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. 2009 May 18;185(4):601-12.
doi: 10.1083/jcb.200901145. Epub 2009 May 11.

Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

Robin W Klemm  1 , Christer S Ejsing, Michal A Surma, Hermann-Josef Kaiser, Mathias J Gerl, Julio L Sampaio, Quentin de Robillard, Charles Ferguson, Tomasz J Proszynski, Andrej Shevchenko, Kai Simons
Affiliations

Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

Robin W Klemm et al. J Cell Biol. .

Abstract

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.

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Figures

Figure 1.
Figure 1.
Intracellular accumulation of the immunoisolation baits at the restrictive temperature 37°C in sec6-4 cells. (A) FusMidGFPLTLM9, the raft-carrier cargo immunoisolation bait, and InvRFP expressed for 45 min. At the permissive temperature 24°C, FusMidGFPLTLM9 reached the PM, whereas InvRFP was secreted and therefore not visible (for InvRFP secretion, see also Fig. S3 B). At the restrictive temperature 37°C, both proteins accumulated intracellularly. (B) Gap1GFPLTLM9, the TGN/E immunoisolation bait, and InvRFP expressed for 45 min. At the permissive temperature 24°C, InvRFP was secreted and, thus, not visible (Fig. S3 B), and Gap1GFPLTLM9 localized to intracellular compartments. At the restrictive temperature 37°C, both proteins accumulated intracellularly. DIC, differential interference contrast. Bars, 2 µm.
Figure 2.
Figure 2.
Accumulation of secretory vesicles in sec6-4 cells at the restrictive temperature 37°C. (A) Transmission electron micrograph of a budding sec6-4 cell cultured at the permissive temperature 24°C. (B) Transmission electron micrograph of a budding sec6-4 cell cultured for 45 min at the restrictive temperature 37°C. White arrowheads indicate accumulated secretory vesicles with a diameter of ∼100 nm. (C) 3D reconstruction of a budding cell with accumulated secretory vesicles (red) in a tomogram recorded from a 200-nm thick section prepared from the same culture of cells as shown in B; the PM is reconstructed in blue, the vacuole is orange, and other organelles such as Golgi cisternae or endosomal structures are in yellow. V, vacuole. Bars, 1 µm.
Figure 3.
Figure 3.
Immunoisolation of FusMidp-vesicles and the TGN/E. (A and B) Several organelle markers were monitored throughout the isolation of FusMidp-vesicles (A) and the TGN/E (B), with GFP detecting the respective baits. InvRFP was used as a specific marker for HDSVs. Gas1p is the major GPI-anchored protein in yeast and is an LDSV marker. Pep12p is the late endosome t-SNARE, Dpm1p is an ER protein, and Tlg1p is a late Golgi marker. Input, sucrose gradient fraction 7 (A) and 4 (B); FT, flow through; Bind, material bound to the immunoadsorbent; Wash, supernatant of the last wash; Remain, material not released from the cellulose fibers after TEV cleavage; ELUATE, eluted material released from the immunoadsorbent after TEV cleavage.
Figure 4.
Figure 4.
Transmission electron micrographs of FusMidp-vesicles in different magnifications. (A) Overview of FusMidp-vesicles in an electron micrograph shows the morphological homogeneity of the FusMidp-vesicle population with a diameter of ∼100 nm. These isolated vesicles have the same morphological characteristics as the structures that accumulate in sec6-4 cells at 37°C; see Fig. 2 (B and C). (B) Transmission electron micrograph of a single FusMidp-vesicle. The 100-nm vesicular structure is formed by a membrane whose electron density pattern shows the integrity of the lipid bilayer. (C) A group of FusMidp-vesicles. The lipid bilayer is visible as in B. Bars: (A) 1,000 nm; (B and C) 100 nm.
Figure 5.
Figure 5.
Molecular lipid species composition of FusMidp-vesicles and the donor TGN/E. Quantitative lipidomic analysis of FusMidp-vesicles and the TGN/E allowed absolute quantification of 83 molecular lipid species. Lipid composition is shown in mole percentage to demonstrate the stoichiometric relationship between the lipid species (n = 4 independent experiments; mean estimate ± SD). Chemical structures of membrane lipids are illustrated on the right. Lipid species are annotated by their molecular composition (see Materials and methods). Cer, ceramide; PG, phosphatidylglycerol.
Figure 6.
Figure 6.
Lipid class composition of FusMidp-vesicles, the TGN/E, and total cell extract. The mole percentage of lipid class was calculated as the sum of the mole percentage of lipid species of the respective lipid class. (n = 4 independent experiments; mean estimate ± SD). Cer, ceramide; PG, phosphatidylglycerol.
Figure 7.
Figure 7.
C-Laurdan spectrophotometry reveals higher membrane order for FusMidp-vesicle as compared with the TGN/E. Immunoisolated membranes were stained with C-Laurdan and subsequently analyzed by fluorescence spectrophotometry. The GP is a relative measure for membrane order (see Materials and methods). FusMidp-vesicles (red bar) exhibited a higher GP value than the TGN/E (blue bar; n = 3 independent experiments; mean estimate ± SD).
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