Stable Cell Surface Expression of GPI-Anchored ... - Wiley Online Library

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/tra.12224

Stable Cell Surface Expression of GPI-Anchored Proteins, but not Intracellular Transport, Depends on their Fatty Acid Structure Nina Jaensch1 , Ivan R. Corrêa Jr2 and Reika Watanabe1,∗,† 1 Department 2 New ∗

of Biochemistry, University of Geneva Sciences II, CH1-1211 Geneva, Switzerland England Biolabs, Ipswich, MA, USA

Corresponding author: Reika Watanabe, [email protected]

Abstract Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a class of

Furthermore, cholesterol extraction drastically releases the unremodeled

lipid anchored proteins expressed on the cell surface of eukaryotes. The

GPI-APs carrying an unsaturated fatty acid from the cell surface, but

potential interaction of GPI-APs with ordered lipid domains enriched in

not remodeled GPI-APs carrying two saturated fatty acids. This under-

cholesterol and sphingolipids has been proposed to function in the intra-

scores the essential role of lipid remodeling to ensure a stable membrane

cellular transport of these lipid anchored proteins. Here, we examined

association of GPI-APs particularly under potential membrane lipid per-

the biological importance of two saturated fatty acids present in the

turbation.

phosphatidylinositol moiety of GPI-APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI-AP

Keywords cholesterol, clathrin-independent carrier, endocytosis, gly-

association within ordered lipid domains. We found that the fatty acid

cosylphosphatidylinositol-anchored proteins, golgi apparatus, GPI-en-

remodeling is not required for either efficient Golgi-to-plasma mem-

riched early endosomal compartment, intracellular protein transport,

brane transport or selective endocytosis via GPI-enriched early endo-

lipid rafts, lipid remodeling, ordered lipid domains, SNAP-tag techno-

somal compartment (GEEC)/ clathrin-independent carrier (CLIC) path-

logy

way, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of

Received 12 December 2013, revised and accepted for publication 2

cholesterol dependence does not appear to be related to the interac-

September 2014, uncorrected manuscript published online 4 September

tion with ordered lipid domains mediated by two saturated fatty acids.

2014, published online 12 October 2014

Glycosylphosphatidylinositol-anchored proteins (GPIAPs) are a class of lipid anchored proteins expressed in the outer leaflet of the plasma membrane in eukaryotes. In mammalian cells, more than 150 proteins including enzymes, receptors, adhesion molecules and complement regulatory proteins have been identified to be GPI-anchored. The GPI anchor is synthesized in the endoplasmic reticulum (ER) and is transferred to the C-terminus of proteins carrying a GPI attachment

signal (1). After attachment to the protein, the GPI

†

Present address, Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA

anchor

undergoes

several

structural

modifications

(2,3). In the ER, post-GPI attachment to proteins 1 (PGAP1) and PGAP5 mediate the removal of the inositol-linked acyl chain and the ethanolamine phosphate group linked to the second mannose, respectively (4,5). In the Golgi apparatus, PGAP2 and PGAP3 mediate the exchange of an unsaturated fatty acid at the sn-2 position of the PI moiety for a saturated fatty acid (6,7). The GPI anchor functions not only as anchor for soluble proteins on the cell surface, but also as sorting signals in www.traffic.dk 1305

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secretory and endocytic pathways. For example, defects in GPI anchoring and anchor remodeling in the ER cause accumulation of the precursor proteins in the ER (2,3). In polarized epithelial cells, most but not all GPI-APs are preferentially targeted to the apical plasma membrane (8–10). Various GPI-APs are internalized primarily by a unique endocytic pathway that is independent of clathrin and dynamin and is regulated by the small G-proteins Arf1 and cdc42, known as the GPI-enriched early endosomal compartment (GEEC)/ clathrin-independent carrier (CLIC) pathway (11–16). As GPI-APs are exoplasmically localized, they are not directly accessible to transport machineries located in the cytoplasm (17). In the ER, the p24 family proteins selectively recognize the precisely remodeled GPI anchor structure and function as cargo receptor for GPI-APs to sort them into COPII vesicles (5,18). In addition to the glycan structure, the long saturated fatty acyl chains found in GPI-APs have been proposed to direct protein sorting by promoting their interaction with ordered lipid domains, also known as ‘nanoclusters’ or ‘lipid rafts’, enriched in sphingolipids and cholesterol (14,19). The examples of lipid-based protein sorting are not only limited to intracellular sorting of GPI-APs. The simian virus 40 (SV40) and cholera toxin both bind the glycosphingolipid GM1 on the cell surface. The length and saturation of fatty acids in GM1 or its analog influences the SV40 internalization step and cholera toxin traffic from the plasma membrane to the trans-Golgi network and the ER (20,21). As a result, the fatty acid composition of cellular GM1 influences the infectivity and toxicity of these pathogenic reagents. To date, possible involvements of lipid-based sorting for GPI-APs have been tested mainly by cholesterol or sphingolipid depletion. For example, sphingolipid or cholesterol depletion causes mistargeting of GPI-APs to the basolateral membranes in polarized epithelial cells (22,23). Cholesterol or sphingolipid depletion also affects the internalization step and sorting of GPI-APs along the endocytic pathway (15,24–27). However, these observations do not specify how these lipids function in the intracellular transport of GPI-APs. Cholesterol or sphingolipids might function in the formation or regulation of the transport machinery involved in each step (15). Alternatively, these lipids, as components of ordered lipid domains, might be involved in sorting 1306

of GPI-APs into transport carriers through interaction with saturated fatty acids in GPI-APs (14,19). It is also important to consider that the depletion of cellular sphingolipids or cholesterol probably causes various pleiotropic effects including changes of general membrane biophysical properties. Cholesterol depletion has been shown to particularly affect the amount of key phospholipids regulating various steps of membrane transport pathways (28), such as phosphatidylinositol 4-phosphate in the Golgi apparatus and phosphatidylinositol 4,5-bisphosphate at the plasma membrane (29,30). Therefore, alternative approaches to test a potential lipid-based sorting mechanism for GPI-APs are required. Recently, mutations in the PGAP3 gene have been found in several individuals presenting severe psychomotor delay, intellectual disability, epilepsy, elevated level of alkaline phosphatase in serum and distinctive facial characteristics (31). Mice defective in fatty acid remodeling have been also reported (32,33). The Pgap3−/− mice show complex phenotypes such as male-specific low birth rate, morphological abnormalities, abnormal reflexes and growth retardation (32). However, molecular mechanisms causing these phenotypes remain largely unknown. In this study, we aim to reveal the structural role of the fatty acid moiety of GPI-APs on their intracellular transport pathways. For that, we established two novel quantitative assays to measure the kinetics of Golgi-to-plasma membrane transport and endocytosis of GPI-APs. By using mutant cells expressing GPI-APs carrying one unsaturated fatty acyl chain in the sn-2 position due to a defect in lipid remodeling, we examined the importance of two saturated fatty acyl chains of GPI-APs and the putative role of the association with ordered lipid domains for protein sorting of GPI-APs in the secretory and endocytic pathways.

Results To test the structural importance of the fatty acid moiety of GPI-APs in their intracellular transport, we used mutant Chinese Hamster Ovary (CHO) cells defective in the lipid remodeling enzymes PGAP2 and PGAP3 (7) stably expressing the SNAP- and FLAG-tagged GPI-AP CD59 (SF-CD59). The SNAP tag is derived from the DNA repair protein O6 -alkylguanine-DNA alkyltransferase and can be Traffic 2014; 15: 1305–1329

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covalently labeled in living cells through the reaction with O6 -benzylguanine derivatives (34). To ensure that phenotypes in the PGAP2/3 double mutant cells result from the absence of lipid remodeling of the GPI anchor but not from clonal variations or differences in expression levels of SF-CD59, we first generated the PGAP2/3 mutant cells expressing different levels of SF-CD59. By restoring the expression of both genes in these mutant cells, we obtained corresponding restored cells used as control cells. As we obtained identical results in two sets of cells in all the experiments described below, we only presented the data obtained from one of two sets of the corresponding mutant and restored cells. Lipid remodeling of the GPI anchor is required for the efficient incorporation of SNAP-FLAG-CD59 into DRMs GPI-APs in wild-type CHO cells contain two saturated fatty acids, stearic or palmitic acid, whereas in PGAP2/3 mutant cells GPI-APs carry one saturated and one unsaturated fatty acyl chains, the latter consisting mainly of arachidonic acid in the sn-2 position (Figure 1A) (7). Lipid remodeling is required for GPI-APs to concentrate in detergent-resistant membranes (DRMs) (7,32,35). Whether DRMs truly reflect specific membrane domains existing in living cells has been questioned (36). Irrespective of that, GPI-APs are concentrated in DRMs through hydrophobic interactions of the saturated fatty acyl chains (7,32,35,37). Therefore, we used the DRM assay in order to indirectly monitor the efficiency of lipid remodeling reaction. In wild-type and restored cells, the highly glycosylated mature form of SF-CD59 produced in the Golgi apparatus was preferentially recovered in the DRM fraction (Figure. 1B,C). This is consistent with the results obtained for the mature form of Decay accelerating factor (DAF), which is another GPI-AP stably expressed in these cell lines. In contrast, in mutant cells, the majority of the mature form of SF-CD59 was localized in the soluble fractions similar to the ER form of SF-CD59 (Figure 1B,C). In all cells, a canonical DRM marker caveolin and the single transmembrane protein transferrin receptor (TfR) were exclusively found in the DRM and soluble fractions, respectively. This verifies a proper fractionation procedure and confirms that the lipid remodeling defect of GPI-APs in mutant cells is almost completely rescued to the level of wild-type cells in restored cells. Traffic 2014; 15: 1305–1329

