Movement of accessible plasma membrane cholesterol by the GRAMD1 lipid transfer protein complex

Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM cholesterol forms complexes with other PM lipids, making it inaccessible for intracellular transport. Transition of PM cholesterol between accessible and inaccessible pools maintains cellular homeostasis, but how cells monitor the accessibility of PM cholesterol remains unclear. We show that endoplasmic reticulum (ER)-anchored lipid transfer proteins, the GRAMD1s, sense and transport accessible PM cholesterol to the ER. GRAMD1s bind to one another and populate ER-PM contacts by sensing a transient expansion of the accessible pool of PM cholesterol via their GRAM domains. They then facilitate the transport of this cholesterol via their StART-like domains. Cells that lack all three GRAMD1s exhibit striking expansion of the accessible pool of PM cholesterol as a result of less efficient PM to ER transport of accessible cholesterol. Thus, GRAMD1s facilitate the movement of accessible PM cholesterol to the ER in order to counteract an acute increase of PM cholesterol, thereby activating non-vesicular cholesterol transport.


Introduction
Sterol is one of the major membrane lipids in eukaryotes. In metazoans, cholesterol represents~20% of total cellular lipids and is therefore essential for the structural integrity of cellular membranes and for cell physiology (van Meer et al., 2008;Vance, 2015). Sterol is distributed among cellular membranes primarily via non-vesicular transport, a process that is independent of membrane traffic (Baumann et al., 2005;Hao et al., 2002;Heino et al., 2000;Ikonen, 2008;Urbani and Simoni, 1990). Levels of sterol vary considerably between different cellular membranes. Between 60% and 80% of total cellular cholesterol is concentrated in the plasma membrane (PM), where it represents up to~45% of total lipids in this bilayer (de Duve, 1971;Lange et al., 1989;Ray et al., 1969). Cellular cholesterol levels are maintained by regulated delivery and production, primarily through receptor-mediated endocytosis of low-density lipoproteins (LDLs) (Goldstein and Brown, 2015) and de novo synthesis in the endoplasmic reticulum (ER) that is controlled by the activation of SREBP transcription factors (Brown et al., 2018;Goldstein and Brown, 1990). Cholesterol is also supplied to cells via high-density lipoproteins (HDL) through the reverse cholesterol flux pathway (Acton et al., 1996;Phillips, 2014).
Cholesterol within the bilayer membranes exists in two distinct chemical states: one being free and 'accessible' (also known as 'unsequestered' or 'chemically active'), and the other being 'inaccessible' (also known as 'sequestered' or 'chemically inactive') owing in part to the formation of complexes with other membrane lipids, including sphingomyelin and phospholipids (Chakrabarti et al., 2017;Das et al., 2014;Gay et al., 2015;Lange et al., 2013;Lange et al., 2004;McConnell and Radhakrishnan, 2003;Ohvo-Rekilä et al., 2002;Radhakrishnan and McConnell, 2000;Sokolov and Radhakrishnan, 2010). Most cholesterol in the PM is sequestered, but a small fraction of PM cholesterol (~15% of PM lipids) remains accessible for extraction and transport (Das et al., 2014). Although the majority of cellular cholesterol resides in the PM, the biosynthesis of cholesterol occurs exclusively in the ER. Thus, the ER must communicate with the PM to monitor levels of PM cholesterol and to adjust cholesterol biosynthesis in order to maintain lipid homeostasis. To achieve this, cells sense transient increases in the accessible pool of PM cholesterol and rapidly transport the newly expanded pool of accessible PM cholesterol to the ER. This suppresses cholesterol biosynthesis by inhibiting SREBP-2, a master regulator of de novo cholesterol synthesis, thereby avoiding cholesterol overaccumulation while maintaining PM cholesterol levels (Das et al., 2014;Infante and Radhakrishnan, 2017;Lange and Steck, 1997;Lange et al., 2014;Scheek et al., 1997;Slotte and Bierman, 1988). Artificially trapping the accessible pool of cholesterol in the PM results in dysregulated activation of SREBP-2 (Infante and Radhakrishnan, 2017;Johnson et al., 2019). Despite its critical importance, the intracellular transport machinery that senses the accessibility of PM cholesterol is unknown. This machinery is likely to respond to a sharp change in the accessibility of cholesterol on the cytoplasmic leaflet of the PM and to facilitate transport of accessible cholesterol from the PM to the ER, thereby helping the ER to communicate with the PM. Such a homeostatic system would also allow cells to monitor PM cholesterol accessibility in order to help to maintain cellular cholesterol homeostasis.
The ER extends throughout the cytoplasm, forming physical contacts with virtually all other cellular organelles and the PM (Phillips and Voeltz, 2016;Wu et al., 2018). Growing evidence indicates eLife digest The human body contains trillions of cells. At the outer edge of each cell is the plasma membrane, which protects the cell from the external environment. This membrane is mostly made of fatty molecules known as lipids and about half of these lipids are specifically cholesterol. Human cells can either take up cholesterol that were obtained via the diet or produce it within a compartment of the cell called the endoplasmic reticulum.
Cells need to monitor the cholesterol levels in both the endoplasmic reticulum and the plasma membrane in order to regulate the uptake or production of this lipid. For example, if there is too much of cholesterol in the plasma membrane, then the cell transports some to the endoplasmic reticulum to tell it to shut down cholesterol production. However, how these different areas of the cell communicate with each other, and transport cholesterol, has remained unclear.
Naito et al. set out to look for key regulators of cholesterol transport and identified a group of endoplasmic reticulum proteins called GRAMD1 proteins. Cholesterol in the plasma membrane is either accessible or inaccessible, meaning it either can or cannot be moved back into the cell. The GRAMD1 proteins sense accessible cholesterol, and experiments with human cells grown in the laboratory showed that, specifically, the GRAMD1 proteins work together in a complex to sense accessible cholesterol at or near the plasma membrane. One particular part of the protein senses when the amount of accessible cholesterol reaches a certain level at the plasma membrane; when this threshold is reached, the complex flips a switch to start the transport of cholesterol to the endoplasmic reticulum and tell it to shut down cholesterol production.
