Abstract
Transport of proteins between intracellular membrane compartments is mediated by a protein machinery that regulates the budding and fusion processes of individual transport steps. Although the core proteins of both processes are defined at great detail, much less is known about the involvement of lipids. Here we report that changing the cellular balance of cholesterol resulted in changes of the morphology of the Golgi apparatus, accompanied by an inhibition of protein transport. By using a well characterized cell-free intra-Golgi transport assay, these observations were further investigated, and it was found that the transport reaction is sensitive to small changes in the cholesterol content of Golgi membranes. Addition as well as removal of cholesterol (10 +/- 6%) to Golgi membranes by use of methyl-beta-cyclodextrin specifically inhibited the intra-Golgi transport assay. Transport inhibition occurred at the fusion step. Modulation of the cholesterol content changed the lipid raft partitioning of phosphatidylcholine and heterotrimeric G proteins, but not of other (non) lipid raft proteins and lipids. We suggest that the cholesterol balance in Golgi membranes plays an essential role in intra-Golgi protein transport and needs to be carefully regulated to maintain the structural and functional organization of the Golgi apparatus.
Highlights
Cholesterol is an essential lipid constituent in the membranes of mammalian cells and has a profound effect on the physical properties of these membranes
We suggest that the cholesterol balance in Golgi membranes plays an essential role in intra-Golgi protein transport and needs to be carefully regulated to maintain the structural and functional organization of the Golgi apparatus
We considered the possibility that a change in cholesterol levels might affect the function of microdomains at the Golgi complex
Summary
Cholesterol is an essential lipid constituent in the membranes of mammalian cells and has a profound effect on the physical properties of these membranes. Terification and subsequent release of cholesterol to the outside circulation or storage in lipid droplets [4] Regulation of these processes keeps total free cholesterol levels in cells precisely controlled. Cholesterol is involved in the stabilization and function of lipid-enriched microdomains (lipid rafts) within a membrane. The scaffold of these microdomains is built by sphingolipids and cholesterol [13,14,15,16]. Cholesterol tightly interacts with sphingolipids that contain predominantly long-chain saturated fatty acids This results in the segregation of these lipids from other membrane lipids into microdomains. The most important role of microdomains may be their function in
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