Unremodeled SF-CD59 is efficiently transported to the cell surface at similar kinetics compared to fully remodeled SF-CD59 Next, using the SNAP-tag technology, we established a quantitative biochemical assay that allows to independently monitor two intracellular transport kinetics: (i) ER-to-Golgi and (ii) Golgi-to-plasma membrane (Figure 2A). The cells were incubated with the non-fluorescent, membrane-permeable substrate, SNAP-Cell Block, to eliminate the reactivity of all existing SF-CD59. After wash out, the cells were incubated in complete medium to allow the synthesis of SF-CD59 for 30 min at 37∘ C. During the following chase steps, 200 μM of cycloheximide (CHX) was added into the complete medium to prevent further synthesis of SF-CD59. For each time point of the chase, one set of wells was incubated with a membrane-impermeable fluorescent substrate, SNAP-Surface 647, which specifically labels SF-CD59 arrived at the cell surface during the chase time. At the end of the chase, both sets of wells (CHX only and CHX + SNAP-Surface 647) were incubated with the membrane-permeable substrate, SNAP-TMR-Star, and were analyzed by SDS–PAGE and in-gel fluorescence scanning (Figure 2B). The fluorescent signals of SF-CD59 labeled only with the permeable substrate correspond to the whole cellular fraction of newly synthesized SF-CD59. The fluorescent signals of SF-CD59 labeled with the permeable substrate after preincubation with impermeable fluorescent substrate correspond to the internal fractions of newly synthesized SF-CD59. The specific surface and internal staining of SF-CD59 by the membrane-impermeable and permeable substrates at different chase times were confirmed by microscopy (Figure 2C). The efficient plasma membrane arrival was revealed by the increased labeling of mature SF-CD59 with the impermeable substrate (Figure 2B,C, impermeable). We noticed that in mutant cells, a certain amount of GPI-APs is released into the medium just after arrival at the cell surface (Figure 2B, secreted, S). Therefore, at the end of the chase, the medium was collected and the secreted fraction of SF-CD59 was immunoprecipitated with anti-FLAG antibody beads and labeled with the membrane-permeable substrate. We observed very subtle delay of maturation of SF-CD59 in the restored cells compared to the mutant cells (Figure 2D). To monitor Golgi-to-plasma membrane transport kinetics independent of the difference in the ER-to-Golgi 1307

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Figure 1: SF-CD59 is not enriched in DRMs in lipid remodeling mutant cells. A) Scheme of the fatty acid remodeling of GPI-APs. After biosynthesis, the GPI-APs carry an unsaturated (e.g. arachidonic acid, C20:4) and a saturated fatty acid (e.g. stearic acid, C18:0). In the Golgi, the unsaturated fatty acid is removed and replaced by a saturated fatty acid. B) DRM assay with wild-type (WT), mutant (MUT) and restored cells (REST) stably expressing SF-CD59. Proteins were fractionated by density gradient centrifugation after solubilization with 1% Triton-X-100 at 4∘ C. Aliquots of the top fraction (DRM) and three additional fractions (detergent soluble) were analyzed by SDS–PAGE followed by in-gel fluorescence scanning (SF-CD59) and western blot detection using antibodies against DAF, Caveolin and TfR. C) Quantitation. The amounts of SF-CD59, DAF, Caveolin and TfR were measured and the percentage of protein in the DRM fraction was calculated. In case of SF-CD59, only the mature form of the protein was taken into account. The quantitation is a summary of all experiments (n = 3). Error bars, SD.

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Figure 2: Legend on Next page. transport, we considered the fluorescence intensities of only the mature form of SF-CD59 labeled with the permeable substrate (Figure 2B). The plasma membrane arrived fraction was calculated by the subtraction of the internal sample from the sum of the cellular and secreted Traffic 2014; 15: 1305–1329

samples. The efficiency of the plasma membrane arrival was calculated by dividing the plasma membrane arrived fraction by the sum of the cellular and secreted fractions (Figure 2A,B). The quantitation revealed that unremodeled and remodeled SF-CD59 show similar transport kinetics 1309

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(Figure 2E), suggesting that two saturated fatty acyl chains in the GPI anchor are not required for the efficient Golgi-to-plasma membrane transport of GPI-APs. Cholesterol depletion impairs Golgi-to-plasma membrane transport of SF-CD59 in both lipid remodeling deficient and restored cells Cholesterol depletion was shown to affect the formation of secretory vesicles at the trans-Golgi network (38). Using the same assay, we also addressed the effect of cholesterol depletion on intracellular transport of fully remodeled SF-CD59 in restored cells. Cholesterol was depleted both by biosynthesis inhibition with compactin and by extraction with methyl-β-cyclodextrin (MβCD), decreasing the amount of free cholesterol by almost 50% relative to control cells (Figure 3A). We observed a lower ratio of the mature form of SF-CD59 in cholesterol depletion conditions compared to control conditions suggesting that cholesterol depletion might cause defects in ER-to-Golgi transport, the intra-Golgi transport and/or glycosylation at the Golgi apparatus of GPI-APs as shown previously (39–41). In addition, DRM association of the mature form of SF-CD59 in restored cells was impaired in agreement with the proposed role of cholesterol to form ordered lipid domains (19) (Figure 3B,C). As the effect of cholesterol depletion could be pleiotropic, it is also possible that cholesterol depletion affects GPI-lipid remodeling at the Golgi apparatus, therefore causes less DRM association of

mature form of SF-CD59. However, we think that this is less likely since our previous study suggests that at least the first step of GPI-lipid remodeling occurs efficiently under cholesterol depletion condition in Madin–Darby canine kidney (MDCK) cells (42). The SNAP-based transport assay revealed that the ER-to-Golgi transport of SF-CD59 was severely delayed in cholesterol-depleted cells compared to the control condition (Figure 3D,E) as mentioned above. In addition, the Golgi-to-plasma membrane transport assay showed that mature SF-CD59 arrived at the plasma membrane was significantly decreased in cholesterol-depleted cells compared to the control cells suggesting that cholesterol is also required for the efficient Golgi-to-plasma membrane transport of fully remodeled GPI-APs (Figure 3D,F). It is important to emphasize again that we did not measure the cell surface arrival by quantifying the newly synthesized population of SF-CD59 labeled with the membrane-impermeable substrate, which would be certainly influenced by the ER-to-Golgi transport. The quantitation of this Golgi-to-PM transport assay includes only the mature form of SF-CD59 labeled with the membrane-permeable substrate at each time point; therefore, it is completely independent from changes in the ER-to-Golgi transport. Cholesterol depletion also decreased the Golgi-to-plasma membrane transport of SF-CD59 in lipid remodeling mutant cells (Figure 4).

Figure 2: Remodeled and unremodeled SF-CD59 show similar Golgi exit kinetics. A) Scheme representing the SNAP-based biosynthetic secretory assay. PM; plasma membrane. B) Biosynthetic secretory assay with mutant cells (MUT) and restored cells (REST). Representative fluorescence scans show cellular (C), internal (I) and secreted (S) SF-CD59 at each time point (0, 20 and 40 min). The three scans correspond to the detection of SNAP-Cell TMR (permeable), SNAP-Surface 647 (impermeable) and the merge. The mature form of SF-CD59 in the cellular sample labeled by the permeable substrate represents newly synthesized SF-CD59 at the Golgi and at the plasma membrane. The mature form of SF-CD59 in the internal sample labeled by the permeable substrate represents newly synthesized SF-CD59 exclusively at the Golgi. The asterisk corresponds to a cleavage product of SF-CD59. C) Morphological validation of the transport assay. Newly synthesized SF-CD59 was labeled with SNAP-Surface 647 (impermeable), followed by a labeling with SNAP-Cell TMR (permeable) at time points 0 and 60 min at 37∘ C. Scale bars, 9 μm. D) Quantitation of the ER-to-Golgi transport. The percentage of the mature form of SF-CD59 was measured from the cellular and secreted samples at each time point. The quantitation includes all experiments (n = 4). Error bars, SEM; *p < 0.05. E) Quantitation of the Golgi-to-PM transport assay. For the fluorescence measurements and calculations, only the mature form of SF-CD59 labeled with the membrane-permeable substrate was analyzed. The plasma membrane arrived fraction was calculated by the subtraction of the internal fraction from the sum of the cellular and secreted fractions. The percentage of newly synthesized mature SF-CD59 at the plasma membrane was calculated by dividing the plasma membrane arrived fraction by the sum of the cellular and secreted fractions; PM [%] = [C + S − I)PM /(C + S) × 100]. The quantitation includes all experiments (n = 4). Error bars, SEM. 1310

