Evidence for segregation of sphingomyelin and cholesterol during formation of COPI-coated vesicles

Abstract

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.

Figure 1

Morphology of CHO and rat liver Golgi membranes by EM. CHO and rat liver Golgi-enriched membranes were isolated and processed for EM, as described in the Materials and Methods. (A) Conventional Epon sections of a CHO Golgi fraction and (B) a rat liver Golgi fraction are shown. Bars: (A) 200 nm, (B) 210 nm.

Figure 5

Comparison of fatty acid composition of SM and PC species in Golgi membranes and Golgi-derived COPI-coated vesicles. Lipid quantification was performed as described in Materials and Methods. The results obtained from the CHO system are depicted at the top; those of the rat liver system are shown at the bottom. The relative amounts of PC (left) and SM (right) species derived from Golgi donor membranes (black bars) were compared with those of COPI-coated vesicles (white bars). Data are expressed as percentage of each lipid molecular species (identical total number of C atoms in both fatty acids as well as identical number of total double bonds, indicated as Σ C atoms:Σ double bonds) of PC or SM to total amount of PC or SM, respectively.

Figure 2

Characterization of in vitro–generated COPI-coated vesicles. Transport vesicles were generated from either CHO (left) or rat liver (right) Golgi membranes in the presence of bovine brain cytosol, ATP-regenerating system, and GTPγS. COPI vesicles were purified on a continuous sucrose gradient. The gradients were fractionated from the bottom into 18 fractions. Aliquots of fractions 3–16 of the isopycnic gradients were chloroform/methanol-precipitated, and analyzed by running on a 13% SDS-PAGE and Western blotting, using antibodies against β-COP and p23 (top). COPI vesicles are recovered in fractions 6–8, corresponding to an average sucrose concentration of 41% (wt/wt). Below are electron micrographs of COPI-coated vesicles present in fractions 6–8. Bars, 90 nm.

Figure 3

(A) Mass spectrometric analysis of donor Golgi membranes and Golgi-derived COPI-coated vesicles. Golgi membranes were isolated from either CHO cells or rat liver and incubated with bovine brain cytosol, ATP-regenerating system, and GTPγS. Vesicles were purified on an isopycnic sucrose gradient. As described in Materials and Methods, lipids from donor Golgi membranes and COPI vesicles were extracted in the presence of internal PC and SM standards. PC and SM quantification was performed in PREC 184 mode. The top panels show PREC 184 spectra of CHO (left) and rat liver (right) donor Golgi membranes. The bottom panels show the corresponding PREC 184 spectra of COPI vesicles generated from CHO (left) and rat liver (right) Golgi membranes. Each spectrum shown was averaged from 100 separate scans, each of 4-s duration. The molecular masses in daltons, the corresponding numbers of total carbon (C) atoms in both fatty acids, and the numbers of double bonds (Σ C atoms:Σ of double bonds) are given for the major peaks. pl, plasmalogen. (B) Quantification of PC, SM, and cholesterol in donor Golgi and COPI vesicle fractions of CHO or rat liver membranes. Lipid quantification was performed as described in Materials and Methods. The results of quantification of PC (black bars), SM (white bars), and cholesterol (gray bars) in CHO (top) and in rat liver (bottom) membranes are shown. The amounts of PC, SM, and cholesterol in donor Golgi membranes are set to 100%. Results are shown as mean ± SD. Reference to absolute amounts of these lipids present in COPI vesicles and their parental membrane fractions is given in Table .

Figure 3

(A) Mass spectrometric analysis of donor Golgi membranes and Golgi-derived COPI-coated vesicles. Golgi membranes were isolated from either CHO cells or rat liver and incubated with bovine brain cytosol, ATP-regenerating system, and GTPγS. Vesicles were purified on an isopycnic sucrose gradient. As described in Materials and Methods, lipids from donor Golgi membranes and COPI vesicles were extracted in the presence of internal PC and SM standards. PC and SM quantification was performed in PREC 184 mode. The top panels show PREC 184 spectra of CHO (left) and rat liver (right) donor Golgi membranes. The bottom panels show the corresponding PREC 184 spectra of COPI vesicles generated from CHO (left) and rat liver (right) Golgi membranes. Each spectrum shown was averaged from 100 separate scans, each of 4-s duration. The molecular masses in daltons, the corresponding numbers of total carbon (C) atoms in both fatty acids, and the numbers of double bonds (Σ C atoms:Σ of double bonds) are given for the major peaks. pl, plasmalogen. (B) Quantification of PC, SM, and cholesterol in donor Golgi and COPI vesicle fractions of CHO or rat liver membranes. Lipid quantification was performed as described in Materials and Methods. The results of quantification of PC (black bars), SM (white bars), and cholesterol (gray bars) in CHO (top) and in rat liver (bottom) membranes are shown. The amounts of PC, SM, and cholesterol in donor Golgi membranes are set to 100%. Results are shown as mean ± SD. Reference to absolute amounts of these lipids present in COPI vesicles and their parental membrane fractions is given in Table .

Figure 4

Generation of COPI vesicles from CHO Golgi membranes in the presence of either GTPγS or GTP. Preparations of COPI vesicles were performed as described in the Materials and Methods. Lipid quantification of the various membrane fractions was done after addition of internal PC and SM standards before extraction. Quantification was performed as described. Illustrated are PREC 184 spectra of (A) CHO Golgi donor membranes, (B) COPI vesicles from GTPγS incubations, and (C) COPI vesicles from GTP incubations. pl, plasmalogen.