October 2009, revised and accepted for pub- pub-lication 10 May 2010, uncorrected manuscript published

Exit of GPI-Anchored Proteins from the ER Differs in Yeast and Mammalian Cells

Received 13 October 2009, revised and accepted for pub- pub-lication 10 May 2010, uncorrected manuscript published

online 11 May 2010

Secretory proteins are synthesized on ribosomes bound to the endoplasmic reticulum (ER) and inserted co-translationally into the ER membrane, from where they are transported via the Golgi apparatus to their final destination on the cell surface or in other intracellular membrane compartments. Export of proteins from the ER is coordinated by assembly of coat protein complex II (COPII) at discrete sites on the ER surface known as ER exit sites (ERES). In the yeastSaccharomyces cerrevisiae

and in mammalian cells, assembly of the COPII coat is initiated by Sec12-dependent GDP–GTP exchange on the small GTPase Sar1 (1). The activated Sar1-GTP sub-sequently recruits the Sec23–Sec24 and Sec13–Sec31 complexes to the exit sites (1).

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are another class of eukaryotic membrane protein, which are held in the membrane by a GPI lipid anchor.

GPI biosynthesis is essential in yeast, and defective biosynthesis in mammals causes embryonic lethality (2,3).

In contrast to other lipid anchors, the GPI anchor has a complex structure, including a conserved glycan core and a phospholipid tail. GPI anchors are synthesized from phosphatidylinositol (PI) and various sugar donors in the ER (4,5). The precursor proteins have an N-terminal signal peptide and a GPI-attachment signal at the C-terminus.

This GPI-attachment signal is subsequently cleaved and a preassembled GPI anchor is transferred in the ER. Without GPI attachment, the precursor proteins are not expressed at the cell surface but instead accumulate mainly in the ER (6–8).

The lipid moieties of GPI-APs are sequentially remodeled both in yeast and mammalian cells. The proteins respon-sible for lipid remodeling were cloned recently from yeast and mammals (9–12). They show significant homologies to each other, indicating the likely conserved biological role of lipid remodeling of the GPI anchor (4,5,13). Inter-estingly, the yeast remodeling enzymes are located in the ER, whereas the mammalian proteins are mainly in the Golgi apparatus (10–12), suggesting that remodel-ing occurs in different membrane compartments in yeast and mammalian cells. We do not fully understand the functional consequences of lipid remodeling of GPI-APs;

however, remodeling confers on GPI-APs the property to co-purify with the detergent-resistant membrane (DRM) fraction (9,10,12). This association with DRMs has been postulated to reflect the incorporation of GPI-APs into lipid rafts, which contain mainly sphingolipids and sterols (14).

Incorporation into lipid rafts may target the GPI-APs to par-ticular regions of the cell. In polarized mammalian epithelial cells, e.g., most GPI-APs are preferentially targeted to the apical domain of the plasma membrane.

In yeast, GPI-APs and other secretory proteins are prefer-entially transported from the ER to the Golgi apparatus in different vesicle populations (15–17). Consistent with this, Castillon et al. (18) observed, by using thermosensitive mutantsec31-1 yeast cells that accumulate cargo proteins in the ERES, that GPI-APs accumulate in ERES that are dis-tinct from those in which other secretory proteins accumu-late. Furthermore, in these cells, concentration of GPI-APs www.traffic.dk 1017

Rivier et al.

in ERES required GPI-lipid remodeling (18). Concentration of transmembrane proteins in ERES, by contrast, was tightly dependent on Sec12p and Sec16p (18). In yeast, efficient ER-to-Golgi transport of GPI-APs requires ongo-ing sphongo-ingolipid synthesis (19–21) and GPI-APs are only loosely associated with the ER membrane in cells mutant for sphingolipid biosynthesis (21). Together, these findings imply that in yeast GPI-APs concentrate at specific ERES by an interaction between their remodeled lipid moieties and sphingolipids, whereas non-GPI-APs concentrate in different ERES by an interaction that requires COPII coat assembly (18).

Given the evidence that in mammalian cells the lipid remodeling enzymes are located in the Golgi apparatus and also the association of GPI-APs into sphingolipid-rich DRM occurs in the Golgi, we set out to investigate whether, as in yeast, the exit of GPI-APs from the ER in mammalian cells depends on interactions with sphingolipids and whether the mechanism involves distinct ERES for GPI-APs and other secretory proteins.