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Figure 3: Cholesterol depletion reduces DRM association and slows down the Golgi exit of remodeled SF-CD59. A) Quantitation of free cholesterol in control cells (CTRL) and cholesterol-depleted cells (CH DEPL). Control cells were cultured in medium containing 5% lipoprotein-depleted serum (LPDS). Cholesterol-depleted cells were incubated in medium containing 5% LPDS and the cholesterol biosynthesis inhibitor compactin for 48 h, followed by cholesterol extraction with 10 mM MβCD for 30 min. The cells were harvested and the whole cell extract was used for Amplex Red cholesterol measurement. The amount of cholesterol in ng per μg of protein was calculated. The quantitation is a summary of all experiments (n = 3). Error bars, SEM. B) DRM assay with cholesterol-depleted cells (CH DEPL) and control cells (CTRL). Representative examples of in-gel fluorescence scan (SF-CD59) and western blot detection of Caveolin and TfR. C) Quantitation for mature SF-CD59 in DRM. The quantitation was done as in Figure 1C and is a summary of all experiments (n = 3). Error bars, SD. D) Biosynthetic secretory assay with cholesterol-depleted restored cells (CH DEPL) and control restored cells (CTRL). Representative fluorescence scans show cellular and internal SF-CD59 at each time point (0, 20 and 40 min). The three scans correspond as in Figure 2B. The asterisk corresponds to a cleavage product of SF-CD59. E) Quantitation of the ER-to-Golgi transport. The quantitation was performed as in Figure 2D and included all experiments (n = 5). Error bars, SEM; *p < 0.05. F) Quantitation of the Golgi-to-PM transport assay. For the fluorescence measurements and calculations, only the mature form of SF-CD59 labeled with the membrane-permeable substrate was analyzed. The percentage of newly synthesized mature SF-CD59 at the plasma membrane was calculated by dividing the plasma membrane arrived fractions by the cellular fractions; PM [%] = (C − I)PM /C × 100. The quantitation includes all experiments (n = 5). Error bars, SEM; *p < 0.05. These results reveal the essential role of cholesterol in the Golgi exit of GPI-APs independent of the fatty acid structure; therefore, it appears that the mechanism of Traffic 2014; 15: 1305–1329

cholesterol dependence for Golgi-to-plasma membrane transport is probably not related to the sorting of GPI-APs into ordered lipid domains at the Golgi apparatus. 1311

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Figure 4: Cholesterol depletion also slows down the Golgi exit of unremodeled SF-CD59. Biosynthetic secretory assay with cholesterol-depleted mutant cells (CH DEPL) and control mutant cells (CTRL). A) Representative fluorescence scans show cellular and internal SF-CD59 at each time point (0, 20 and 40 min). The three scans correspond as in Figure 2B. The asterisk corresponds to a cleavage product of SF-CD59. B) Quantitation of the Golgi-to-PM transport assay. The quantitation was done as in Figure 3F and included all experiments (n = 4). Error bars, SEM; *p < 0.05. Note that the secreted fraction was not considered for quantification because the secreted fraction was hardly detected in both conditions. This is likely due to the very slow transport kinetics of GPI-APs in LPDS containing media. Unremodeled SF-CD59 is as efficiently endocytosed as fully remodeled SF-CD59 and the internalization of both GPI-APs is sensitive to cholesterol extraction As most GPI-APs do not have a natural ligand to allow visualization of endocytosis events, we utilized a labeling strategy with membrane-impermeable SNAP-tag substrates containing cleavable fluorescent groups (43) to follow the endocytosis of SF-CD59 (Figure 5A). SF-CD59 at the cell surface was labeled with a membrane-impermeable, fluorescent S-S-cleavable SNAP substrate at 10∘ C. After the excess of substrate was washed out, the cells were incubated at 37∘ C to initiate endocytosis. At different incubation times, the cell surface fluorescent molecules were cleaved by the action of the membrane-impermeable reducing reagent sodium 2-mercaptoethane-sulfonate (MesNa) to specifically detect the internalized fraction of SF-CD59. At time point 0 min, the strong cell surface fluorescence detected in the non-MesNa treated sample was diminished almost completely by MesNa treatment (Figure 5B). This demonstrates that the fluorescent labels on the cell surface are efficiently cleaved off by MesNa treatment and the internalization of SF-CD59 does not occur during labeling at 10∘ C. After 45 min at 37∘ C, we observed a significant internalized fraction of SF-CD59 in the MesNa-treated sample (Figure 5B). In order to measure the efficiency of endocytosis of SF-CD59 quantitatively, the fluorescence levels of individual cells were analyzed by flow cytometry. At each time point, the 1312

mean fluorescence intensity of the MesNa-treated sample corresponding to the amount of internalized SF-CD59 was divided by the mean fluorescence intensity of the non-treated sample corresponding to the total SF-CD59. Using this assay, we compared the endocytosis efficiency of unremodeled and remodeled SF-CD59 expressed in mutant and restored cells, respectively (Figure 5C,D). The endocytosis efficiencies of SF-CD59 in both cell lines were identical, suggesting that changes of fatty acid composition of the GPI anchor do not have an impact on the endocytosis efficiency of GPI-APs (Figure 5D). Since endocytosis of various GPI-APs via the CLIC/GEEC pathway is highly sensitive to cholesterol depletion (15), we tested whether cholesterol extraction with 10 mM MβCD for 30 min affect the endocytosis of SF-CD59. Upon cholesterol extraction, the internalization of both unremodeled and remodeled SF-CD59 was significantly reduced to a similar extent compared to that of control cells (Figure 6A–C). Dil-LDL uptake was not inhibited by cholesterol extraction, demonstrating that clathrin-mediated endocytosis occurs normally under this condition (Figure 6D,E). It is important to note that we obtained the same conclusions from two endocytosis assays using either suspension cells or attached cells (Figures 5 and 6: suspension cells and Figure S1A–C: attached cells). Taken together, our results reveal that cholesterol depletion affects endocytosis of GPI-APs independent of the fatty acid structure; therefore, it appears that the mechanism of Traffic 2014; 15: 1305–1329

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Figure 5: Lipid remodeling is not required for the efficient internalization of SF-CD59. A) Scheme representing the endocytosis assay B) Morphological representation of the endocytosis assay. Restored cells stably expressing SF-CD59 were labeled with SNAP-Surface-SS-488 at 10∘ C and treated with (+) or without (−) MesNa after 0 and 45 min of internalization at 37∘ C. C) Endocytosis assay with mutant (MUT) and restored (REST) cells. The histograms represent the data obtained by flow cytometry of one experiment and show the fluorescence distribution of non-treated (dotted lines) and MesNa-treated (solid lines) for mutant (dark-colored) and restored (light-colored) cells at 0, 15, 30 and 45 min of internalization at 37∘ C. The filled histograms show the fluorescence distribution of labeled cells without SF-CD59 expression. D) Quantitation. At each time point, the percentage of internalized SF-CD59 compared to the total SF-CD59 was calculated. The quantitation includes all experiments (n = 3). Error bars, SD. cholesterol dependence for endocytosis is not related to the sorting of GPI-APs into ordered lipid domains similar to the Golgi-to-plasma membrane transport shown above. The internalization of both remodeled and unremodeled SF-CD59 is insensitive to dynamin inhibition and requires Arf1 activity and actin dynamics We noticed that the effects of cholesterol extraction on the internalization of SF-CD59 were milder compared to previously published data showing a severe inhibition Traffic 2014; 15: 1305–1329

of CLIC/GEEC pathway by cholesterol extraction (15). A possible explanation for this could be that we measured progressive accumulation of internalized SF-CD59 over time, rather than measuring the internalization at early time points (15). Therefore, we further investigated whether SF-CD59 uses CLIC/GEEC pathway for internalization. The CLIC/GEEC pathway is independent of clathrin and dynamin and is regulated by the small G-proteins Arf1 and cdc42 (11–16). To examine whether the fatty acid structure determines the internalization of 1313

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Figure 6: Cholesterol extraction similarly reduces the internalization of remodeled and unremodeled SF-CD59. A and B) Endocytosis assay with mutant (MUT) and restored (REST) cells in control (CTRL) and cholesterol extracted (MβCD) conditions. Control cells were cultured in medium containing 5% LPDS, cholesterol-extracted cells were incubated in medium containing 5% LPDS and 10 mM MβCD for 30 min. The histograms show the fluorescence distribution of non-treated (dotted lines) and MesNa-treated (solid lines) in control (dark-colored) and cholesterol-extracted (light-colored) cells at 0, 15, 30 and 45 min of internalization at 37∘ C. The filled histograms show the fluorescence distribution of labeled cells without SF-CD59 expression. C) Quantitation was done as in Figure 5D and includes all experiments (n = 3). Error bars, SD. D) Dil-LDL uptake in mutant (MUT) and restored (REST) cells in control condition (CTRL) and cholesterol extracted condition (MβCD). Cells were incubated with 10 μg/mL of Dil-LDL for 1 h at 10∘ C, followed by an uptake for 15 min at 37∘ C. Scale bars, 10 μm. E) Quantitation. The mean intensity of internalized Dil-LDL was measured and normalized to control conditions. The quantitation includes the data of two independent experiments. A total of 100–120 cells were measured for each condition. Error bars, SEM. 1314

