A Novel Contact by a Novel Protein Complex Supports Cholesterol Transport to the Endoplasmic Reticulum

Abstract

Cholesterol is an essential component of membrane lipids and a starting material for hormone synthesis. After cholesterol is delivered to the cell as low-density lipoprotein, it is endocytosed and degraded in lysosomes to liberate free cholesterol. Free cholesterol is transported to the endoplasmic reticulum (ER) and esterified for further use. However, the mechanisms that transport cholesterol from lysosomes to the endoplasmic reticulum are poorly understood. We searched for binding proteins of a small GTP-binding protein, Rab11, and identified a novel protein, Rab11-binding protein containing LisH, coiled coil, and heat repeats (RELCH). RELCH also binds to oxysterol-binding protein (OSBP), an essential protein for nonvesicular cholesterol transport. The Rab11-RELCH-OSBP complex was found to tether to recycling endosomes and the trans-Golgi network, thereby mediating nonvesicular cholesterol transport between them. This pathway is distinct from the cholesterol transport pathway identified previously. In the absence of this complex, cholesterol accumulates in lysosomes in vitro and in vivo, suggesting the involvement of this complex in diseases associated with cholesterol transport.

Cholesterol is an essential molecule for living organisms. It serves as a major building block for the lipid bilayer of organelle membranes. Cholesterol is delivered to cells in our bodies as low-density lipoprotein (LDL) through the blood stream. LDL binds to the LDL receptor and is endocytosed and transported to lysosomes, where the esterified cholesterol within LDL is degraded by acid lipase to liberate free cholesterol molecules. Free cholesterol molecules are integrated into the lysosomal membrane by the concerted action of Niemann–Pick type C1 and 2 (NPC1 and 2) gene products. Integrated free cholesterol molecules are transported to the endoplasmic reticulum (ER) though various vesicular and nonvesicular pathways, as described later. Free cholesterol in the ER is transported to various destinations or is reesterified in the ER for storage in lipid droplets.

Cholesterol is transported from lysosomes to the ER through a number of pathways (Chu et al., 2015; Luo, Jiang, Yang, & Song, 2017; Pfisterer. Peranen, & Ikonen, 2016). However, these pathways are classified into two main groups (Figure 1). The first classification is the pathway that transmits cholesterol from lysosomes to the ER via other organelles. The second classification is the pathway thought to be mediated by direct contacts between lysosomes and the ER. Recently, many molecules have been found to be involved in the second pathway. For example, a rab-interacting lysosomal protein (Rab7/RILP) complex on the lysosomal membrane is considered to form membrane contact sites (MCSs) with the ER by binding vesicle-associated membrane protein-associated protein (VAP) and phosphatidylinositol 4-phosphate (PI4P) on the ER membrane and ORP1L protein to transfer cholesterol directly from lysosomes to the ER (Zhao & Ridgeway, 2017). It has also been proposed that NPC1 in the lysosome and ORP5 in the ER are important for cholesterol transport (Du et al., 2011).

Figure 1.

(a) Intracellular transport of cholesterol. The area in the red square indicates the cholesterol transport mediated by RELCH discovered by the authors. (b) Enlarged view of cholesterol transfer between the RE and the TGN. The area in the red square is enlarged to show cholesterol transfer in detail. RELCH tethers the RE and the TGN to transfer cholesterol from the former to the latter. (c) Interactions between RELCH and its binding proteins. cc = coiled-coil domain; LisH = Lis1homology domain; PH = pleckstrin homology domain; FFAT = FFAT (two phenylalanines in an acidic track) motif; ORD = ORP-related domain; LDL = low-density lipoprotein; TGN = trans-Golgi network; OSBP = oxysterol binding protein; RELCH = Rab11-binding protein containing LisH, coiled coil, and heat repeats; ER = endoplasmic reticulum; EE = early endosome; LE/LY = late endosome/lysosome.

For the first pathway, several organelles, such as the plasma membrane, peroxisomes, and the trans-Golgi network (TGN), have been proposed as intermediates between lysosomes and the ER. In the case of peroxisomes, lysosomal synaptotagmin VII and peroxisomal PI(4,5) P₂ are necessary to form the MCS between lysosomes and peroxisomes. In the case of the TGN, a SNARE complex composed of VAMP4, syntaxin 6, and syntaxin 16 or a Golgi-associated retrograde protein (GARP) complex is thought to be essential for the vesicular transport between lysosomes and the TGN. However, in this case, inhibiting the function of these molecules did not completely block cholesterol transport (Urano et al., 2008). This result suggests the presence of additional pathway(s) mediated by other molecules from lysosomes to the ER.

We have been studying the function of molecules involved in cell polarity by knocking out Rab and SNARE molecules in the mouse. Rab11a is localized in the recycling endosome (RE) and is known to be involved in vesicular transport. RE was initially named for its function in the recycling of endocytosed molecules. However, it has recently been considered to be involved in the biosynthetic pathway from the TGN as well. When we generated intestine-specific Rab11a knockout mice, they showed abnormality in the localization of apical proteins (Sobajima et al., 2014).

To elucidate the molecular mechanism of Rab11a for this phenotype, we searched for binding proteins for Rab11a and identified a protein, Rab11-binding protein containing LisH, coiled coil, and heat repeats (RELCH), whose function had been unknown so far (Sobajima et al., 2018). Thus, to understand the function of RELCH, we searched for binding proteins for RELCH and identified oxysterol-binding protein (OSBP) as a binding protein. According to the literature, OSBP is known to transfer cholesterol in a nonvesicular manner at the MCS between the ER and the Golgi (Mesmin et al., 2017). It was also reported to function at the endosome-ER contacts for PI4P transfer (Dong et al., 2016).

When we knocked down Rab11, RELCH, or OSBP in HeLa cells, the number of lysosomes significantly increased and cholesterol accumulated in these lysosomes. We confirmed biochemically that cholesterol increased in lysosomes and decreased in the TGN and ER. Using an in vitro cholesterol transfer assay between Rab11-positive RE-like vesicles and OSBP-positive TGN-like vesicles, we proved that cholesterol is transferred from RE-like vesicles to TGN-like vesicles by fluorescence-resonance energy transfer (FRET). We also observed an increased number of contacts between the beads loaded with Rab11 and the ones loaded with OSBP on the surface in the presence of RELCH. From these findings, the Rab11-RELCH-OSBP complex is likely to mediate direct cholesterol transfer from the RE to the TGN at the MCS. Previously, cholesterol transport from lysosomes to the ER was thought to be mediated by vesicular transport from NPC-positive endosomes/lysosomes to the Golgi (Figure 1(a)). Thus, we identified an additional novel nonvesicular pathway of cholesterol transfer from the RE to the TGN that is mediated by a novel complex, Rab11-RELCH-OSBP. However, implicating the presence of multiple pathways for intracellular cholesterol transport, RELCH knockout mice appeared healthy. In spite of their healthy appearance, they showed an increased number of lysosomes and increased cholesterol content in lysosomes in the liver or tail fibroblasts, suggesting that RELCH deficiency led to fatty liver or other human diseases associated with cholesterol transport deficits, such as NPC1, 2. Thus, close examination of RELCH knockout mice might be beneficial for the prevention, diagnosis, and treatment of these diseases in the future. We are currently analyzing the lipid and protein contents in the liver of knockout mice in detail.