This coupling of sensing and transporting lipids by one protein complex also helps maintain the right ratio of accessible and inaccessible cholesterol in the plasma membrane to prevent cells from activating unwanted cell-signaling events. Getting rid of the GRAMD1 proteins in cells, or removing sensing part of these proteins, leads to inefficient transport of cholesterol. A better understanding of how GRAMD1 proteins sense the accessibility of cholesterol could potentially help identify new approaches to control cholesterol transport inside cells. This may in turn eventually lead to new treatments that counteract the defects in cholesterol metabolism seen in some forms of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. that these membrane contact sites play critical roles in cellular physiology, including lipid exchange and delivery via non-vesicular lipid transport that is facilitated by lipid transfer proteins (LTPs) (Antonny et al., 2018;Drin, 2014;Elbaz and Schuldiner, 2011;Holthuis and Menon, 2014;Jeyasimman and Saheki, 2019;Kumar et al., 2018;Lahiri et al., 2015;Lev, 2012;Luo et al., 2019;Nishimura and Stefan, 2019;Petrungaro and Kornmann, 2019;Saheki et al., 2016;Saheki and De Camilli, 2017a;Saheki and De Camilli, 2017b;Wong et al., 2018). Thus, LTPs may participate in intracellular cholesterol transport and may help to maintain PM cholesterol homeostasis by regulating non-vesicular cholesterol transport between the PM and the ER at ER-PM contact sites.
Decades of biochemical and genetic research into cholesterol metabolism has identified several key LTPs that bind to cholesterol and mediate its non-vesicular transport (Luo et al., 2019;Wong et al., 2018). These proteins include a family of 15 proteins that contain a StAR-related lipid transfer (StART) domain, which binds and transports a wide variety of lipids, including cholesterol, glycerolipids, and sphingolipids (Alpy and Tomasetto, 2014). Five members of this family, namely STARD1, STARD3, STARD4, STARD5, and STARD6, bind and transport cholesterol (Alpy et al., 2013;Iaea et al., 2017;Lin et al., 1995;Mesmin et al., 2011;Soccio et al., 2002;Stocco, 2001;Wilhelm et al., 2017), but they are not conserved in yeast. This lack of conservation suggests that there may be a more ancient family of sterol transfer proteins that control cholesterol homeostasis in all eukaryotes.
A bioinformatics search for proteins that possess StART-like domains identified a novel family of evolutionarily conserved proteins that includes six Lam/Ltc proteins in budding yeast (Gatta et al., 2015;Murley et al., 2015), and five GRAM domain-containing proteins (GRAMDs) in metazoans. These GRAMDs include the StART-like domain-containing GRAMD1s, also known as Asters (GRAMD1a/Aster-A, GRAMD1b/Aster-B, and GRAMD1c/Aster-C), and two highly related proteins that lack a StART-like domain (GRAMD2 and GRAMD3). Lam/Ltc proteins and GRAMDs all possess an N-terminal GRAM domain, which has structural similarity to the PH domain and thus may sense or bind lipids (Begley et al., 2003;Tong et al., 2018), and a C-terminal transmembrane domain, which anchors the proteins to the ER. Structural and biochemical studies of yeast and mammalian StARTlike domains have identified a hydrophobic cavity that can bind sterol Horenkamp et al., 2018;Jentsch et al., 2018;Sandhu et al., 2018;Tong et al., 2018). The StARTlike domains of GRAMD1s bind and transport sterols in vitro Sandhu et al., 2018). Recent studies have demonstrated that some GRAMDs, including GRAMD1a, GRAMD1b, and GRAMD2, localize to ER-PM contact sites (Besprozvannaya et al., 2018;Sandhu et al., 2018). GRAMD2 facilitates STIM1 recruitment to ER-PM contacts and potentially regulates Ca 2+ homeostasis (Besprozvannaya et al., 2018), whereas GRAMD1b facilitates the transport of HDL-derived cholesterol to the ER in the adrenal glands of mice . By contrast, yeast Lam/Ltc proteins sense cellular stress and potentially regulate cholesterol exchange between the ER and other membranes (Gatta et al., 2015;Murley et al., 2015;Murley et al., 2017). However, the role of these proteins in PM cholesterol or sterol homeostasis has been elusive. In this study, we provide evidence that GRAMD1s sense a transient expansion of the accessible pool of PM cholesterol and facilitate its transport to the ER at ER-PM contact sites, thereby contributing to PM cholesterol homeostasis.
We found that GRAMDs form homo-and heteromeric complexes via their transmembrane domains and predicted the existence of luminal amphipathic helices that interact with each other within these complexes. We also found that GRAMD1s rapidly move to ER-PM contacts upon acute hydrolysis of sphingomyelin in the PM. We characterized the mechanisms of this acute recruitment and found that the GRAM domain acts as a coincidence detector of unsequestered/accessible cholesterol and anionic lipids in the PM, including phosphatidylserine, allowing the GRAMD1s to sense a transient expansion of the accessible pool of PM cholesterol once it increases above a certain threshold. We generated HeLa cells that lacked GRAMD1a/1b/1c (i.e., all of the GRAMDs that contain a StART-like domain) and determined the effect of knocking out these proteins on cholesterol metabolism, using a combination of cholesterol-sensing probes for live cell imaging and lipidomics of membrane extracts. Upon treatment with sphingomyelinase, which liberates the sphingomyelinsequestered pool of PM cholesterol into the 'accessible' pool and thus stimulates its PM to ER transport, GRAMD1 triple knockout (TKO) cells exhibited exaggerated accumulation of the accessible pool of PM cholesterol and reduced suppression of SREBP-2 cleavage compared to wild-type control cells. This accumulation resulted from less efficient transport of accessible cholesterol from the PM to the ER.
Using structure-function analysis, we demonstrated that GRAMD1s couple their PM-sensing property and cholesterol-transport function via their GRAM and StART-like domains, and that GRAMD1 complex formation ensures the progressive recruitment of GRAMD1 proteins to ER-PM contacts. Finally, we observed striking expansion of the accessible pool of PM cholesterol in GRAMD1 TKO cells at steady state. Drug-induced acute recruitment of GRAMD1b to ER-PM contacts was sufficient to facilitate removal of the expanded pool of accessible cholesterol from the PM in GRAMD1 TKO cells. Collectively, our findings provide evidence for novel cellular mechanisms by which GRAMD1s monitor and help to maintain PM cholesterol homeostasis in mammalian cells. As one of the key homeostatic regulators, GRAMD1s sense a transient expansion of the accessible pool of PM cholesterol and facilitate its transport to the ER, thereby contributing to PM cholesterol homeostasis at ER-PM contact sites.

