Interplay between Asters/GRAMD1s and phosphatidylserine in intermembrane transport of LDL cholesterol.

Low-density lipoprotein (LDL) delivers cholesterol to mammalian cells through receptor-mediated endocytosis. The LDL cholesterol is liberated in lysosomes and transported to the plasma membrane (PM) and from there to the endoplasmic reticulum (ER). Excess ER cholesterol is esterified with a fatty acid for storage as cholesteryl esters. Recently, we showed that PM-to-ER transport of LDL cholesterol requires phosphatidylserine (PS). Others showed that PM-to-ER transport of cholesterol derived from other sources requires Asters (also called GRAMD1s), a family of three ER proteins that bridge between the ER and PM by binding to PS. Here, we use a cholesterol esterification assay and other measures of ER cholesterol delivery to demonstrate that Asters participate in PM-to-ER transport of LDL cholesterol in Chinese hamster ovary cells. Knockout of the gene encoding PTDSS1, the major PS-synthesizing enzyme, lowered LDL-stimulated cholesterol esterification by 85%, whereas knockout of all three Aster genes lowered esterification by 65%. The reduction was even greater (94%) when the genes encoding PTDSS1 and the three Asters were knocked out simultaneously. We conclude that Asters participate in LDL cholesterol delivery from PM to ER, and their action depends in large part, but not exclusively, on PS. The data also indicate that PS participates in another delivery pathway, so far undefined, that is independent of Asters.

Last step in the path of LDL cholesterol from lysosome to plasma membrane to ER is governed by phosphatidylserine.

Animal cells acquire cholesterol from receptor-mediated uptake of low-density lipoprotein (LDL), which releases cholesterol in lysosomes. The cholesterol moves to the endoplasmic reticulum (ER), where it inhibits production of LDL receptors, completing a feedback loop. Here we performed a CRISPR-Cas9 screen in human SV589 cells for genes required for LDL-derived cholesterol to reach the ER. We identified the gene encoding PTDSS1, an enzyme that synthesizes phosphatidylserine (PS), a phospholipid constituent of the inner layer of the plasma membrane (PM). In PTDSS1-deficient cells where PS is low, LDL cholesterol leaves lysosomes but fails to reach the ER, instead accumulating in the PM. The addition of PS restores cholesterol transport to the ER. We conclude that LDL cholesterol normally moves from lysosomes to the PM. When the PM cholesterol exceeds a threshold, excess cholesterol moves to the ER in a process requiring PS. In the ER, excess cholesterol acts to reduce cholesterol uptake, preventing toxic cholesterol accumulation. These studies reveal that one lipid-PS-controls the movement of another lipid-cholesterol-between cell membranes. We relate these findings to recent evidence indicating that PM-to-ER cholesterol transport is mediated by GRAMD1/Aster proteins that bind PS and cholesterol.

Lysosomal cholesterol export reconstituted from fragments of Niemann-Pick C1.

Niemann-Pick C1 (NPC1) is a polytopic membrane protein with 13 transmembrane helices that exports LDL-derived cholesterol from lysosomes by carrying it through the 80 Å glycocalyx and the 40 Å lipid bilayer. Transport begins when cholesterol binds to the N-terminal domain (NTD) of NPC1, which projects to the surface of the glycocalyx. Here, we reconstitute cholesterol transport by expressing the NTD as a fragment separate from the remaining portion of NPC1. When co-expressed, the two NPC1 fragments reconstitute cholesterol transport, indicating that the NTD has the flexibility to interact with the remaining parts of NPC1 even when not covalently linked. We also show that cholesterol can be transferred from the NTD of one full-length NPC1 to another NPC1 molecule that lacks the NTD. These data support the hypothesis that cholesterol is transported through interactions between two or more NPC1 molecules.

3.3 Å structure of Niemann-Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport.

Niemann-Pick C1 (NPC1) and NPC2 proteins are indispensable for the export of LDL-derived cholesterol from late endosomes. Mutations in these proteins result in Niemann-Pick type C disease, a lysosomal storage disease. Despite recent reports of the NPC1 structure depicting its overall architecture, the function of its C-terminal luminal domain (CTD) remains poorly understood even though 45% of NPC disease-causing mutations are in this domain. Here, we report a crystal structure at 3.3 Å resolution of NPC1* (residues 314-1,278), which-in contrast to previous lower resolution structures-features the entire CTD well resolved. Notably, all eight cysteines of the CTD form four disulfide bonds, one of which (C909-C914) enforces a specific loop that in turn mediates an interaction with a loop of the N-terminal domain (NTD). Importantly, this loop and its interaction with the NTD were not observed in any previous structures due to the lower resolution. Our mutagenesis experiments highlight the physiological relevance of the CTD-NTD interaction, which might function to keep the NTD in the proper orientation for receiving cholesterol from NPC2. Additionally, this structure allows us to more precisely map all of the disease-causing mutations, allowing future molecular insights into the pathogenesis of NPC disease.

