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.

Intra-Golgi Protein Transport Depends on a Cholesterol Balance in the Lipid Membrane

“…Vesicular transport processes do not seem to play a major role in intracellular cholesterol transport (57)(58)(59). This is in agreement with the recent observation that cholesterol segregates from COPI-coated vesicles during budding from Golgi membranes (36). Current candidates for mediating intracellular cholesterol transport include caveolin (26), possibly in a complex with other proteins (60).…”

“…Microdomains have also been suggested to occur as a result of the cholesterol and sphingomyelin gradient along the secretory pathway to explain protein sorting at the Golgi complex (3). Recently, it was found that cholesterol and sphingomyelin are segregated from Golgi-derived COPI-coated vesicles, implicating a segregation process that might involve microdomains within the Golgi complex (36). By detergent extraction, Golgi-derived detergent-soluble complexes (GIC mi- 2, 7, and 8) or presence of 2 mM M␤CD (lanes 3 and 4) or 2 mM M␤CD⅐Chol (4:1) for 2 h at 30°C.…”

“…For quantitation, an internal standard was added to the samples, and after sulfatation of the cholesterol, cholesterol was measured with ESI-MS/MS by scanning in the negative mode for the parent ions of m/z ϭ 97 (HSO 4 Ϫ ) or 80 (SO 3 Ϫ ), depending on the internal standard ( [3,[4][5][6][7][8][9][10][11][12][13] C 2 ]cholesterol and 2,2,3,4,4,6-D 6 -cholesterol, respectively). Phosphatidylcholine, sphingomyelin, and glucosylceramide analysis by ESI-MS/MS was performed as described previously (36).…”

“…Cholesterol is a major lipid in CHO Golgi membranes (0.57 mol of cholesterol/mol of phospholipid) (36) and has been implicated in the formation of microdomains (13)(14)(15)(16). Golgi-derived microdomains can be isolated as detergent-resistant membranes, enriched in sphingolipids and cholesterol (30).…”

Molecular recognition of a single sphingolipid species by a protein’s transmembrane domain

“…Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane 1 Lipidomics of Golgi-derived COPI vesicles unravelled a partial segregation of cholesterol and most SM species except for one (N-stearoyl SM) from the vesicle fraction 5 . In order to understand the molecular mechanisms of SM 18 enrichment in vesicles, we investigated in vivo binding of lipids to p24 and p23, membrane proteins involved in COPI vesicle biogenesis ( Supplementary Fig.…”