We report here that neither is the case: transport of mammalian GPI-APs out of the ER does not depend on ongoing sphingolipid synthesis nor are the GPI-APs segregated from other secretory proteins into distinct COPII vesicles. These findings suggest that yeast and mammals use different mechanisms to concentrate their GPI-APs into COPII vesicles.

Results

Sphingolipid is not required to transport mammalian GPI-APs from the ER to the Golgi

In yeast, the efficient ER-to-Golgi transport of GPI-anchored proteins from the ER to the Golgi apparatus specifically requires ongoing sphingolipid biosynthesis.

The first step in the ceramide synthesis is mediated by Lcb1p in yeast and serine palmitoyltransferase (SPT) in mammalian cells. To test whether efficient ER-to-Golgi transport of GPI-APs in mammals requires ongoing sphingolipid biosynthesis, we examined the kinetics of ER-to-Golgi transport of a GPI-AP, human placental alkaline phosphatase (PLAP) by using pulse-chase metabolic labeling with ³⁵S-methionine and cysteine followed by endoglycosidase H (Endo H) treatment in an SPT mutant CHO cell line, LY-B (22). The acquisition of Endo H

resistance by newly synthesized N-linked glycoproteins is a marker for arrival of the glycoprotein in the medial Golgi apparatus (23). In this experiment, in which the medium was depleted of sphingolipid, ceramide in the LY-B cells was undetectable and levels of sphingolipids such as sphingomyelin and glycosphingolipids were less than 20%

of wild-type cells (Table 1). Under these conditions, we observed no significant delay in the ER-to-Golgi transport of PLAP in LY-B cells when compared to the wild-type cells (Figure 1A,B). Furthermore, when we treated wild-type CHO-K1 cells with myriocin, a specific inhibitor of SPT (24) used to reduce significantly the amount of cellular sphingolipid in mammalian cells (25), we also saw no difference in the transport kinetics of PLAP when compared with untreated cells (Figure 1C,D), as reported previously for HeLa cells (26). These data demonstrate that, in contrast to yeast, in mammalian cells transport of GPI-APs from the ER to the medial Golgi does not require ongoing sphingolipid synthesis.

In vitrobudding of cargo proteins from the ER in mammalian cells

To study the incorporation of transmembrane secretory proteins and GPI-APs into transport vesicles that bud from the ER, we used anin vitro assay that reconstitutes cargo protein incorporation into COPII vesicles that bud from the ER. First, we tried to detect endogenous GPI-APs, such as μPar1, expressed in CHO-K1 cells; however, insufficient label was incorporated by metabolic labeling to detect a significant signal in the vesicle fraction with the monoclonal antibody we used (data not shown).

We, therefore, expressed two exogenous FLAG-tagged GPI-anchored proteins: the folate receptor (F-FR1) and CD59 (F-CD59). We compared the expression level of these GPI-APs with endogenous GPI-APs in CHO-K1 cell by affinity purification using alpha-toxin, which binds GPI-anchored proteins (27), and found that the expression level of the exogenous GPI-APs was comparable to that of an unknown endogenous GPI-AP in CHO-K1 cell (Figure S1A).

To follow transport of cargo proteins from the ER, we first pulse-labeled cells with ³⁵S-methionine and cysteine, then we perforated the pulse-labeled cells with a low concentration of digitonin and incubated these

‘semi-intact’ cells with exogenous rat liver cytosol in the presence of an ATP regenerating system and GTP (28,29).

Table 1:Phospholipid and sphingolipid levels in CHO cells

Strain PC PE PS/PI SM G_M3 GlcCer Ceramide

(nmol/mg protein)

wt/PLAP 104.7±5.7 49.5±2.1 22.4±2.2 25.3±0.8 3.3±1.1 1.0±0.1 1.14±0.07

LY-B/PLAP 122.6±5.3 44.1±1.5 20.3±0.7 2.6±1.0 0.6±0.3 <0.2 <0.05

CHO-K1 cells were cultured in Nutridoma-BO medium at 37^◦C for 2 days. Lipids were extracted from the cells, separated by thin layer chromatography and the amount of each lipid was determined. The data shown are the mean values±SD from triplicate experiments.

PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; SM, sphingomyelin; GM3, N-acetylneuraminyl lactosylceramide; GlcCer, Glucosylceramide.

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ER Exit of GPI-AP in Mammalian Cell

Figure 1: Sphingolipid is not required for ER-to-Golgi transport of GPI-APs in mammalian cells.Cells were pulse-labeled for 15 min with³⁵S-Met and³⁵S-Cys and chased for the indicated times. Immunoprecipitates were not treated (−) or treated (+) with Endo H for 16 h and separated by SDS–PAGE. The intensities of the Endo H-resistant and cleaved forms were quantified with a phosphoimager.

R, Endo H-resistant form; S, Endo H-sensitive form; C, Endo H cleaved form. (A) Pulse-chase experiment with wild-type CHO-K1 and LY-B mutant cells defective in the first step of sphingolipid biosynthesis, both stably expressing the human GPI-AP PLAP cultured in Nutridoma-BO for two days before the experiment to deplete exogenous sphingolipids from the normal medium. (B) Quantification of A.

(C) Pulse-chase experiment with wild-type CHO-K1 cells expressing PLAP preincubated in Nutridoma-BO for 1 h with (+myr) or without (−myr) 2.5μ^Mmyriocin, a specific inhibitor of the first step of sphingolipid biosynthesis. (D) The quantification of C.

Because we aimed to reconstitute incorporation of various cargo proteins into COPII vesicles, we chose rat liver cytosol, which contains the various isoforms of COPII typical of mammalian cells (30), as a source of COPII components. Vesicles formed in vitro during the assay were separated from donor membranes by centrifugation, and the amount of pulse-labeled cargo proteins in the vesicle fraction was quantified. We observed cytosol-dependent incorporation into the vesicle fraction of the FLAG-tagged GPI-APs, F-FR1 and F-CD59 (Figure 2A, lanes 1 versus 2), and the transmembrane proteins vesicular stomatitis virus G protein (VSV-G; Figure 2A, lanes 1 versus 2), EGFP-tagged Kit ligand (KitL-EGFP;

data not shown) and EGFP-tagged low-density lipoprotein receptor (LDLR-EGFP; Figure 2B, lanes 1 versus 2). There were two bands of VSV-G, F-FR1 and F-CD59 in both total and vesicle fractions (Figure 2A). We examined the

sensitivity of these bands by Endo H treatment and confirmed that the most proteins observed in the vesicle fractions were indeed derived from the ER (Figure S1).

Incorporation of these cargo proteins was significantly inhibited by addition of recombinant GTP-locked form of hamster Sar1A protein, Sar1AH79G, which cause a dominant negative effect in the function of Sar1p (31) (Figure 2A,B,D), demonstrating that incorporation of the cargo proteins into the vesicle fraction is COPII dependent.

By analyzing ER-resident proteins such as calnexin and Sec61α, we estimated that there was very little passive incorporation of ER membrane proteins into the vesicle fraction (Figure 2C). Because the signal from these ER-resident proteins in the vesicle fraction was low, we could only estimate the percentage in the vesicle fractions by comparison with the titrated amount of total

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Rivier et al. F-FR1 and F-CD59 and (B) LDLR-EGFP. Lanes 1–3, vesicle frac-tion; lanes 4, 14% of total reac-tion. Italic numbers correspond to the percentage of each pro-tein found in the vesicle frac-tion. (C) Immunoblot analysis of endogenous ER-resident proteins calnexin and Sec61αin vesicles prepared under various several cargo proteins in the semi-intact cell assay with CHO-K1 cells. Relative budding efficiency was normalized to budding in the presence of rat liver cytosol alone [+cyto-Sar1A(H79G)].n₌5.

signal: 0.2–0.6% of calnexin and Sec61α were found in the vesicle fraction, which is substantially lower than the amounts of the cargo proteins (Figure 2A,B and Table 2).

GPI-APs, F-FR1 and F-CD59 budded less efficiently than VSV-G in our in vitro assay (Table 2). We measured the in vivo kinetics of ER-to-Golgi transport of VSV-G, F-FR1 and F-CD59 by pulse-chase experiments followed by Endo H treatment. The two GPI-APs showed much slower ER-to-Golgi transport than VSV-G (Figure S1B,C).