GRAMD proteins form homo-and heteromeric complexes
Previous studies identified GRAMD1s as ER-resident proteins that are distributed throughout ER structures in a punctate pattern . GRAMDs (namely GRAMD1a, GRAMD1b, GRAMD1c, GRAMD2, and GRAMD3) all possess an N-terminal GRAM domain and a C-terminal transmembrane domain. In addition, the three GRAMD1 proteins (GRAMD1s) possess a StART-like domain ( Figure 1A). Some LTPs are known to form homo-and heteromeric complexes. Thus, we reasoned that GRAMD1s may also interact with one another to form complexes. To further analyze the dynamics of these proteins on the ER at high spatial resolution, we tagged the GRAMD1s, as well as GRAMD3, with fluorescent proteins and analyzed their localization using spinning disc confocal microscopy coupled with structured illumination (SDC-SIM). Analysis of COS-7 cells expressing individual EGFP-tagged GRAMD1s or GRAMD3 (EGFP-GRAMD1a, EGFP-GRAMD1b, EGFP-GRAMD1c, or EGFP-GRAMD3) and a general ER marker (RFP-tagged Sec61b) revealed enrichment of GRAMD1s and GRAMD3 in similar discrete patches along ER tubules. By contrast, RFP-Sec61b localized to all domains of the ER, including the nuclear envelope and the peripheral tubular ER network (Hoyer et al., 2018) ( Figure 1B and Figure 1-figure supplement 1A). When individual EGFP-GRAMD1s and either mRuby-tagged GRAMD1b (mRuby-GRAMD1b) ( Figure 1C) or mCherrytagged GRAMD3 (mCherry-GRAMD3) (Figure 1-figure supplement 1B) were co-expressed in COS-7 cells, the patches of EGFP and mRuby/mCherry significantly overlapped, indicating potential complex formation between these proteins on tubular ER.
To test whether these proteins form complexes, we examined biochemical interactions between GRAMD1s and GRAMD3 using co-immunoprecipitation assays. HeLa cells co-transfected with individual EGFP-GRAMD1s together with either myc-tagged GRAMD1b (Myc-GRAMD1b) ( Figure 1D and Luminal helices and transmembrane domains of GRAMD proteins are important for their complex formation The formation of homo-and heteromeric complexes between GRAMD1s and GRAMD3 suggested the presence of amino-acid sequence within these proteins that facilitate their interaction. Secondary structure predictions indicated the presence of a conserved alpha helix within the luminal region of GRAMD1s ( Figure 2A). Furthermore, helical wheel analysis of the luminal helix from GRAMD1b predicted that this protein contained an amphipathic helix, with charged and hydrophobic amino acids occupying opposite sides of the helix ( Figure 2B and   Confocal images of live COS-7 cells expressing the ER membrane marker RFP-Sec61b and EGFP-GRAMD protein constructs as indicated. Insets show at higher magnification the regions indicated by white dashed boxes. Note the presence of the patches of EGFP-GRAMDs throughout the tubular ER. Scale bars, 10 mm. (C) Confocal images of live COS-7 cells expressing mRuby-GRAMD1b and EGFP-GRAMD1s as indicated. Note the presence of mRuby-GRAMD1b patches that partially overlap with the patches of EGFP-GRAMD1s. Scale bars, 1 mm. (D, E) Extracts of HeLa cells transfected with the indicated constructs were subjected to anti-GFP immunoprecipitation (IP) and then processed for SDS-PAGE and immunoblotting (IB) with anti-GFP and anti-Myc antibodies. Inputs are 1% of the total cell lysates. Note the strong biochemical interaction between GRAMD1b and GRAMD1s (D) and between GRAMD3 and GRAMD1s (E). Immunoprecipitated EGFP-GRAMD1s, Myc-GRAMD1b and Myc-GRAMD3 are indicated by arrows. The online version of this article includes the following figure supplement(s) for figure 1:   . This region is predicted by Phyre2 to contain an amphipathic helix (Kelley et al., 2015) as indicated. Blue and red asterisks mark hydrophobic amino acid residues that are partially conserved in GRAMD1s. The shared identities of the amino acid sequences of the amphipathic helices predicted by BLAST analysis were: 75% (GRAMD1a vs. GRAMD1b); 75% (GRAMD1a vs. GRAMD1c); and 80% (GRAMD1b vs. GRAMD1c). The effects of the mutations of these residues to glutamic acid (4E in the case of blue marks; 5E in the case of red marks) were tested in GRAMD1b. Black, red, blue, and pink/purple colors denote hydrophobic, acidic, basic, and hydrophilic amino acid residues, respectively.  Figure 2 continued on next page hydrophobic surfaces (Segrest et al., 1990). Therefore, we first asked whether the luminal helix was necessary for these proteins to form discrete patches on tubular ER. We focused on GRAMD1b as a model protein for analysis of the properties of the GRAMD1 luminal helices, generating a version of GRAMD1b that lacked the luminal helix (Dhelix), and a second version in which the five hydrophobic residues within the luminal helix were mutated to glutamic acid (5E), thereby disrupting the hydrophobic surface ( Figure 2B and Figure 2-figure supplement 1C). Whereas GRAMD1b (wild-type control) formed patches on tubular ER, both GRAMD1b (Dhelix) and GRAMD1b (5E) exhibited diffuse localization patterns, with fewer discrete patches on tubular ER ( Figure 2C). By contrast, a version of GRAMD1b in which the four hydrophobic residues preceding the luminal helix were mutated to glutamic acid (4E) formed patches that were similar to those formed by the control ( Figure 2C), demonstrating that the 5E mutation specifically disrupted patch formation. The potential ability of the luminal helices to interact directly with one another was examined using cell-free assays. Wild-type luminal helices (GRAMD1b 674-718 ) and luminal helices with the 5E mutation (GRAMD1b 674-718 5E ) were purified individually as EGFP fusion proteins and analyzed by size exclusion chromatography (SEC). Whereas the predicted molecular weights of the fusion proteins were the same (~35 kDa), wild-type luminal helices (EGFP-helix: EGFP-GRAMD1b 674-718 ) eluted at a much lower elution volume compared to 5E mutant luminal helices [EGFP-helix (5E): EGFP-GRAMD1b 674-718 5E ] ( Figure 2D). Blue native PAGE analysis (BN-PAGE) of the purified proteins revealed that wild-type helices migrated slower than the 5E mutants, indicating that interaction between luminal helices depended on the hydrophobic surface of GRAMD1b ( Figure 2E). By contrast, in the presence of SDS, the denatured forms of these proteins migrated similarly (SDS-PAGE). Slightly slower migration of 5E mutants on the gel was possibly due to the increased hydrophilicity of this fragment compared to wild-type (Guan et al., 2015) ( Figure 2E). These results suggest that the luminal helix is probably amphipathic and is important for the formation of GRAMD1b complexes through its hydrophobic surface.
