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

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.

Order of lipid phases in model and plasma membranes

“…1B). This trend could also be confirmed for more complex samples such as yeast membranes (37). Moreover, GP s values were not discrete for the phase states but changed with lipid composition, decreasing in value from Lo phases to coexisting Lo-Ld phases and Ld phases (Fig.…”

“…It cannot be ruled out that the partial loss of membrane asymmetry dampens more pronounced differences present in native membranes (44). However, we propose that this small energetic gap in order is rather the result of the compositional complexity of the two phases in the plasma membrane preparations, as similar differences have also been reported for other domains in intact biomembranes such as the T cell synapse (45) and Golgi-derived transport vesicles (37). In addition to the interaction of cholesterol with saturated hydrocarbon chains that brings about Lo-Ld-type phase separation in model membranes, additional lateral associations come into play in cell membranes: (i) hydrogen bond networks mediated by the backbone chemistry of sphingolipids (1,(46)(47)(48), (ii) specific protein-lipid interactions (18,49,50), (iii) homo-and heterotypic protein interactions (51,52), and (iv) glycan-based interaction with proteins and other glycans (53,54).…”

Membrane raft association is a determinant of plasma membrane localization

“…membrane domain | endocytosis | trafficking | phase separation | microdomains R ecent advances in superresolution microscopy (1), lipid analysis (2,3), and plasma membrane (PM) isolation (4,5) have confirmed the coexistence of lipid-driven, fluid domains in biological membranes. The relatively ordered domains, known as "membrane rafts," have been proposed to be involved in protein sorting (6), viral/pathogen trafficking (3,7), and PM signaling in a variety of contexts (8).…”

“…Lipid-mediated domains have been implicated as a mechanism for protein sorting in the latter stages of the secretory pathway (trans-Golgi network to the PM) (2,6,(10)(11)(12), with analogous pathways mediating endosomal sorting/recycling (13,14). Raft lipids (i.e., sterols and sphingolipids) are significantly enriched at the PM (15)(16)(17), and recent observations confirm that these lipids also are enriched in sorting vesicles destined for the PM (2,11).…”

COPI Budding within the Golgi Stack

“…Much less is known, however, of how a living cell can maintain the identities of its various organelles with respect to their unique lipid composition, although lipidomes of a few membrane carriers have become available recently (Brü gger et al 2000;Takamori et al 2006;Klemm et al 2009). Lipidomic analysis of COPI vesicles revealed a depletion of cholesterol and sphingomyelin (SM), two lipids characteristic of the liquid-ordered (Lo) phase, when compared with the donor Golgi membrane (Brü gger et al 2000).…”