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Figure 7: The internalization of remodeled and unremodeled SF-CD59 is insensitive to dynamin inhibition. Endocytosis assays with mutant (MUT) and restored (REST) cells in the presence or absence of the dynamin inhibitor dyngo-4a. A) The histograms represent the data of one experiment and show the fluorescence distribution of non-treated (dotted lines) and MesNa-treated (solid lines) in mutant (dark-colored) and restored (light-colored) cells at 0, 15, 30 and 45 min of internalization at 37∘ C. The filled histograms show the fluorescence distribution of labeled cells without SF-CD59 expression (light color, without dyngo; dark color, with dyngo). B) Quantitation was done as in Figure 5D and includes all experiments (n = 3). Error bars, SD. C) Mutant (MUT) and restored (REST) cells stably expressing SF-CD59 were labeled with SNAP-Surface-SS-488 and 10 μg/mL of Dil-LDL for 1 h at 10∘ C in the absence (CTRL) or presence of dyngo-4a (Dyngo) and then incubated for 25 min at 37∘ C. The MesNa-treated and fixed cells were analyzed by confocal microscopy. Scale bars, 7 μm. D) The quantitation of Dil-LDL uptake was done as in Figure 6E and includes the data of two independent experiments. A total of 60–80 cells were measured for each condition. Error bars, SEM. Traffic 2014; 15: 1305–1329

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Figure 8: The internalization of remodeled and unremodeled SF-CD59 is dependent on Arf1. A) Mutant (MUT) and restored (REST) cells stably expressing SF-CD59 were transiently transfected with GFP or Arf1T31N-GFP, a dominant-negative form of Arf1. The cells were labeled with SNAP-Surface-SS-549 at 10∘ C and incubated at 37∘ C for 30 min. The cells were treated with MesNa, fixed, and without permeabilization, labeled with a mouse anti-FLAG antibody (1:1000), followed by a cy5-conjugated anti-mouse antibody. The cells were analyzed by confocal microscopy and displayed as a z-projection. Scale bars, 10 μm. B) Quantitation. The intensity of internalized SF-CD59 in transfected cells was measured and normalized to the FLAG surface labeling and to untransfected cells in the same field. The quantitation includes the data of two independent experiments. A total of 40–60 cells were measured for each condition. Error bars, SEM. C) The cells were incubated with 10 μg/mL of Dil-LDL at 10∘ C for 1 h and incubated at 37∘ C for 15 min. The cells were fixed and analyzed by confocal microscopy and displayed as a z-projection. Scale bars, 10 μm. D) Quantitation. The intensity of internalized Dil-LDL in transfected cells was measured and normalized to untransfected cells. The quantitation includes the data of two independent experiments. A total of 30–50 cells were measured for each condition. Error bars, SEM. GPI-APs via the CLIC/GEEC pathway, we performed the endocytosis assay in the presence of 20 μM dyngo-4a, a potent dynamin inhibitor (44). The endocytosis of both unremodeled and remodeled SF-CD59 was completely insensitive to dyngo-4a treatment (Figure 7A,B: suspension cells and Figure S1D: attached cells). Under the same condition, Dil-LDL uptake, representing the action of clathrin-mediated endocytosis was inhibited by more than 80% in both mutant and restored cells compared to control conditions, demonstrating the efficient inhibition 1316

of dynamin activity by dyngo-4a (Figure 7C,D). To test the involvement of the CLIC/GEEC pathway more directly, we transiently expressed a dominant negative T31N form of Arf1 (Arf1T31N) (16,45,46) in mutant and restored cells and measured the endocytosis efficiency of SF-CD59 (Figure 8). Considering the function of Arf1 in the biosynthetic secretory pathway (47,48), we performed a surface labeling of SF-CD59 with an anti-FLAG antibody to estimate the amount of SF-CD59 at the cell surface after the endocytosis assay (Figure 8A) and the internalization Traffic 2014; 15: 1305–1329

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Figure 9: The internalization of remodeled and unremodeled SF-CD59 is dependent on actin dynamics. Endocytosis assays with mutant (MUT) and restored (REST) cells in control (CTRL) and latrunculin B-treated (LatB) cells. A) The cells were labeled and analyzed as in Figure 8. Scale bars, 10 μm. B) Quantitation. The intensity of internalized SF-CD59 was measured. The quantitation includes the data of two independent experiments. A total of 60–80 cells were measured for each condition. Error bars, SEM. *p < 0.05. C) The cells were incubated with 10 μg/mL of Dil-LDL at 10∘ C and incubated at 37∘ C for 10 min. The cells were fixed and analyzed by confocal microscopy and displayed as a z-projection. Scale bars, 10 μm. D) Quantitation was done as in Figure 6E and includes the data of two independent experiments. A total of 50–70 cells were measured for each condition. Error bars, SEM. efficiency of SF-CD59 was normalized with the surface amount of SF-CD59 in individual cells. We observed that the internalization efficiencies of both unremodeled and remodeled SF-CD59 were clearly reduced to the same degree by the expression of Arf1T31N-GFP, but not by the expression of green fluorescent protein (GFP) alone (Figure 8A,B). As it is reported that CLIC/GEEC pathway is highly sensitive to the disturbance of actin dynamics (15), we tested the effect of latrunculin B, an inhibitor of actin polymerization (Figure 9). The endocytosis of both unremodeled and remodeled SF-CD59 was significantly affected by latrunculin B treatment to a similar extent (Figure 9A,B). In contrast, Dil-LDL uptake was not inhibited by either the expression of Arf1T31N-GFP or the latrunculin B treatment, demonstrating that clathrin-mediated endocytosis occurs normally under both conditions (Figures 8C,D and 9C,D, respectively). These results strongly support the idea that the saturated fatty acid structure of GPI-APs is not required for the selective endocytosis via the dynamin-independent and actin- and Arf1-dependent CLIC/GEEC pathway. Traffic 2014; 15: 1305–1329

Endocytosed remodeled and unremodeled SF-CD59 are preferentially localized in recycling endosomes It has been suggested that the association with ordered lipid domains dictates the fate of internalized GPI-APs to late endosomes or recycling endosomes (13,26,27). We examined the localization of the endocytosed fraction of SF-CD59 after 30 min of internalization at 37∘ C (Figure 10A). We observed that both unremodeled and remodeled SF-CD59 preferentially co-localized with the recycling endosomal marker, TfR, but not with the late endosomal marker Lamp1 (Figure 10B), suggesting that the steady-state localization of endocytosed GPI-APs at the recycling endosome is not dependent on the saturated fatty acid structure. Lipid remodeling of the GPI anchor is required for the stable association of SF-CD59 at the plasma membrane During the course of this study, we noticed that upon arrival at the cell surface, unremodeled GPI-APs are partially released to the medium (Figure 2B). We also observed a significant decrease of surface SF-CD59 in 1317

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Figure 10: Internalized remodeled and unremodeled SF-CD59 colocalize with TfR-positive endosomes. A) Mutant (MUT) and restored (REST) cells stably expressing SF-CD59 were labeled with SNAP-Surface-SS-488 at 10∘ C and incubated at 37∘ C for 30 min. The MesNa-treated, fixed and saponin-permeabilized cells were labeled with a mouse anti-TfR antibody and anti-Lamp1 antibody, followed by a cy3-conjugated anti-mouse antibody. Scale bars, 7 μm. B) Quantification of the colocalization of SF-CD59 with recycling endosome marker (TfR) and late endosome marker (Lamp1), respectively. The Pearson correlation coefficient was calculated with JACoP (IMAGE J). 1 = perfect colocalization, 0 = no colocalization, −1 = perfect exclusion. A total of 20 cells were measured for each condition. Error bars, SEM. lipid remodeling mutant cells after cholesterol extraction (Figure 6A). It has been reported that the surface expression of different GPI-APs expressed in various cells derived from Pgap3−/− mice is reduced compared to that of Pgap3+/− mice (32). Therefore, we tested whether lipid remodeling is important for the stability of GPI-APs at the plasma membrane (Figure 11). Firstly, SF-CD59 on the cell surface in wild-type, mutant and restored cells was labeled 1318

by a membrane-impermeable SNAP substrate. The cells were incubated with or without 10 mM MβCD for 30 min. In the absence of MβCD, the surface level of SF-CD59 in the mutant and corresponding restored cells was similar (Figures 5C and 11A). Upon cholesterol extraction with MβCD, we observed around 60% reduction of the surface SF-CD59 in mutant cells, but much less in wild-type and restored cells (Figure 11A,B). The levels of free cholesterol Traffic 2014; 15: 1305–1329