Finally, the formation of GRAMD1 complexes was examined biochemically in cells using co-immunoprecipitation assays. Homomeric interactions between GRAMD1bs and heteromeric interactions between GRAMD1b and GRAMD1a were greatly reduced when the luminal helix of GRAMD1b was either removed (Dhelix) or mutated to the 5E version, supporting the important role of the luminal helix in homo-and heteromeric interactions of the GRAMD1s ( Figure 2F,G). Residual interactions were mediated by the transmembrane domain of GRAMD1b, as replacing this domain and its luminal region with those from Sec61b (TM swap) ( Figure 2J) completely abolished the ability of GRAMD1b to form homo-and heteromeric complexes ( Figure 2F,G). Accordingly, GRAMD1b with GRAMD1b (EGFP-helix) and EGFP-helix with the 5E mutations [EGFP-helix (5E)]. Note the difference in elution volumes, indicating the formation of complexes mediated by the wild-type luminal helix. (E) Blue native (BN)-PAGE analysis (left panel) and SDS-PAGE analysis (right panel) of SEC-purified EGFP-helix and EGFP-helix (5E). Black and red arrows indicate the major bands for EGFP-helix and EGFP-helix 5E, respectively. Note the difference in their migration pattern in BN-PAGE. CB, Colloidal blue staining. (F) Extracts of HeLa cells transfected with the constructs as indicated were subjected to anti-GFP immunoprecipitation (IP) and then processed for SDS-PAGE and immunoblotting (IB) with anti-GFP and anti-Myc antibodies. Inputs are 5% of the total cell lysates. Note that the interaction of GRAMD1b or GRAMD1a is much reduced in GRAMD1b Dhelix or 5E mutants and abolished in the GRAMD1b (TM swap) mutant (GRAMD1b with its transmembrane domain and luminal region replaced with those of Sec61b) when compared to the levels of interactions seen in cells with wild-type GRAMD1b. This reduction is smaller in the GRAMD1b 4E mutant. (G) Quantification of the coimmunoprecipitation experiments shown in (F). The ratio of the band intensity of the co-immunoprecipitated Myc-GRAMD1b (left) or Myc-GRAMD1a (right) over that of the indicated immunoprecipitated EGFP-tagged proteins were calculated. The values were then normalized by the ratio of the band intensity of Myc-GRAMD1b over that of EGFP-GRAMD1b (WT) (left) or by the ratio of the band intensity of Myc-GRAMD1a over that of EGFP-  the TM swap exhibited a diffuse localization pattern compared to that of wild-type GRAMD1b ( Figure 2H), and failed to interact with wild-type GRAMD1b on tubular ER ( Figure 2I). Thus, both transmembrane domains and luminal helices contributed to the formation of GRAMD1 complexes ( Figure 2J). Taken together, these results revealed the biochemical mechanisms by which GRAMDs form homo-and heteromeric complexes. As key residues contributing to the hydrophobic surface of the luminal helix are conserved among GRAMD1s (Figure 2A and Figure 2-figure supplement 1A), they probably play a role in the heteromeric interactions of all of these proteins.
The GRAM domain of GRAMD1s acts as a coincidence detector of unsequestered/accessible cholesterol and anionic lipids, and senses the accessibility of cholesterol Recent studies demonstrated that 'cholesterol loading' leads to the accumulation of GRAMD1s at ER-PM contact sites . Within 20 min of treating cells with a complex of cholesterol and methyl-b-cyclodextrin (cholesterol/MCD), GRAMD1b was indeed recruited to the PM ( Figure 3A,B; Video 1). In addition, we found that GRAMD1a, GRAMD1c, and GRAMD3 were all recruited to ER-PM contacts upon cholesterol loading, with kinetics similar to GRAMD1b recruitment ( Figure 3B). However, a version of GRAMD1b that lacked the GRAM domain (GRAMD1b DGRAM) failed to localize to the PM, even after 30 min, indicating the essential role of this domain in sensing PM cholesterol (Figure 3-figure supplement 1A; Video 2). Although these results suggest that PM cholesterol plays a critical role in recruiting GRAMDs to ER-PM contacts, all of the GRAMDs localize to tubular ER at rest, even though a significant amount of cholesterol is already present in the PM (Lange et al., 1989;Ray et al., 1969). Thus, their GRAM domains may possess unique abilities to sense the accessibility of PM cholesterol, rather than detecting the total levels of PM cholesterol. However, it is not known whether the GRAM domains are able to sense accessible cholesterol in the PM.
To elucidate the biochemical properties of the GRAMD1 GRAM domain, we first purified the GRAM domain of GRAMD1b and performed liposome sedimentation assays to test its ability to bind lipids. In this assay, purified GRAM domains were mixed with sucrose-loaded heavy liposomes in sucrose-free buffer. After incubation, free liposomes and the liposomes that bound to GRAM domains (P) were pelleted by centrifugation; the supernatant contained only unbound GRAM domains (S) ( Figure 3C . Thus, we explored the possibility that the GRAM domain may bind to membranes more efficiently in the presence of both lipids, thereby acting as a coincidence detector of unsequestered/accessible cholesterol and phosphatidylserine.
Little binding was observed when the liposomes contained 50% cholesterol or 20% phosphatidylserine ( Figure 3C and domains and the PM could be suppressed by 'the factors that sequester cholesterol' in this bilayer in cells. One of the major factors that mediate the direct sequestration of PM cholesterol is sphingomyelin, which forms a complex with cholesterol and makes it inaccessible Finean, 1953;McConnell and Radhakrishnan, 2003;Radhakrishnan and McConnell, 2000;Slotte, 1992). The sphingomyelin-sequestered pool of PM cholesterol consists of~15% of PM lipids, while the rest of the inaccessible pool is sequestered by other membrane factors (Das et al., 2014). To test whether the sequestration of cholesterol by sphingomyelin affects the binding of GRAM domains to artificial membranes, we incorporated increasing amounts of sphingomyelin (SM) (10% or 25%) into liposomes that contained 50% cholesterol and 20% phosphatidylserine ( Figure 3E). When these liposomes contained 25% sphingomyelin, the percentage of GRAMD1b GRAM domains that bound to the liposomes decreased from~80% to~45% ( Figure 3E and      https://elifesciences.org/articles/51401#video1 membranes that contain cholesterol and phosphatidylserine can be modulated by the presence of sphingomyelin (Figure 3-figure supplement 1C). These results suggest that sphingomyelin helps to suppress the binding of GRAM domains to the PM at rest by reducing the accessibility of the cholesterol in this bilayer.
