Sphingomyelin-enriched Microdomains at the Golgi Complex

Abstract

Sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes from total cell extracts (total-DRM). It is generally believed that this total-DRM represents microdomains of the plasma membrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipids, containing 25% of total Golgi phospholipids including 67% of Golgi-derived sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to total-DRM, GICs contain only 10 major proteins, present in nearly stoichiometric amounts, including the α- and β-subunits of heterotrimeric G proteins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morphological data show a brefeldin A-sensitive and temperature-sensitive localization to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cells, GICs can be isolated from a fused Golgi-endoplasmic reticulum organelle. This indicates that GIC microdomains are not in a dynamic equilibrium with neighboring membrane proteins and lipids. After disruption of the microdomains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indicates that several of the identified GIC proteins localize to the same microdomains and that the microdomain scaffold is not required for protein interactions between these GIC proteins but instead might modulate their affinity.

Insights into the Role of Specific Lipids in the Formation and Delivery of Lipid Microdomains to the Plasma Membrane of Plant Cells

“…This article clearly demonstrates that DRMs can be isolated from the GA of plant cells, a result in line with the presence of lipid microdomains in the GA of animal cells (Hansen et al, 2000;Gkantiragas et al, 2001) and yeast (Bagnat et al, 2000). However, although the presence of lipid microdomains in the ER of animal cells (Sevlever et al, 1999) and yeast (Bagnat et al, 2000;Lee et al, 2002) has been reported, our attempts to isolate significant amounts of DRMs from the ER of leek cells were unsuccessful.…”

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

“…We considered the possibility that a change in cholesterol levels might affect the function of microdomains at the Golgi complex. Recently we obtained evidence for the existence of such microdomains at the early Golgi complex (30). We now show that changing the cholesterol content of membranes specifically affects the phase partitioning of heterotrimeric G proteins, suggesting a possible involvement of microdomains in the regulation of protein transport through the Golgi apparatus.…”

“…Golgi-derived microdomains can be isolated as detergent-resistant membranes, enriched in sphingolipids and cholesterol (30). To determine the amount of cholesterol that partitions in microdomains, Triton X-100 was added to solubilize Golgi membranes.…”

Enlargeosome, an Exocytic Vesicle Resistant to Nonionic Detergents, Undergoes Endocytosis via a Nonacidic Route

“…Extensive evidence in various organelles and processes (especially the TGN; endocytosis; constitutive exocytosis) has demonstrated the key role of rafts in membrane dynamics (fusions, fissions, trafficking, and interactions: for reviews, see Nichols, 2003;Helms and Zurzolo, 2004;Mayor and Rao, 2004). The abundance of rafts, as observed in enlargeosomes, is not common among the organelles competent for regulated exocytosis and could therefore play specific roles at various steps of the new vesicle life, including (in addition to exocytosis, which is discussed below): generation, presumably at specific domain(s) of the TGN (Gkantiragas et al, 2001;Maxfield, 2002;Gleeson et al, 2004); traffic and possible interactions, homologous and possibly also heterologous with other membrane systems. The recognition of the extensive detergent resistance, the first general property of the enlargeosome membrane so far identified, could serve in the study of these processes, for example, by providing criteria for the isolation and characterization of the vesicles together with important cues and tools for the interpretation of future results.…”