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Figure 11: Cholesterol extraction preferentially reduces surface expression of unremodeled SF-CD59. Membrane association assay with wild-type (WT), mutant (MUT) and restored (REST) cells stably expressing SF-CD59. A) Cells were first labeled with SNAP-Surface 647 at 10∘ C (to block endocytosis and biosynthetic transport), and incubated either with 10 mM MβCD (MβCD) or mock-treated (CTRL) for 30 min at 37∘ C, and then analyzed by flow cytometry. The histograms show the fluorescence distribution of control cells (solid lines) and MβCD-treated cells (dotted lines). B) Quantitation. The quantitation represents the data of all experiments (n = 2) and shows the percentage of the surface expression of SF-CD59 upon treatment with MβCD. Range bars. C) Quantitation of free cholesterol in control (CTRL) and cholesterol-depleted (MβCD) cells. (n = 3) Error bars, SD. between mutant and restored cells were comparable before and after cholesterol extraction (Figure 11C). This rules out the possibility that the different sensitivities of surface amount of SF-CD59 by cholesterol extraction among these cells are a consequence of different cholesterol levels or different cholesterol extraction efficiencies between mutant and restored cells. On the basis of our finding that there is no striking difference in the intracellular transport of GPI-AP along biosynthetic secretory and endocytic pathway among lipid remodeling deficient mutant and restored cells under control and cholesterol depleted or extracted condition (Figures 2–6 and S1), the results suggest that unremodeled SF-CD59 expressed on the cell surface is susceptible to be released into the medium by membrane perturbation through cholesterol extraction. We then investigated the released fraction of SF-CD59 after immunoprecipitation with an anti-FLAG antibody (Figure 12). With MβCD treatment, more than 70% of cell surface SF-CD59 was released into the media compared to 10–30% of released fractions from wild-type and restored cells (Figure 12A,B). The released fraction Traffic 2014; 15: 1305–1329

primarily partitioned into the detergent phase after TX-114 partition, a classic criterion of membrane association (49), demonstrating that released SF-CD59 contains at least one fatty acid within GPI anchor (Figure 12C,D). Next, we tested the effect of sphingolipid depletion on the surface level of SF-CD59 (Figure 13). Usually, CHO cells predominantly express GM3 as endogenous glycosphingolipids. However, both mutant and restored cells express mainly GD3 as their cellular glycosphingolipids due to the stable expression of GD3 synthase gene introduced during the establishment of mutant cells (6,7). We observed similar surface amounts of GD3 in mutant and restored cells by flow cytometry in the control condition (Figure 13A). By treating cells with myriocin, the inhibitor of the first step of sphingolipid biosynthesis for 24 h, surface amount of GD3 decreased by more than 60% in both mutant and restored cells (Figure 13A,B). Under this condition, SF-CD59 surface expression decreased by only 20% similarly in both mutant and restored cells (Figure 13C,D) suggesting that unlike cholesterol depletion, sphingolipid depletion affects the surface expression of GPI-APs regardless of their 1319

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Figure 12: Released unremodeled SF-CD59 contains GPI lipid moiety. A) Representative fluorescence scans of biochemical membrane association assay. Cells were labeled with SNAP-Surface 647 at 10∘ C and then treated with 10 mM MβCD for 30 min. SF-CD59 was immunoprecipitated from the medium (secreted fraction) or from cellular fractions of control cells (−CTRL) and treated cell (+MβCD). B) Quantitation of secreted and cellular SF-CD59. The fluorescence intensities of SF-CD59 were measured and the percentages of secreted and cellular fractions were calculated. (n = 3). Error bars, SD. C) Representative fluorescence scan of Triton-X-114 partition assay of the secreted SF-CD59. Cells were labeled with SNAP-Surface 647 at 10∘ C, and then incubated with 10 mM MβCD (+) or without (−) for 30 min. The medium was collected and supplemented with Triton-X-114. The aqueous (A) and detergent (D) fractions were separated and followed by immunoprecipitation for SF-CD59. D) Quantitation of Triton-X-114 partition assay. The fluorescence intensities of SF-CD59 were measured, and the percentages of aqueous and detergent phases were calculated. Secreted SF-CD59 in control conditions in wild-type and restored cells were not detectable (n.d.). The quantitation includes two experiments. Range bars. fatty acid structure. Taken together, we conclude that the lipid remodeling reaction replacing an unsaturated fatty acid with a saturated fatty acid does not affect the overall amount of cellular cholesterol and glycosphingolipids as previously shown for the other lipid remodeling step (50) but is specifically required for the stable association of GPI-APs particularly under cholesterol extraction.

Discussion Advantages of SNAP-based secretory and endocytic transport assays With the use of the SNAP-tag labeling technology, we developed novel quantitative assays to measure the transport of newly synthesized GPI-AP, SF-CD59, from the 1320

ER to the Golgi apparatus and from the Golgi to the plasma membrane, and the overall endocytosis efficiency. In contrast to surface biotinylation assays combined with pulse labeling to measure the surface arrival of newly synthesized proteins (51), the SNAP-tag technology enables continuous labeling of SNAP-tagged proteins with membrane-impermeable substrates in live cells under physiological conditions. This allows us to measure the efficiency of surface arrival of newly synthesized proteins independent of the subsequent endocytosis of proteins of interest. Given the fact that most GPI-APs do not have natural ligands useful to follow the endocytic pathway and that cross-linking of GPI-APs with antibodies leads to their artificial concentration in caveolae (52), monovalent SNAP labeling is a particularly Traffic 2014; 15: 1305–1329

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Figure 13: Sphingolipid depletion affects the surface expression of GPI-APs regardless of their fatty acid structure. Mutant (MUT) and restored (REST) cells were cultured in Nutridoma-BO medium with (myriocin) or without (CTRL) 2.5 μM myriocin for 24 h and then analyzed by flow cytometry. A) Cells were labeled with a mouse anti-glycosphingolipid GD3 antibody and a secondary APC-conjugated anti-mouse antibody. The histograms show the fluorescence distribution of control cells (solid lines) and myriocin-treated cells (dotted lines) for mutant (dark-colored) and restored (light-colored) cells. The filled histograms show the fluorescence distribution of cells labeled only with secondary antibody. B) Quantitation. The quantitation represents the data of all experiments (n = 5) and shows the percentage of the surface expression of GD3 normalized to control conditions. Error bars, SEM. C) Cells were labeled with SNAP-Surface 488 at 10∘ C for 1 h. The filled histograms show the fluorescence distribution of labeled cells without SF-CD59 expression. D) Quantitation. The quantitation represents the data of all experiments (n = 5) and shows the percentage of the surface expression of SF-CD59 normalized to control conditions. Error bars, SEM. beneficial technique to study the endocytic pathway of GPI-APs (53). Significance of fatty acid structure and incorporation into ordered lipid domains for the intracellular transport of GPI-APs Similar to other structural modifications of the GPI anchor, fatty acid remodeling is conserved among species, which suggests important physiological roles for GPI-APs (2,3,54). In yeast, lipid remodeling, which takes place in the ER, is critical for the concentration of GPI-APs into the ER exit sites, and therefore required for the efficient ER-to-Golgi transport of GPI-APs (55–57). In contrast, in mammalian cells, lipid remodeling occurs in the Golgi apparatus (6,7). It has been hypothesized that the association of GPI-APs with ordered lipid domains through the two saturated fatty acids is required for their Traffic 2014; 15: 1305–1329

segregation from other proteins and/or concentration upon Golgi exit, particularly in polarized epithelial cells (19). The existence of such domains in living cells has been continuously challenged by many studies (36,58,59). This hypothesis, however, has been attractive as an example of lipid-based protein sorting, particularly due to the topology of GPI-APs lacking a cytoplasmic domain to be recognized directly by coat proteins localized in the cytoplasm. In this study, we found that the Golgi-to-plasma membrane transport of SF-CD59 is impaired by cholesterol depletion, partially supporting the lipid-based protein sorting. However, SF-CD59 transport kinetics from the Golgi apparatus to the plasma membrane in lipid remodeling mutant cells were identical to that of restored cells. Previously, the Kinoshita group described that a significant transport delay of GPI-APs from the ER to the surface was not observed in PGAP2/3 mutant cells (7). These results 1321

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suggest that the difference in fatty acid structure which influences the association of GPI-APs with ordered lipid domain does not affect the Golgi exit efficiency of GPI-APs in CHO cells. The fact that cholesterol depletion similarly affects remodeled and unremodeled SF-CD59 transport suggests that the mechanism of cholesterol dependence on this pathway do77es not appear to be related to the potential interaction with ordered lipid domains. Based on this finding, we hypothesize alternative mechanisms for the Golgi exit of GPI-APs in CHO cells. One possibility is that GPI-APs exit the Golgi apparatus without particular concentration or segregation mechanisms from other proteins in non-polarized cells. However, this is unlikely as it has been reported that artificial GPI-APs (e.g. a fluorescent protein fused with a GPI attachment signal from lymphocyte function-associated antigen 3) and a transmembrane protein vesicular stomatitis virus glycoprotein segregate progressively in the Golgi apparatus excluding resident proteins and exit in separate transport containers in non-polarized rat kangaroo kidney epithelial cells (60). Several studies suggest that GPI-APs might have an intrinsic property to form homodimers/oligomers through protein–protein and/or GPI–GPI interactions (23,35,61–63). This property might be involved in segregation and/or concentration of GPI-APs required for their efficient exit from Golgi apparatus. Alternatively, there might be unknown transmembrane receptors responsible for the sorting of GPI-APs for their Golgi exit, similar to p24 family proteins that are responsible for the ER exit of GPI-APs (18,41,64). Further analysis will be required for the identification of such molecules to reveal the molecular mechanisms for the Golgi exit of GPI-APs. Interestingly, a recent study from the Zurzolo group has shown that the cholesterol dependence of the organization of GPI-APs is very different in non-polarized CHO cells and highly polarized MDCK cells suggesting that different mechanisms might operate for the selective Golgi exit in non-polarized and polarized cells (65). We previously showed that lyso forms of GPI-APs (lipid remodeling intermediates containing only one saturated fatty acid; Figure 1A) are apically sorted independent of DRM association and are sensitive to cholesterol depletion in MDCK cells (42). Further study testing the behavior of unremodeled GPI-APs in polarized cells will be required to clarify the role of lipid remodeling in polarized transport of GPI-APs. 1322