In addition to sphingomyelin, phospholipid acyl chain saturation has profound effects on the accessibility of cholesterol in membranes (Chakrabarti et al., 2017;Gay et al., 2015;Lange et al., 2013;Radhakrishnan and McConnell, 2000;Sokolov and Radhakrishnan, 2010). If the GRAM domain binds to the PM by sensing the accessibility of cholesterol, its binding to artificial membranes should also be influenced by the acyl chain diversity of the phospholipids. To test this possibility, we generated liposomes containing fixed amounts of phosphatidylserine (20%) with varying ratios of cholesterol and phosphatidylcholine ( Branched (DPhyPC) and more unsaturated (DOPC) acyl chains lower the tendency to form ordered conformation in the membranes, and thus, POPC has the strongest cholesterol sequestration effect of these three lipids, followed by DOPC and DPhyPC (Sokolov and Radhakrishnan, 2010). The binding of the GRAM domain of GRAMD1b to liposomes shifted to lower cholesterol concentration as the ordering tendency of phosphatidylcholine is lowered (i.e. as the cholesterol sequestration effect is reduced) (Figure 3-figure supplement 2B). These results are consistent with the ability of the GRAM domain to sense the accessibility of cholesterol in membranes.
Finally, to determine whether GRAM domains bind more broadly to other anionic lipids, we replaced phosphatidylserine with other anionic lipids, namely phosphatidic acid (PA), PI(4)P, and PI (4,5)P 2 , and asked whether they affected GRAM domain binding similarly. In this assay, we used 5% anionic lipids, including phosphatidylserine, because even 5% phosphatidylserine was sufficient to mediate the binding of the GRAM domain to liposomes that also contained 50% free cholesterol, albeit less efficiently than 20% phosphatidylserine (Figure 3-figure supplement 1D). No or little binding was observed when GRAM domains were mixed with liposomes that contained 5% of these anionic lipids (each was tested individually) ( As anionic lipids, including phosphatidylserine, are enriched in the inner leaflet of the PM (Yeung et al., 2008), these results indicate that the recruitment of GRAMD1s to the PM is regulated by interactions between GRAM domains and anionic lipids, and that these interactions are enhanced by the additional presence of accessible/unsequestered cholesterol in the PM.

Liberation of sphingomyelin-sequestered pool of cholesterol induces acute recruitment of GRAMD1b to the PM
To examine the physiological role of sphingomyelin in GRAM domain-dependent recruitment of GRAMD1s to the PM, HeLa cells expressing either EGFP-GRAMD1b or EGFP-GRAMD1b DGRAM were treated with sphingomyelinase, which hydrolyzes PM sphingomyelin, and imaged under total internal reflection fluorescence (TIRF) microscopy. Although sphingomyelin is enriched in the outer leaflet of the PM bilayer, it also contributes to suppressing the accessibility of cholesterol in the inner Video 2. GRAMD1b DGRAM is not recruited to ER-PM contacts upon cholesterol loading. HeLa cells expressing EGFP-GRAMD1b DGRAM were imaged under TIRF microscopy. Images were taken every 20 s, and 200 mM cholesterol/MCD was added at the 5 min time point. Image size, 66.1 mm x 66.1 mm. https://elifesciences.org/articles/51401#video2 leaflet of the PM, because unsequestered cholesterol can spontaneously flip flop between the outer and inner leaflets of this bilayer (Leventis and Silvius, 2001;Steck and Lange, 2018). Within 30 min of sphingomyelinase treatment, GRAMD1b was indeed recruited to the PM ( Figure 3G), albeit this recruitment was less efficient than cholesterol loading to the PM ( Figure 3B). GRAMD1b DGRAM, however, failed to localize to the PM, even after 60 min ( Figure 3G). EGFP-tagged GRAMD1 GRAM domains (namely EGFP-GRAM 1a , EGFP-GRAM 1b , and EGFP-GRAM 1c ) were all recruited to the PM upon sphingomyelinase treatment ( Figure 3H), revealing a direct role of the GRAM domain in detecting the unsequestered/accessible pool of PM cholesterol in cells. These results are also consistent with the lack of enrichment of GRAMD1s at ER-PM contact sites at rest ( Figure 1B, Figure 3A and Figure 4-figure supplement 3B). Taken together, these data demonstrate that GRAMD1s are recruited to the PM by sensing an increase in the accessibility of PM cholesterol (i.e. acute expansion of the accessible pool of PM cholesterol that exceeds a certain threshold at which the GRAM domain interacts with the PM). Furthermore, the data show that this recruitment depends on the GRAM domain, which acts as a coincidence detector for both unsequestered/accessible cholesterol and anionic lipids in the PM ( Figure 3I).

Deletion of GRAMD1s results in exaggerated accumulation of the accessible pool of cholesterol in the PM
As GRAMD1s move to ER-PM contact sites upon acute expansion of the accessible pool of PM cholesterol ( Figure 3G,H), they may also contribute to the extraction of accessible PM cholesterol in order to maintain homeostasis. To investigate the potential functions of GRAMD1s in this process, we used the CRISPR/Cas9 system to disrupt GRAMD1 function by targeting all three GRAMD1 genes (GRAMD1A, GRAMD1B and GRAMD1C) in HeLa cells. Guide RNAs specific to exon 13 of GRAMD1A and GRAMD1B and to exon 11 of GRAMD1C were chosen, as they encode the lipid-harboring StART-like domains ( Figure 4A). After transfection of plasmids expressing GRAMD1-specific guide RNAs and Cas9 protein, two independent isolates of GRAMD1a/1b double knockout cell clones (DKO #38 and DKO #40) and two independent isolates of GRAMD1a/1b/1c triple knockout cell clones (TKO #1 and TKO #15) were selected. The absence of GRAMD1a and GRAMD1b was confirmed by western blotting and genomic sequencing ( Figure 4B and No obvious defects in cell viability or overall morphology were observed for these KO cells, with the exception that KO cells grew slightly slower than parental HeLa cells. Subsequent experiments were performed using GRAMD1a/1b/1c TKO #15 cells (hereafter referred to as GRAMD1 TKO cells).
