent of fluorescence reduction (yYop1 and yTE) obtained using different volumes (v) of sample for reconstitution, subtracted the extent of reduction obtained with protein-free liposomes (yo), and divided by sample volume to get a specific activity measure (i.e. SAYop1 = (yYop1 − yo)/v and SATE = (yTE − yo)/v. The volumes of TE chosen were such that (yTE − yo) was between 5 and 15% (i.e. on the rising portion of the protein dependence plot shown in A). The bar chart presents the average of the ratio SAYop1/SATE. The specific activity of the flow-through fraction obtained after quantitative depletion of Yop1-HA from TE was the same as that obtained for TE. Error bars, S.E.
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