We also presented evidence that the lipid remodeling of GPI-APs does not appear to be required for the selective endocytosis via the CLIC/GEEC pathway, and for the targeting of internalized GPI-APs to recycling endosome. This is consistent with previous findings that phosphatidylethanolamine-polyethylene glycol (PE-PEG)-APs mimicking GPI-APs are internalized via the CLIC/GEEC pathway and colocalize with internalized GPI-anchored folate receptor, regardless of the length and saturation of the fatty acyl chains (45). In contrast, the same group demonstrated that the rate of recycling of internalized PE-PEG proteins to the plasma membrane is influenced by the fatty acyl chain structure (66). Internalized PE-PEG protein conjugates with long saturated chains recycle to the plasma membrane at a slow rate comparable to GPI-anchored folate receptor, whereas conjugates with short or unsaturated chains recycle at a faster rate (66). This supports a previous proposal that the association of GPI-APs with ordered lipid domains slows down recycling to the plasma membrane (26). Our endocytosis assay measures progressive accumulation of internalized SF-CD59 resulting from the equilibration of endocytosis and recycling events. In order to precisely measure the recycling efficiency of SF-CD59 without the contribution of continuous endocytosis, the cleavage of the surface SNAP-tag substrate has to be continuous during the recycling assay. As an efficient cleavage is obtained by the incubation of cells with 500 mM MesNa under alkaline pH (pH 8.6), which is not suitable for a continuous incubation during the chase, we could not address this particular point. Nonetheless, in our endocytosis assay, there is no difference in the efficiency of endocytosis of fully remodeled and unremodeled SF-CD59 suggesting that the overall internalization efficiency of GPI-APs via the CLIC/GEEC pathway does not depend on the precise fatty acid structure of GPI-APs. What could be the determinant for the selective endocytosis of GPI-APs via the CLIC/GEEC pathway? There are several potential mechanisms that are not mutually exclusive. As mentioned above, the potential intrinsic ability of GPI-APs to form homodimers/oligomers (23,35,61–63) might be involved in their incorporation into the CLIC/GEEC pathway. The conserved glycan structure of GPI-APs might be recognized by unknown receptors and responsible for an efficient and selective endocytosis. The finding that the replacement of the GPI anchor of folate receptor with a transmembrane Traffic 2014; 15: 1305–1329

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domain leads to the exclusion from the CLIC/GEEC pathway (13) supports this model. In contrast, the artificial PE-PEG proteins, which do not possess any structural similarity to the conserved GPI anchor, are selectively internalized via the CLIC/GEEC pathway. The presence of a bulky protein attached to the lipid anchor appears to be the key requirement for the preferential uptake into this pathway (45). Similarly, endogenous GPI-APs might also be internalized without an active sorting mechanism via this pathway, which is believed to be responsible for the uptake of bulk membrane and solutes. (67). Further biochemical and morphological analyses using the SNAP-tag system could be useful to address these open questions. In this work, we exclusively examined CD59 as a model GPI-AP to study intracellular transport. Therefore, our results do not exclude the possibility that some other GPI-APs might behave differently in mutant cells. Furthermore, the sorting and fate of internalized GPI-APs appear to be different in different cell types. For example, internalized GPI-APs accumulate in the recycling endosome in CHO cells, whereas in Baby Hamster kidney cells GPI-APs are transported to late endosomes (68). Therefore, it is important to confirm whether the contributions of lipid remodeling for the internalization or post-Golgi transport of other GPI-APs are conserved among cell types. Two saturated fatty acids of GPI-APs are required for the stable association of GPI-APs at the plasma membrane The lyso forms of GPI-APs are released into the medium just after their arrival at the cell surface, possibly by the reaction of an unknown lipase, as released GPI-APs no longer display hydrophobic properties by TX-114 partition assay (6,42). The surface expression of various GPI-APs in the corresponding mutant cells is drastically decreased (6,42). In contrast, we did not observe any obvious decrease of surface SF-CD59 containing an unsaturated fatty acid in PGAP2/3 mutant cells relative to restored cells at steady-state. We, however, observed a drastic release of surface SF-CD59 into the medium during cholesterol extraction in mutant cells, but much less in wild-type and restored cells. The released SF-CD59 showed hydrophobic characteristic by TX-114 partition assay suggesting that they contain at least one fatty acyl chain. Interestingly, the hydrophobic characteristic of released Traffic 2014; 15: 1305–1329

SF-CD59 was observed only under lipoprotein-depleted serum (LPDS) containing medium. The limited released fraction of SF-CD59 in PGAP2/3 mutant cells observed under complete medium (Figure 2B) was preferentially partitioned into the aqueous phase (data not shown). These results suggest that SF-CD59 expressed in PGAP2/3 mutant cells is sensitive to the cleavage reaction mediated in normal fetal calf serum (FCS) but not in LPDS. In contrast, the much larger amount of SF-CD59 released during cholesterol extraction in LPDS medium does not appear to be dependent on such cleavage, but more likely occur as spontaneous release. Even though the exact mechanisms responsible for the release and cleavage of unremodeled GPI-APs are not completely understood, our study demonstrates the importance of two saturated fatty acids in the stability of GPI-APs at the plasma membrane, particularly during cholesterol extraction. In yeast, significant fractions of GPI-APs are released from the membrane not only in mutant cells defective in different steps of lipid remodeling, but also defective in the first step of sphingolipid biosynthesis (55,56,69). Therefore, we originally speculated that two saturated fatty acids of GPI-APs might be important to form tight interaction with sphingolipids which often contain saturated acyl chain (70). Under control conditions, unremodeled GPI-APs might still associate with the membrane due to the support of cholesterol in the membrane. However, once cellular cholesterol is extracted, unremodeled GPI-APs (but not remodeled GPI-APs associating tightly with sphingolipid) might be no longer retained in the membrane and released from the cell surface. However, glycosphingolipids depletion by 60% showed only modest and similar effects on surface expression of unremodeled and remodeled GPI-APs suggesting that both forms of GPI-APs appear to be equally dependent on sphingolipid for their membrane association. Examining mobility and distribution of GPI-APs in these mutant cells using various advanced microscopy techniques (61–63,65,71) and the identification of molecules specifically interacting with remodeled GPI-APs but not with unremodeled GPI-APs will help our further understanding on how the fatty acid structure influences the association with membranes. The surface expression of different GPI-APs expressed in various cells derived from Pgap3−/− mice is reduced compared to that of Pgap3+/− mice. Although various complex 1323

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phenotypes of Pgap3−/− mice have been reported (32,33), the molecular mechanisms underlying these phenotypes remain largely unknown. It is highly possible that the precise fatty acid structure of GPI-APs might be important for the various functions of different GPI-APs. In addition to this, the lack of stability of GPI-APs especially upon membrane perturbation, as demonstrated in this study, is probably to cause considerable impact on the steady-state expression of various GPI-APs in different tissues, particularly in living environments. This could account for some of the phenotypes observed in PGAP3-deficient patients and Pgap3−/− mice. Our results highlight a widely conserved role of lipid remodeling in the stability of GPI-APs at the plasma membrane in eukaryotic cells.

Materials and Methods Cell lines, cell culture, plasmids and reagents CHO cells were cultured in Ham F12 medium (Invitrogen) supplemented with 10% FCS and penicillin-streptomycin (Invitrogen) in an incubator at 37∘ C with 5% CO2 . Wild-type GD3S-C37 and PGAP2&3 double-mutant DM2&3-C cells were obtained from Prof. T. Kinoshita (Research Institute for Microbial Diseases, Osaka University). The clones stably expressing SF-CD59 were generated by transfection (electroporation) of pME-hygro-SNAP-FLAG-CD59 and the selection was made with 400 μg/mL of hygromycin in complete F12 medium. The PGAP2&3 restored cells were generated by retroviral infection using pMSCV-rPGAP2-zeo-hPGAP3 obtained from Prof. T. Kinoshita and the selection was made with 250 μg/mL of zeocin in complete F12 medium. The vector to transiently express dominant negative Arf1(T31N)-GFP was provided by Prof. J. Gruenberg (Biochemistry Department, University of Geneva). To construct pME-hygro-SNAP-FLAG-CD59, a BamHI fragment containing a cDNA encoding SNAP-FLAG-CD59 derived from pME-puro-SNAP-FLAG-CD59 (42) was ligated with a BamHI fragment of pME-hygro (+) provided by Dr. Y. Maeda (Osaka University). The SS-cleavable SNAP-substrates (BG-SS-PEG488 and BG-SS-PEG549) were provided by New England Biolabs. The other SNAP substrates (SNAP-Cell Block, SNAP-Cell TMR Star, SNAP-Surface 488 and SNAP-Surface 647) were purchased from New England Biolabs. The mouse monoclonal antibodies against Lamp1 and DAF were provided by Prof. J. Gruenberg and by Prof. T. Kinoshita, respectively. The mouse monoclonal antibodies against TfR (13-6800) and FLAG M2 were purchased from Invitrogen and Sigma, respectively. The rabbit polyclonal antibody against Caveolin (610059) was purchased from BD Biosciences. The mouse monoclonal anti-Ganglioside GD3 antibody (R24) was purchased from Abcam. The cy3, cy5 or allophycocyanine (APC)-conjugated anti-rabbit or mouse IgG were purchased from Jackson ImmunoResearch.