The lower incorporation efficiency of GPI-APs than VSV-G into COPII vesicles in thein vitro assay seems to agree well with their respective in vivo ER-to-Golgi transport kinetics. Thus, we have reconstituted physiological and COPII-dependent incorporation of various transmembrane secretory proteins and GPI-APs into ER-derived vesicles in vitro.

Budding of GPI-APs from the yeast ER depends only partially on Sar1p

Castillon et al. recently reported that in yeast the GPI-lipid moiety of GPI-APs must be remodeled before the proteins can concentrate at ERES and incorporate into COPII vesicles in vivo (18). The transmembrane secretory protein, hexose transporter 1, requires COPII coat assembly mediated by Sec12p and Sec16p for its concentration in the ER, whereas GPI-APs do not, suggesting that different concentration mechanisms coexist upon ER exit in yeast (18). To examine directly the requirement for COPII coat assembly at the ER budding step in yeast, we used an assay in semi-intact yeast cells (15,32). This assay is similar to the mammalian semi-intact cell assay described above; it uses pulse-labeled semi-intact yeast spheroplasts and a yeast cytosol fraction to reconstitute vesicle formation from the ER.

We found that yeast GPI-APs and other secretory

Table 2:Efficiencies of secretory cargo protein incorporation into ER-derived vesiclesin vitro

Membrane fraction VSV-G KitL-EGFP LDLR-EGFP F-FR1 F-CD59

(Percentage in vesicle fractions)

Semi-intact cells 13.8±5.7 (5) 10.9±2.2 (4) 8.1±2.7 (2) 2.6±0.8 (5) 2.6 ±1.3 (5)

Microsomes 12.0±4.2 (9) 7.2±1.4 (2) n.d. 3.5±0.4 (4) n.d.

The data shown are the mean values±SD or range (number of independent experiments). Variations in the incorporation efficiency are mostly because of the different batches of rat liver cytosol and membrane fractions used. n.d, not determined.

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Figure 3: Partial Sar1p dependence of GPI-APs ER budding in yeast.(A) The semi-intact cell-based budding assay with wild-type yeast membranes and cytosol with or without the GTP-locked form of yeast Sar1 (H77L). Lanes 1–3, vesicle fraction; lanes 4, 7%

or 4% of total the reaction where cytosol was added. Italic numbers correspond to the percentage of each protein found in the vesicle fraction. (B) Relative budding efficiency quantified from A similarly done as Figure 2D.n₌10 for Gap1p and Gas1p;n₌3 for Ccw14p-Venus;n₌4 for Cwp2p-Venus. (C) The semi-intact cell-based budding assay with yeast membrane and cytosol derived from temperature-sensitive mutant cells ofSAR1, sar1E112Kcells in the absence or presence of recombinant wild-type yeast Sar1p. Lanes 1–4, vesicle fraction; lanes 5, 7% of the total reaction where cytosol was added. Italic numbers correspond to the percentage of each protein found in the vesicle fraction. (D) Relative budding efficiency quantified from C. Relative budding efficiency was normalized to the budding efficiency in the presence of mutant cytosol and wild-type Sar1p (+cyto+Sar1p). The results correspond to the average of two independent experiments.

proteins had differential requirements for Sar1p in this assay (Figure 3A,B). The recombinant GTP-locked form of Sar1p (H77L) (33) strongly inhibited incorporation of non-GPI-anchored secretory proteins, such as general amino acid permease (Gap1p) and glycosylated pro-α−factor, into the vesicle fraction (data not shown), whereas it only partially inhibited incorporation of the GPI-APs Gas1p, Venus-tagged Ccw14p and Cwp2p into ER-derived vesicles (Figure 3A,B). Furthermore, when we used cytosol and semi-intact cells derived from cells with a temperature-sensitive allele of sar1E112K (34), incorporation of Gap1p was tightly dependent on addition of recombinant wild-type Sar1p, whereas Gas1p

incorporation into vesicles was quite efficient even without the addition of Sar1p (Figure 3C,D). These data contrast remarkably with those we obtained in experiments with mammalian cells (Figure 2D), in which GPI-APs were as tightly dependent on Sar1 as the other transmembrane cargo proteins for their incorporation into ER-derived vesicles. Together with the morphological observations of yeast cell mutants in different COPII components reported by Castillon et al. in vivo (18), our biochemical results support the idea that yeast GPI-APs seem to use a unique different mechanism for concentration upon ER exit compared to other secretory proteins for their concentration at ERES.