The incubation of cells with sphingomyelinase reduces the sequestration of PM cholesterol, resulting in a transient expansion of the accessible pool of cholesterol in the PM (Das et al., 2014;Endapally et al., 2019). The newly expanded pool of accessible cholesterol is then extracted and transported to the ER (Das et al., 2014;Lange and Steck, 1997;Scheek et al., 1997;Slotte and Bierman, 1988). Based on the ability of the GRAM domain to sense expansion of the accessible pool of PM cholesterol ( Figure 3H,I), EGFP-GRAM 1b was used as a probe to detect acute increases in the accessible pool of PM cholesterol. Without stimulation, cytosolically expressed EGFP-GRAM 1b was distributed throughout the cytoplasm without particular enrichment in the PM in both wild-type and GRAMD1 TKO HeLa cells (Figure 4-figure supplement 2A). Treatment with sphingomyelinase for 1 hr led to only modest recruitment of EGFP-GRAM 1b to the PM in wild-type HeLa cells ( Figure 4D). By contrast, the same treatment lead to much more prominent recruitment of EGFP-GRAM 1b to the PM in GRAMD1 TKO cells ( Figure 4D). TIRF microscopy of cells expressing EGFP-GRAM 1b revealed that PM recruitment of EGFP-GRAM 1b upon sphingomyelinase treatment was significantly enhanced in GRAMD1 TKO cells, compared to wild-type control cells, over the entire 1 hr treatment ( Figure 4E). Importantly, additional treatment of GRAMD1 TKO cells with methyl-b-cyclodextrin (MCD), which extracts cholesterol from cellular membranes, resulted in acute loss of the PM recruitment of EGFP-GRAM 1b within 2 min (Figure 4-figure supplement 2B). However, the same treatment resulted in only modest changes in the binding of the phosphatidylserine biosensor (the mCherry-tagged C2 domain of lactadherin, mCherry-LactC2) or the PI(4,5)P 2 biosensor (the iRFP-tagged PH domain of PLCd, iRFP-PH PLCd ), confirming the specificity of EGFP-GRAM 1b in sensing the newly expanded pool of accessible cholesterol in the PM upon sphingomyelinase treatment (Figure 4-figure supplement 2B). Taken together, these results demonstrate an exaggerated accumulation of the accessible pool of PM cholesterol in GRAMD1 TKO cells upon sphingomyelinase treatment, and suggest that the extraction and transport of this acutely expanded accessible pool may be impaired in the absence of GRAMD1s.
We also assessed the role of GRAMD1s in regulating steady-state PM cholesterol levels by separating and purifying PMs from cultured cells using poly-D-lysine-coated dextran beads (Saheki et al., 2016). Cultured cells were attached to the beads and osmotically lysed by vigorous vortexing. Brief sonication was used to remove most organelles, whereas PM sheets remained attached to the bead surface (visualized by BODIPY-labeled ceramide) (      western blotting, the PM sheets that remained bound to the beads were highly enriched for PM marker proteins (such as CD44) relative to the starting material. The endosomal marker, EEA1, was greatly depleted, whereas small amounts of ER proteins (such as VAPA and VAPB) were recovered in the PM (Figure 4-figure supplement 3B). This probably reflected the tight attachment of cortical ER (Saheki et al., 2016). Importantly, the levels of endogenous GRAMD1a and GRAMD1b on bead-attached PM sheets were similar to those seen for the integral ER protein, VAP (Figure 4-figure supplement 3B). This confirmed that the majority of these two proteins are distributed throughout the ER, with only a very small fraction localizing to ER-PM contact sites at rest ( Figure 1B and Figure 3A). Mass spectrometry analysis of whole-cell and purified PM lipid extracts from wild-type control and GRAMD1 TKO HeLa cells did not reveal significant changes in cholesterol and other major lipids, except for very minor increases in cholesterol esters (Figure 4-figure supplement 3C, D). Thus, GRAMD1s are not essential for maintaining total levels of PM cholesterol. This result is also consistent with very little enrichment of GRAMD1s at ER-PM contacts at the steady state. Collectively, these results indicate that GRAMD1s may contribute to PM cholesterol homeostasis by counteracting acute increases in the accessible pool of PM cholesterol through its extraction and transport to the ER.
The cholesterol transporting property of the StART-like domain of GRAMD1s is critical for the removal of an acutely expanded pool of accessible PM cholesterol Although GRAMD1 StART-like domains transport cholesterol in vitro, it remains unclear whether this property is relevant to cellular physiology. Our live-cell imaging analysis of EGFP-GRAM 1b (which is a novel biosensor for detecting acute expansion of the accessible pool of PM cholesterol that we identified in this study) allowed us to conduct a structure-function analysis of GRAMD1s in the context of cellular functions for the first time. We first asked whether the sterol-binding pocket of the StART-like domain is required for the cellular functions of GRAMD1s. As a first step, we characterized the cholesterol-transporting properties of individual StART-like domains in vitro and generated a series of structure-guided mutations in order to identify key amino-acid residues that are essential for cholesterol transport. We purified StART-like domains from all three GRAMD1s and performed cell-free liposome-based lipid transfer assays. In this assay, the amount of dehydroergosterol (DHE) (a fluorescent analog of cholesterol) in liposomes was quantitatively measured using fluorescence resonance energy transfer (FRET) between DHE and Dansyl-PE (DNS-PE) ( Figure 5A). DHE was initially loaded only into donor liposomes, and its transfer from donor to DNS-PE-containing acceptor liposomes was monitored over time by measuring FRET between transferred DHE and DNS-PE in acceptor liposomes ( Figure 5A and    Building upon our newly designed 5P mutation, which eliminated the ability of StART-like domains to transport cholesterol, we asked whether the exaggerated accumulation of the accessible pool of PM cholesterol that was observed in GRAMD1 TKO cells upon sphingomyelinase treatment (using the EGFP-GRAM 1b biosensor) could be rescued by re-expressing wild-type or mutant versions of GRAMD1b. Strikingly, the enhanced PM recruitment of EGFP-GRAM 1b was dramatically suppressed by expressing wild-type mRuby-GRAMD1b but not by expressing a mutant version of mRuby-GRAMD1b that is defective in cholesterol transport [mRuby-GRAMD1b (5P)] ( Figure 5E). By contrast, PM recruitment of mRuby-GRAMD1b upon sphingomyelinase treatment of TKO cells was higher for the 5P mutant GRAMD1b than for wild-type GRAMD1b ( Figure 5F). These results suggest that the StART-like domain-dependent extraction and transport of accessible PM cholesterol to the ER facilitates the dissociation of GRAMD1b from the PM, as the interaction of the GRAM domain of GRAMD1b with the PM is weakened, owing to a reduction in accessible cholesterol in the PM.
Our results to date suggest that GRAMD1b may play a unique role in sensing and controlling the movement of accessible PM cholesterol. To further support this notion, we used GRAMD1 TKO cells to examine whether overexpression of other known cholesterol-transfer proteins, such as STARD4 (Iaea et al., 2017;Mesmin et al., 2011) and some ORPs [including OSBP (Antonny et al., 2018), ORP4 (Charman et al., 2014) and ORP9 (Ngo and Ridgway, 2009)] could substitute the function of GRAMD1s. Specifically, we examined whether their overexpression rescue exaggerated accumulation of the accessible pool of PM cholesterol observed in GRAMD1 TKO cells, as monitored by the EGFP-GRAM 1b biosensor, upon sphingomyelinase treatment ( Figure 4D,E). Transiently transfected mCherry-tagged STARD4 (mCherry-STARD4) and mRuby-tagged ORPs (mRuby-OSBP, mRuby-ORP4, and mRuby-ORP9) were all well expressed in TKO cells ( Taken together, our results suggest a critical role of the GRAMD1s in controlling the movement of the accessible pool of PM cholesterol between the PM and the ER via their StART-like domains.
