Transport of LDL-derived cholesterol from the NPC1 compartment to the ER involves the trans-Golgi network and the SNARE protein complex

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 105; no. 43; pp. 16513 - 16518
• Main Authors: Urano, Yasuomi, Watanabe, Hiroshi, Murphy, Stephanie R, Shibuya, Yohei, Geng, Yong, Peden, Andrew A, Chang, Catherine C.Y, Chang, Ta Yuan
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 28.10.2008; National Acad Sciences
• Subjects: Adenosine Triphosphate; Animals; Antibodies; Biochemistry; Biological Sciences; Cell Fractionation; Cell membranes; Cells; CHO Cells; Cholesterol; Cholesterol, LDL - metabolism; Cholesterols; Cricetinae; Cricetulus; Endoplasmic Reticulum - metabolism; Endosomes; Endosomes - metabolism; Hepatocytes; Lipids; Mammals; Membrane Proteins - analysis; Metabolism; Neuroglia; Protein Transport; Proteins; RNA, Small Interfering - pharmacology; Small interfering RNA; SNARE Proteins - genetics; SNARE Proteins - metabolism; Sterols; trans-Golgi Network - metabolism; Tritium
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0807450105

Mammalian cells acquire cholesterol mainly from LDL. LDL enter the endosomes, allowing cholesteryl esters to be hydrolyzed by acid lipase. The hydrolyzed cholesterol (LDL-CHOL) enters the Niemann-Pick type C1 (NPC1)-containing endosomal compartment en route to various destinations. Whether the Golgi is involved in LDL-CHOL transport downstream of the NPC1 compartment has not been demonstrated. Using subcellular fractionation and immunoadsorption to enrich for specific membrane fractions, here we show that, when parental Chinese hamster ovary (CHO) cells are briefly exposed to ³H-cholesteryl linoleate (CL) labeled-LDL, newly liberated ³H-LDL-CHOL appears in membranes rich in trans-Golgi network (TGN) long before it becomes available for re-esterification at the endoplasmic reticulum (ER) or for efflux at the plasma membrane. In mutant cells lacking NPC1, the appearance of newly liberated ³H-LDL-CHOL in the TGN-rich fractions is much reduced. We next report a reconstituted transport system that recapitulates the transport of LDL-CHOL to the TGN and to the ER. The transport system requires ATP and cytosolic factors and depends on functionality of NPC1. We demonstrate that knockdown by RNAi of 3 TGN-specific SNAREs (VAMP4, syntaxin 6, and syntaxin 16) reduces greater-than-or-equal50% of the LDL-CHOL transport in intact cells and in vitro. These results show that vesicular trafficking is involved in transporting a significant portion of LDL-CHOL from the NPC1-containing endosomal compartment to the TGN before its arrival at the ER.