DRM assay The assay was performed as previously described (72). Cells were seeded on 14 cm dishes the day before the experiment. On the day of the

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experiment, the cells were 80–90% confluent. The cells were washed with PBS containing Ca2+ and Mg2+ (PBS+ ) and scraped. After centrifugation, the cells were resuspended in TNE + PIs [150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA) and 50 mM Tris–HCl at pH 7.4 containing the protease inhibitors leupeptin, pepstatin and aprotinin]. The cell extract was homogenized by passing 20 times through a 25-G needle. Three hundred micrograms of protein was diluted into 180 μL of TNE + PIs and incubated with 20 μL of 10% Triton-X-100 for 30 min on ice. Four hundred microliters of 60% optiprep was added and the extract was transferred to centrifuge tubes (Beckman Coulter, No. 347356). The extract was overlaid with 1.2 mL of 30% optiprep in TNE and 200 μL of TNE buffer. The gradient was centrifuged for 2 h at 4∘ C at 250 000 × g in a TLS 55 rotor (Beckman Coulter). Four 500 μL fractions were taken from the top of the gradient. For western blotting, the fractions were either mixed with 2× sample buffer with or without 10% β-mercaptoethanol (reducing conditions suitable for all antibodies except for DAF). The first antibody was diluted in 5% milk in TBS-T (DAF 1:2500, Caveolin 1:5000 and TfR 1:2500) and incubated at 4∘ C overnight. The membrane was washed three times with TBS-T and incubated with the horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse antibody diluted in 5% milk in TBS-T (1:2500). The chemiluminescence was detected with the CCD camera for quantitation (Syngene GeneGnome) or X-ray films (Fujifilm). For in-gel fluorescence analysis, the fractions were labeled with SNAP-Cell TMR Star (0.3 μM) for 30 min at room temperature, mixed with 2× sample buffer including 10% β-mercaptoethanol and heat-treated at 95∘ C for 5 min. All fluorescent samples were analyzed by SDS–PAGE followed by an in-gel fluorescence scan (Ettan DIGE fluorescence scanner, GE Healthcare) and analyzed and quantified with IMAGE J. The fluorescent scanner provided a very high dynamics range and we did not use any data with saturated signals for all experiments presented here. For western blotting, membranes were exposed for at least five different lengths of time with a CCD camera. To avoid a quantitation of saturated bands, we analyzed several images for quantitation. The most suitable image showing linear range of the intensity for proteins of interest were quantified and used in the figures.

Biosynthetic transport assay Cells were seeded in the six-well plates 24 or 72 h (in the case of cholesterol depletion) prior to the experiments. On the day of the experiment, the cells were 70–80% confluent. Two wells of a separate plate were prepared for each time point. A separate plate was used for each time point. The cells were incubated with SNAP-Cell Block (0.4 μM) at 37∘ C for 30 min, then washed three times with warm medium and incubated for 30 min at 37∘ C. All following steps were performed in the presence of 200 μg/mL of CHX to prevent further protein synthesis. One of the wells was incubated with and the other without SNAP-Surface 647 (0.2 μM) at 10∘ C for the time point 0 min for 40 min and at 37∘ C for the 20 and 40 min time points during chase time. Finally, all wells of all three plates were incubated with medium containing with 0.12 μM SNAP-Cell TMR and 10 mM HEPES (pH 7.4) for 30 min at room temperature. The cells were washed with PBS+ and harvested by scraping. After centrifugation, the cell pellet was resuspended in 120 μL of 2× samples buffer, heat-treated at 95∘ C for 5 min and sonicated for 10 min. For the analysis of the secreted

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proteins, the medium was taken after each time point and centrifuged at 20 000× g at 4∘ C for 10 min to remove cell debris. The supernatant

antibiotics, in the presence or absence of 2 μM compactin, also known

was mixed with anti-FLAG M2 agarose beads (Sigma) equilibrated in PBS+ and incubated overnight at 4∘ C. The beads were washed three times

as Mevastatin, and 0.25 mM mevalonate for 48 h. On the day of the experiment, cells were further incubated with or without 10 mM MβCD in F12 medium containing 5% LPDS for 30 min at 37∘ C. For cholesterol

with PBS+ , labeled with 0.3 μM SNAP-Cell TMR in TNE + PIs for 30 min at room temperature, mixed with 120 μL of 2× sample buffer and heat-treated at 95∘ C for 10 min. All samples were analyzed by SDS–PAGE

extraction, cells were cultured normally and treated with 10 mM MβCD in F12 medium containing 5% LPDS for 30 min at 37∘ C just before the experiment.

followed by an in-gel fluorescence scan. The scans were analyzed and quantified with IMAGE J. For morphological analysis, the transport assay was performed similarly except that cells were seeded on coverslips and

Amplex red cholesterol assay kit

fixed with 4% PFA in PBS at the end of the assay.

The Amplex Red Cholesterol Assay from Invitrogen was performed according to the manufacturer’s protocol. Cells were washed and scraped

Endocytosis assay

in PBS+ and the protein concentration was determined by Bradford assay. The cholesterol amounts were measured using duplicates of 5 and 10 μg protein. The samples were measured with a SpectraMax fluorometer

For suspension cell conditions, the cells were seeded in the six-well plates 24 h prior to the experiment. On the day of the experiment, the cells were 80–90% confluent. The cells were harvested with trypsin–EDTA (Invitrogen) and then collected with complete medium, transferred to 1.5 mL eppendorf tubes and centrifuged. The cells were resuspended in 250 μL of complete medium containing 0.2 μM SS-cleavable SNAP-substrate (BG-SS-PEG488 or BG-SS-PEG549) and were divided into four wells in a 96-well plate, each well corresponding to one time point (0, 15, 30 and 45 min). The cells were labeled for 1 h at 10∘ C. All centrifugation steps were performed with a 96-well plate rotor at 2000× g at 4∘ C. After two washes with medium, the cells were incubated at 37∘ C for 15, 30 and 45 min and then put on ice or directly put on ice for time point 0 min. The cells were washed twice with opti-MEM (Invitrogen) and split into two wells. They were incubated with 75 μL of alkaline TNE (100 mM NaCl, 50 mM Tris–HCl at pH 8.6 and 1 mM EDTA) with or without 500 mM MesNa (Sigma) for 5 min on ice. One hundred fifty microliters of opti-MEM was added to each well and the cells were centrifuged. Finally, the cells were resuspended in 200 μL of PBSazBSA (PBS− , 0.1% BSA and 0.1% NaN3 ) and then transferred into FACS tubes containing 300 μL of PBSazBSA and analyzed with Gallios Flow cytometry (Beckman Coulter). Five thousand cells were counted for each sample. To determine the fluorescence background of the cells, we labeled control CHO cells without SF-CD59

(Molecular Devices).

Dil-LDL uptake Cells were grown on coverslips. Four to six hours before the experiment, the cells were washed and the medium was changed to F12 medium containing 5% LPDS. Dil-LDL (Invitrogen) was diluted to 10 μg/mL in F12 medium containing 5% LPDS and applied to the cells for 1 h at 10∘ C separately or together with SS-cleavable SNAP-substrates. The unbound Dil-LDL was washed out with PBS+ . The cells were then incubated with F12 containing 5% LPDS for 15 min (Dil-LDL uptake alone) or 25 min (Dil-LDL uptake together with SF-CD59 internalization) at 37∘ C and further processed as described for the endocytosis assay.

Dynamin inhibition The dynamin inhibitor dyngo-4a (Abcam) was diluted to 20 μM in complete F12 medium and applied during labeling with SS-cleavable SNAP-substrates and Dil-LDL binding for 1 h at 10∘ C. The same dyngo-4a concentration was used during internalization at 37∘ C.

expression. The data was analyzed with FLOWJO.

Actin manipulation For attached cell condition, the cells were seeded in 24-well plates for 24 h. The cells were incubated in 150 μL of complete medium containing 0.2 μM SS-cleavable SNAP-substrate (BG-SS-PEG488). One well corresponds to one time point (0, 15, 30 and 45 min). The cells were labeled for 1 h at 10∘ C. After two washes with medium, the cells were incubated at 37∘ C for 15, 30 and 45 min and then put on ice, or directly put on ice for time point 0 min. The cells were harvested with trypsin–EDTA (Invitrogen) and then collected with complete medium and further processed for incubation with or without 500 mM MesNa as shown for suspension cell condition. For morphological analysis, the endocytosis assay was performed similarly, except that cells were cultured on coverslips and remained attached throughout the experiment, and were fixed with 4% PFA in PBS at the end of the assay.