GRAMD1s play a role in accessible cholesterol transport from the PM to the ER during acute expansion of the accessible pool of PM cholesterol Acute expansion of the accessible pool of PM cholesterol results in the suppression of SREBP-2 cleavage and the inhibition of cholesterol biosynthesis as a result of transport of accessible cholesterol from the PM to the ER. However, the intracellular transport machinery by which accessible cholesterol is transported from the PM to the ER remains unknown. GRAMD1s may play a role in this process, as they are able to sense and counteract the acute expansion of the accessible pool of PM cholesterol.
TIRF microscopy of cells expressing EGFP-GRAMD1b revealed that sphingomyelinase treatment led to sustained recruitment of GRAMD1b to the PM (during 3 hr of imaging) ( Figure 6A; Video 3). As GRAMD1 TKO cells show exaggerated accumulation of the accessible pool of PM cholesterol upon sphingomyelinase treatment compared with wild-type cells ( Figure 4D,E), GRAMD1s may be involved in PM to ER transport of the accessible pool of cholesterol via their GRAM and StART-like domains. To examine the role of GRAMD1s in this process, we determined a time-course for the suppression of SREBP-2 cleavage upon sphingomyelinase treatment in wild-type control and GRAMD1 TKO cells as an estimate of the efficiency of the transport of accessible cholesterol from the PM to the ER. In this assay, we first depleted most of the accessible cholesterol from control and  TKO cells by treating them with a combination of lipoprotein-deficient serum (LPDS) and mevastatin, an HMG-CoA reductase inhibitor, for 16 hr (a treatment designed to induce maximum SREBP-2 cleavage by cholesterol starvation). We then stimulated the cells with sphingomyelinase and, using total cell lysates, we monitored over time the suppression of SREBP-2 cleavage, which results from the PM to ER transport of accessible cholesterol in response to the liberation of the sphingomyelinsequestered pool of PM cholesterol by sphingomyelinase. Cell lysates were collected at different time points (0, 30, 60, 90, 120, 150, and 180 min) and analyzed by SDS-PAGE followed by immunoblotting against SREBP-2 ( Figure 6B). At time 0, there were no detectable changes in the cleavage of SREBP-2 in GRAMD1 TKO cells compared to wild-type control cells. Suppression of SREBP-2 cleavage was observed in control cell lysates within 90 min; however, such suppression was delayed and reduced (but not eliminated) in TKO cells. Even after 180 min, TKO cells were not able to suppress SREBP-2 cleavage to levels similar to those observed in wild-type control cells ( Figure 6B,C). Importantly, re-expression of GRAMD1b in TKO cells was sufficient to suppress SREBP-2 cleavage to an extent similar to that observed in wild-type control cells at the 180 min time point, thereby rescuing the phenotype ( Figure 6D,E and Figure 6-figure supplement 1A,B). We hypothesized that both the recruitment of GRAMD1s to ER-PM contact sites and their ability to transport cholesterol are critical for the suppression of the cleavage of SREBP-2 by facilitating transport of the newly expanded pool of accessible PM cholesterol to the ER. To test this hypothesis, we used a GRAMD1b mutant that lacks the GRAM domain (GRAMD1b DGRAM), which cannot be recruited to the PM (Figure 3G), and a GRAMD1b with the mutated StART-like domain, which is defective in cholesterol transport (5P) ( Figure 5B-D). The expression of GRAMD1b DGRAM or GRAMD1b 5P in TKO cells failed to rescue the phenotype ( Figure 6D and Figure 6-figure  supplement 1A). These data demonstrate that GRAMD1s play a role in the transport of accessible cholesterol from the PM to the ER upon acute expansion of the accessible pool of PM cholesterol and help to suppress SREBP-2 activity. Furthermore, the data show that such functions require the recruitment of GRAMD1s to ER-PM contact sites, which is Figure 6 continued condition, Dunnett's multiple comparisons test, *p=0.0162; (E): mean ± SEM, n = 5 lysates (independent experiments) for each condition, Dunnett's multiple comparisons test, **p<0.0001; n.s. denotes not significant]. (F) Representative TIRF images of live HeLa cells expressing EGFP-GRAMD1b (Wild-type) and EGFP-GRAMD1b TM swap (TM swap) treated as described for panel (G). Note the differences in how these proteins are recruited to the PM. Wild-type GRAMD1b accumulated progressively at ER-PM contacts, forming patches by the end of the 3 hr imaging period, whereas the GRAMD1b TM swap mutant remained diffuse on the tubular ER, even at the end of the 3 hr imaging period. Scale bars, 1 mm. (G) Left: time course of normalized EGFP signal, as assessed by TIRF microscopy, from HeLa cells expressing EGFP-GRAMD1b or EGFP-GRAMD1b TM swap in response to SMase treatment (100 mU/ml). Note the reduced recruitment of EGFP-GRAMD1b TM swap to the PM compared to EGFP-GRAMD1b after 3 hr of the SMase treatment. Right: values of DF/F 0 at a time point corresponding to the end of the experiment (as indicated by the arrow) [mean ± SEM, n = 72 cells (GRAMD1b), n = 69 cells (GRAMD1b TM swap); data are pooled from three independent experiments for each condition; two-tailed unpaired Student's t-test, **p<0.0001]. The online version of this article includes the following source data and figure supplement(s) for figure 6: Source data 1. Dataset for Figure 6.     In order to measure changes in the accessible pool of PM cholesterol, we took advantage of the cholesterol-binding domain 4 (D4) of bacterial Perfringolysin O (PFO), which has been widely used as a probe to measure the accessible pool of PM cholesterol (Das et al., 2013;Gay et al., 2015;Shimada et al., 2002;Sokolov and Radhakrishnan, 2010). Wild-type control and GRAMD1 TKO cells that had been pre-treated with a combination of LPDS and mevastatin for 16 hr were stimulated with sphingomyelinase for a fixed period of time (0, 30, 60, 90, 120, 150, and 180 min) and washed. Cells were then incubated with recombinant EGFP-tagged D4 (EGFP-D4) proteins for 15 min at room temperature. After washing, cell lysates were collected and analyzed by SDS-PAGE followed by immuno-blotting against GFP to detect EGFP-D4 proteins that were bound to accessible cholesterol in the PM (Figure 6-figure supplement 2A). At time 0, there were no detectable changes in EGFP-D4 signals in TKO cells compared to wild-type control cells. A 30 min treatment with sphingomyelinase induced a similar increase in the binding of EGFP-D4 to both control and TKO cells. A gradual decrease of EGFP-D4 signals was observed in control cell lysates over the time course of 180 min, similar to that reported in a previous report that utilized a mutant form of PFO to assess changes in accessible cholesterol in the PM upon sphingomyelinase treatment (Das et al., 2014). TKO cells, however, showed continuous increase in binding of EGFP-D4 to the PM even after 180 min ( Figure 6-figure supplement 2A,B), suggesting a sustained accumulation of accessible cholesterol in the PM of TKO cells, due to less efficient transport of accessible cholesterol from the PM to the ER, that does not occur in wild-type control cells.
