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

Abstract

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 3 H-cholesteryl linoleate (CL) labeled-LDL, newly liberated 3 H-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 3 H-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 ≥50% 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.

Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

“…ORP9L depletion did not affect ER cholesterol homeostatic responses, potentially indicating no change in ER cholesterol content, but significantly increased cholesterol deposition in the endosomal/lysosomal compartment, as measure by filipin binding (Figure 8). The TGN is an important delivery site for endocytosed cholesterol (Urano et al, 2008) that, in the absence of ORP9L, appeared to backup into the endosomes and lysosomes. This suggests that ORP9L maintains cholesterol flux from the endosomal pathway to the transGolgi/TGN by transferring cholesterol to the ER or other organelles.…”

Role of cholesterol in SNARE-mediated trafficking on intracellular membranes

“…The majority of late-endosome-derived cholesterol is then delivered to the plasma membrane, while some of it is transported to the ER to enable feedback control or undergoes esterification for storage in lipid droplets in the form of cholesteryl esters. The routes and mechanisms by which late endosome-originating cholesterol reaches the plasma membrane are not clear, but as shown by our laboratory (Cubells et al, 2007;Reverter et al, 2014) and others (Kanerva et al, 2013;Urano et al, 2008), might involve transport through the ER, the transGolgi network (TGN) and recycling endosomes (Fig. 1A).…”

“…Niemann Pick C1 protein (NPC1) and Niemann Pick type C2 protein homolog (NPC2) bind to late-endosome-associated cholesterol and facilitate its export from late endosomes (Ikonen, 2006;Vance and Karten, 2014). Mutations in the genes encoding NPC1 or NPC2 inhibit the egress of cholesterol from late endosomes (Klein et al, 2006) and reduce its delivery to the Golgi, plasma membrane and recycling endosomes (Cubells et al, 2007;Kanerva et al, 2013;Reverter et al, 2014;Urano et al, 2008). Together, this triggers a dysfunction of membrane trafficking, which is associated with cardiovascular, neurological and lysosomal storage diseases (Cortes et al, 2013;De Matteis and Luini, 2011;Ikonen, 2006;Maxfield and Tabas, 2005).…”

“…1B,C). In addition, membrane contact sites between late endosome or lysosomes and other compartments, such as the ER, appear to enable cholesterol transfer (Alpy et al, 2013;Chang et al, 2006;Ikonen, 2008;Rowland et al, 2014;van der Kant and Neefjes, 2014).…”

Niemann-Pick Type C2 protein contributes to the transport of endosomal cholesterol to mitochondria without interacting with NPC1

“…Next, we asked whether endosome-to-mitochondria movement of cholesterol required additional, cytosolic carrier proteins and/ or passage through other subcellular membranes, such as ER or plasma membrane. To address these questions, we used CHO-F2 cells that were semi-permeabilized to remove cytosolic proteins following a protocol that was used previously to investigate endosome-to-trans Golgi cholesterol traffi cking ( 34 ). Treatment with 20 µg/ml digitonin permeabilized the plasma membrane of 90% of cells, as shown by the uptake of Trypan blue ( Fig.…”