Cholesterol manipulation For cholesterol depletion, 24 h after the cells were seeded, the cells were washed twice and cultured in F12 medium containing 5% LPDS,

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Latrunculin B (Sigma) was added at 1 μM in F12 medium containing 5% LPDS toward the end of the labeling with SS-cleavable SNAP-substrates and Dil-LDL binding for 15 min at 10∘ C. The same concentration was used throughout the washing and the incubation periods at 37∘ C.

Immunofluorescence and microscopy The cells were seeded on glass coverslips 48 h prior to the experiment. The confluency on the day of the experiment was 50–60%. After endocytosis assay under attached cells, cells were fixed with 4% PFA in PBS for 15 min followed by permeabilization and blocking with 3% BSA and 0.05% saponin in PBS+ for 30 min. The first antibodies were diluted in 3% BSA in PBS+ (TfR, 1:250; Lamp1, 1:50) and incubated for 1 h. After three washes with PBS+ , the cells were incubated with cy3-conjugated anti-mouse IgG (1:200) in 3% BSA in PBS+ for 30 min. All steps were performed at room temperature. The images were taken with a ZEISS LMS 700 confocal and analyzed and quantified with IMAGE J using JACoP.

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Membrane association assay

500 μL of TNE was added to the detergent phase and 50 μL of 11%

For flow cytometry analysis, the cells were seeded in 24-well plates 24 h before the experiment. The confluency on the day of the experiment was 60–80%. For surface labeling of SF-CD59, cells were incubated in

Triton-X-114 to the aqueous phase and the samples were incubated for 5 min on ice. The samples were incubated for 5 min at 32∘ C and then centrifuged at 3000× g for 3 min at room temperature. The aqueous and

200 μL of complete F12 medium containing 0.2 μM SNAP-Surface 647 for 1 h at 10∘ C. After three washes with PBS+ , cells were incubated in F12 containing 5% LPDS in the presence or absence of 10 mM MβCD for 30 min at 37∘ C. The cells were washed twice with PBS− , detached

detergent phases were separated. Detergent phases and aqueous phases from the first and the second separations were combined. The detergent phases were supplemented with 1 mL of RIPA buffer containing protease

with 150 μL of 0.5% trypsin–EDTA and then collected with F12 medium, transferred into eppendorf tubes and centrifuged. After two washes with F12 medium and once with PBSazBSA, the cells were resuspended in 200 μL of PBSazBSA and transferred into FACS tubes containing 300 μL of PBSazBSA and analyzed with a Gallios Flow cytometer. Five thousand cells were counted for each sample. The data was analyzed with FLOWJO. For biochemical analysis, the cells were seeded in six-well plates 24 h before the experiment. The confluency on the day of the experiment was 60–80%. For surface labeling of SF-CD59, cells were incubated in 750 μL of complete F12 medium containing 0.2 μM SNAP-Surface 647 for 1 h at 10∘ C. After three washes with PBS+ , cells were incubated in F12 containing 5% LPDS in the presence or absence of 10 mM MβCD for 30 min at 37∘ C. To examine released fraction, the medium was collected. The cells were washed twice with PBS+ and harvested by scraping, transferred into eppendorf tubes and centrifuged. The medium and the cell pellets were solubilized in RIPA buffer (50 mM Tris–HCl at pH 7.5, 150 mM NaCl, 1% TX-100, 0.5% sodium deoxycholate and 0.1% SDS) containing protease inhibitors and incubated for 30 min at 25∘ C. The samples were centrifuged 10 min at 20 000 × g to remove insoluble material. The supernatants were mixed with anti-FLAG M2 agarose beads and incubated overnight at 4∘ C to immunoprecipitate SF-CD59. The beads were washed twice with RIPA buffer, mixed with 2× sample buffer including 10% β-mercaptoethanol and heat-treated at 95∘ C for 10 min. All samples were analyzed by SDS–PAGE followed by an in-gel fluorescence scan. The scans were analyzed and quantified with IMAGE J.

inhibitor, the aqueous phases with 100 μL of 10× RIPA buffer containing protease inhibitors and then incubated at 25∘ C for 30 min. The samples were centrifuged at 20 000 × g for 10 min at 4∘ C. The supernatant was mixed with anti-FLAG M2 agarose beads and incubated overnight at 4∘ C to immunoprecipitate SF-CD59. The beads were washed with RIPA buffer, mixed with 2× sample buffer including 10% β-mercaptoethanol and heat-treated at 95∘ C for 10 min. All samples were analyzed by SDS–PAGE followed by an in-gel fluorescence scan. The scans were analyzed and quantified with IMAGE J.

Sphingolipid depletion Cells were seeded on 24-well plates and cultured for 24 h in Nutridoma-BO containing medium [Ham F12, 1% Nutridoma-SP (Roche), 0.1% FCS and 10 μM sodium oleate complexed with bovine serum albumin] with or without 2.5 μM myriocin (Sigma). The cells were 50% confluent on the day of the experiment. The surface levels of sphingolipids were measured by flow cytometry. The cells were detached with trypsin–EDTA and incubated for 2 h with a monoclonal mouse anti-GD3 antibody (1:400) at 4∘ C in PBSazBSA. The cells were washed with PBSazBSA and incubated with a secondary APC-conjugated anti-mouse IgG antibody (1:200) at 4∘ C for 1 h. After one wash with PBSazBSA, the cells were resuspended in 200 μL of PBSazBSA and transferred into FACS tubes containing 300 μL of PBSazBSA and analyzed with a Gallios Flow cytometer. Analysis of the steady-state SF-CD59 surface expression under sphingolipid depletion was done as following. After 24 h of myriocin or mock treatment, the cells were detached with trypsin–EDTA and incubated with Nutridoma-BO medium containing 0.5 μM SNAP-surface 488 for 1 h at 10∘ C. After one wash with PBSazBSA, the cells were resuspended in 200 μL of PBSazBSA and transferred into FACS tubes containing 300 μL of PBSazBSA and analyzed with a Gallios

Triton-X-114 partition assay

Flow cytometer. Five thousand cells were counted for each sample. The data was analyzed with FLOWJO.

The cells were seeded in six-well plates 24 h before the experiment. The confluency on the day of the experiment was 60–80%. For surface labeling of SF-CD59, cells were incubated in 750 μL of complete F12 medium containing 0.2 μM SNAP-Surface 647 for 1 h at 10∘ C. After three

Acknowledgments

washes with PBS+ , cells were incubated in F12 containing 5% LPDS in the presence or absence of 10 mM MβCD for 30 min at 37∘ C. The medium was

We thank T. Kinoshita, Y. Maeda and J. Gruenberg for cells and reagents;

collected. Four hundred microliters of medium was supplemented with 50 μL of TNE (10 mM Tris–HCl at pH 7.4, 150 mM NaCl, 5 mM EDTA and protease inhibitors) and 50 μL of 11% Triton-X-114. The samples were

D. Garcin for retrovirus production; C. Bauer and J. Bosset for assistance with imaging; B. Baker for assistance with the synthesis of SS-cleavable SNAP-substrates; M.-Q. Xu for assistance with conditions for MesNa

mixed and incubated on ice for 30 min. To remove insoluble material, the samples were centrifuged for 15 min at 20 000 × g at 4∘ C and the supernatant was transferred to new eppendorf tubes. The samples were incubated for 5 min in a 32∘ C water bath to induce phase separation.

treatment; G. Castillon, D. Abegg, J. Gruenberg, M. Fujita, A. Roux and H. Riezman for discussion. This study was supported by the Swiss National Science Foundation through a professorship to R. W. I. R. C is an employee

After centrifugation for 3 min at 3000× g at room temperature, the upper aqueous phase was transferred to a new eppendorf tube. For re-separation,

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of New England Biolabs that manufactures and sells the SNAP-tag system, components of which are used in this study. The other authors declare that no competing interests exist.

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Role of Fatty Acid Moiety of GPI-APs

Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1: Endocytosis assay with attached cells. A) Endocytosis assay with mutant (MUT) and restored (REST) cells. The histograms represent the data obtained by flow cytometry of one experiment and show the fluorescence distribution of non-treated (dotted lines) and MesNa-treated (solid lines) in mutant (dark-colored) and restored (light-colored) cells at 0, 15, 30, and 45 min of internalization at 37∘ C. The filled histograms show the fluorescence distribution of labeled cells without SF-CD59 expression. B) Quantitation. At each time point, the percentage of internalized SF-CD59 compared to the total SF-CD59 was calculated. The quantitation includes all experiments (n = 3). Error bars, SEM. C) Endocytosis assay with mutant (MUT) and restored (REST) cells in control (CTRL) and cholesterol depletion (MβCD) conditions. Control cells were cultured in medium containing 5% LPDS. Cholesterol-extracted cells were incubated in medium containing 5% LPDS and 10 mM MβCD for 30 min. At 45 min, the percentage of internalized SF-CD59 compared to the total SF-CD59 was calculated. The quantitation includes all experiments (n = 5). Error bars, SEM. *p < 0.05. D) Endocytosis assay with mutant (MUT) and restored (REST) cells in control condition (CTRL) and dyngo-treated condition (Dyngo). At 45 min, the percentage of internalized SF-CD59 compared to the total SF-CD59 was calculated. The quantitation includes all experiments (n = 5). Error bars, SD.

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