Triazoles inhibit cholesterol export from lysosomes by binding to NPC1.

Niemann-Pick C1 (NPC1), a membrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly inhibited by itraconazole, a triazole that is used as an antifungal drug [Xu et al. (2010) Proc Natl Acad Sci USA 107:4764-4769]. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also cross-linked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterol-sensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.

Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection.

Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry.

Structure and mechanism of the uracil transporter UraA.

The nucleobase/ascorbate transporter (NAT) proteins, also known as nucleobase/cation symporter 2 (NCS2) proteins, are responsible for the uptake of nucleobases in all kingdoms of life and for the transport of vitamin C in mammals. Despite functional characterization of the NAT family members in bacteria, fungi and mammals, detailed structural information remains unavailable. Here we report the crystal structure of a representative NAT protein, the Escherichia coli uracil/H(+) symporter UraA, in complex with uracil at a resolution of 2.8 Å. UraA has a novel structural fold, with 14 transmembrane segments (TMs) divided into two inverted repeats. A pair of antiparallel ß-strands is located between TM3 and TM10 and has an important role in structural organization and substrate recognition. The structure is spatially arranged into a core domain and a gate domain. Uracil, located at the interface between the two domains, is coordinated mainly by residues from the core domain. Structural analysis suggests that alternating access of the substrate may be achieved through conformational changes of the gate domain.

Structure of a fucose transporter in an outward-open conformation.

The major facilitator superfamily (MFS) transporters are an ancient and widespread family of secondary active transporters. In Escherichia coli, the uptake of l-fucose, a source of carbon for microorganisms, is mediated by an MFS proton symporter, FucP. Despite intensive study of the MFS transporters, atomic structure information is only available on three proteins and the outward-open conformation has yet to be captured. Here we report the crystal structure of FucP at 3.1 Å resolution, which shows that it contains an outward-open, amphipathic cavity. The similarly folded amino and carboxyl domains of FucP have contrasting surface features along the transport path, with negative electrostatic potential on the N domain and hydrophobic surface on the C domain. FucP only contains two acidic residues along the transport path, Asp 46 and Glu 135, which can undergo cycles of protonation and deprotonation. Their essential role in active transport is supported by both in vivo and in vitro experiments. Structure-based biochemical analyses provide insights into energy coupling, substrate recognition and the transport mechanism of FucP.

Mechanism of substrate recognition and transport by an amino acid antiporter.

In extremely acidic environments, enteric bacteria such as Escherichia coli rely on the amino acid antiporter AdiC to expel protons by exchanging intracellular agmatine (Agm(2+)) for extracellular arginine (Arg(+)). AdiC is a representative member of the amino acid-polyamine-organocation (APC) superfamily of membrane transporters. The structure of substrate-free AdiC revealed a homodimeric assembly, with each protomer containing 12 transmembrane segments and existing in an outward-open conformation. The overall folding of AdiC is similar to that of the Na(+)-coupled symporters. Despite these advances, it remains unclear how the substrate (arginine or agmatine) is recognized and transported by AdiC. Here we report the crystal structure of an E. coli AdiC variant bound to Arg at 3.0 A resolution. The positively charged Arg is enclosed in an acidic binding chamber, with the head groups of Arg hydrogen-bonded to main chain atoms of AdiC and the aliphatic portion of Arg stacked by hydrophobic side chains of highly conserved residues. Arg binding induces pronounced structural rearrangement in transmembrane helix 6 (TM6) and, to a lesser extent, TM2 and TM10, resulting in an occluded conformation. Structural analysis identified three potential gates, involving four aromatic residues and Glu 208, which may work in concert to differentially regulate the upload and release of Arg and Agm.

Structure and mechanism of an amino acid antiporter.

Virulent enteric pathogens such as Escherichia coli strain O157:H7 rely on acid-resistance (AR) systems to survive the acidic environment in the stomach. A major component of AR is an arginine-dependent arginine:agmatine antiporter that expels intracellular protons. Here, we report the crystal structure of AdiC, the arginine:agmatine antiporter from E. coli O157:H7 and a member of the amino acid/polyamine/organocation (APC) superfamily of transporters at 3.6 A resolution. The overall fold is similar to that of several Na+-coupled symporters. AdiC contains 12 transmembrane segments, forms a homodimer, and exists in an outward-facing, open conformation in the crystals. A conserved, acidic pocket opens to the periplasm. Structural and biochemical analysis reveals the essential ligand-binding residues, defines the transport route, and suggests a conserved mechanism for the antiporter activity.