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Sar1p is required for ER-to-Golgi transport of GPI-APs in yeast in vivo

To examine whether Sar1p is required in vivo for ER-to-Golgi transport of GPI-APs in yeast, we performed pulse-chase experiments to follow the transport of the GPI-AP Gas1p and the non-GPI-anchored protein Carboxypeptidase Y (CPY) in sar1E112K mutant cells.

We used various temperatures in an attempt to reveal a differential effect of the sar1 mutation on the two proteins in vivo. Transport of both Gas1p and CPY from the ER to the Golgi was slower insar1E112K mutant cells than in wild-type cells at all temperatures (Figure 4A–C). Because the transport kinetics of CPY differs from that of Gas1p in wild-type cells, we cannot conclude whether or not the two cargo proteins have different degrees of dependence on Sar1p. Nonetheless, these data demonstrate that Sar1p is required for ER-to-Golgi transport of Gas1p in vivo. We present several explanations for the apparent discrepancies between the requirement for Sar1pin vitro andin vivo later in the Discussion section.

Mammalian GPI-APs and transmembrane proteins are concentrated in the same ERES

The different requirements of yeast and mammalian GPI-APs for ongoing sphingolipid synthesis (Figure 1) and Sar1 protein for their incorporation into ER-derived vesicles (Figures 2 and 3) suggest that yeast and mammalian cells use different mechanisms to export GPI-APs from the ER.

To investigate this further, we tested whether mammalian cells segregate their GPI-APs from other transmembrane secretory proteins upon ER exit as yeast does (18). To do so, we used a morphological analysis to visualize various cargo proteins’ localization in the ERESin vivo.

Incubation at 10^◦C has been used to visualize cargo protein accumulation in the ERES in mammalian cells (35). We used this strategy to examine whether transmembrane secretory proteins and GPI-APs accumulate in different ERES prior to their incorporation into COPII vesicles in CHO-K1 cells. At steady state, a single-spanning transmembrane protein, KitL-EGFP, localized mainly at the plasma membrane and in the perinuclear region and did not colocalize with the ERES marker Sec13 (Figure S2A). After 2 h incubation at 10^◦C, KitL-EGFP accumulated in intracellular punctate structures that colocalized almost perfectly with Sec13 (Figure 5A). As a control, we used a mutant KitL-EGFP that lacks the terminal valine residue in the cytoplasmic tail, KitL-EGFP(delV); cell surface expression of KitL-EGFP(delV) is reduced and the protein accumulates in the ER (36).

At steady state, KitL-EGFP(delV) exhibited a typical reticular ER pattern (Figure S2B). Even after temperature shift to 10^◦C, as predicted (36), KitL-EGFP(delV) did not accumulate in the ERES (Figure S2C), demonstrating that wild-type KitL-EGFP accumulates in ERES dependant on the terminal valine signal.

At steady state, a GPI-AP, Venus-tagged CD59 (Venus-CD59), localized mainly at the plasma membrane and in

the perinuclear region and did not colocalize with Sec13 (Figure S3A). After temperature shift to 10^◦C, some dot-like structures appeared that partially colocalized with Sec13 (data not shown). In most cells, however, because of the high amount of cell surface Venus-CD59, we could not observe significant ERES accumulation even after blocking transport at 10^◦C. We therefore removed the cell surface Venus-CD59 with a phosphatidylinositol-specific phospholipase C (PI-PLC), drastically decreasing the total Venus-CD59 signal. At steady state, we observed strong perinuclear and faint reticular localization without affecting ERES morphology (Figure S3B). Under this condition, and after temperature shift to 10^◦C for 1 h, Venus-CD59 accumulated in punctate structures, which significantly colocalized with Sec13 (Figure 5B). In a mutant CHO-K1 cell line defective for GPI biosynthesis (37), the signal from Venus-CD59 was much weaker and typical of a reticular ER pattern (Figure S4A). Even after temperature shift to

Dans le document The intracellular transport of glycosylphosphatidylinositol-anchored proteins in mammalian Chinese Hamster Ovary cells (Page 31-48)