Together with the results obtained with the cytosolically expressed EGFP-GRAM 1b biosensor ( Figure 4D,E), these data strongly indicate that the extraction and transport of accessible PM cholesterol to the ER by GRAMD1s is able to counteract with acute expansion of the accessible pool of PM cholesterol (e.g. acute expansion induced by sphingomyelinase treatment) to prevent the accumulation of accessible cholesterol in the PM in wild-type control cells, and that this homeostatic response is impaired in GRAMD1 TKO cells. It is also important to note that there might be other intracellular cholesterol transport mechanisms that may act in parallel with GRAMD1s to facilitate accessible cholesterol extraction from the PM for its transport to the ER, as suppression of SREBP-2 cleavage is not eliminated even in the total absence of GRAMD1s (see Discussion). . TIRF microscopy analysis of HeLa cells expressing the GRAMD1b TM swap mutant, however, revealed major differences in how this protein was recruited to the PM compared to wild-type GRAMD1b ( Figure 6F). GRAMD1b TM swap remained diffusely distributed on the tubular ER (which is closely attached to the PM) even at the end of the 180 min imaging period. By contrast, wild-type GRAMD1b progressively accumulated at ER-PM contacts as discrete patches with much stronger PM recruitment ( Figure 6F,G; Video 3). These results support an important role for GRAMD1 complex formation in facilitating the progressive accumulation of GRAMD1s at ER-PM contacts, thereby supporting efficient accessible cholesterol transport at these contacts. Taken together, we conclude that GRAMD1s play a role in PM to ER transport of the accessible pool of PM cholesterol upon acute expansion of this pool. Loss of GRAMD1 function leads to sustained accumulation of accessible cholesterol in the PM, resulting in less effective suppression of SREBP-2 cleavage and possibly dysregulation of cellular cholesterol homeostasis.

Chronic expansion of the accessible pool of PM cholesterol in GRAMD1 TKO cells
Distinct pools of cholesterol co-exist in the PM at steady state: a major pool is 'inaccessible' (i.e., sequestered or chemically inactive) and a smaller pool is 'accessible' (i.e., unsequestered or chemically active). Given the role of GRAMD1s in facilitating the transport of accessible cholesterol from the PM to the ER, the impact of GRAMD1 deficiency on steady-state levels of accessible PM cholesterol was examined.
We purified EGFP-tagged D4 mutant (D434S) proteins (EGFP-D4H), which have a lower threshold for binding to accessible cholesterol compared to D4 in vitro (Johnson et al., 2012;Maekawa and Fairn, 2015). Wild-type control and GRAMD1 TKO HeLa cells that express a PM marker (iRFP-PH PLCd ) were incubated with buffer containing purified recombinant EGFP-D4H proteins for 15 min at room temperature and washed, and then imaged under spinning disc confocal microscopy. D4H binding was assessed by line scan analysis. Strikingly, EGFP-D4H proteins bound more strongly to the PM of GRAMD1 TKO cells compared to that of control cells ( Figure 7A Taken together, these results suggest the importance of GRAMD1s in maintaining steady-state levels of accessible PM cholesterol by facilitating its transport from the PM to the ER.

Acute recruitment of GRAMD1b to ER-PM contacts facilitates removal of the expanded pool of accessible PM cholesterol in GRAMD1 TKO cells
Chronic expansion of the accessible pool of PM cholesterol in GRAMD1 TKO cells at steady state, revealed by increased PM binding of the EGFP-D4H probe, indicates that GRAMD1s are important for maintaining PM cholesterol homeostasis through their functions in sensing a transient expansion of the accessible pool of PM cholesterol and by facilitating the transport of accessible PM cholesterol to the ER at ER-PM contact sites. If this is the case, artificial forced recruitment of re-expressed GRAMD1s to ER-PM contacts in GRAMD1 TKO cells should mediate the extraction and transport of accessible cholesterol from the PM to the ER and reduce the binding of the EGFP-D4H probe to the PM.
To test whether GRAMD1s can directly act at ER-PM contact sites, rapamycin-induced dimerization of the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) (Muthuswamy et al., 1999) was used to recruit GRAMD1b to these sites acutely. In this assay, GRAMD1 TKO cells were co-transfected with a version of GRAMD1b in which the N-terminus, which contains the GRAM domain, was replaced by a miRFP-tagged FKBP module (miRFP-FKBP-GRAMD1b) and a PM-targeted FRB module (PM-FRB-mCherry) ( Figure 7C). TIRF microscopy revealed rapid recruitment of miRFP-FKBP-GRAMD1b to the PM within 10 min of rapamycin treatment ( Figure 7D and On the basis of these results, we conclude that GRAMD1s play a direct role in facilitating the transport of accessible PM cholesterol to the ER at ER-PM contact sites.

Discussion
We have demonstrated that the evolutionarily conserved family of ER-anchored GRAMD1s       https://elifesciences.org/articles/51401#video4 contribute to PM cholesterol homeostasis by sensing a transient expansion of the accessible pool of PM cholesterol and facilitating its transport to the ER at ER-PM contact sites. We have also identified the molecular mechanisms by which GRAMD1s interact with one another to form a complex, and how they are recruited to the PM. Key findings of the current study are the following: (1) We found that GRAMD1s and GRAMD3 form homo-and heteromeric complexes and localize to discrete patches on tubular ER in mammalian cells at rest. We identified that their transmembrane domains and luminal helices, which are predicted to form amphipathic surfaces, mediated the formation of protein-protein complexes and regulated their progressive recruitment to ER-PM contacts and their functions.
(2) Using in vitro liposome sedimentation assays and live-cell imaging, we found that the GRAM domain of GRAMD1s acts as a coincidence detector, tuned to the presence of both 'unsequestered/accessible' cholesterol and anionic lipids, including phosphatidylserine, in the PM ( Figure 3I). Importantly, the binding of the GRAM domain to membranes requires that unsequestered/accessible cholesterol exceeds a certain threshold. As the majority of cholesterol in the PM is sequestered (i.e., inaccessible), this switch-like property allows GRAMD1s to move to ER-PM contact sites only when the accessible pool of PM cholesterol transiently expands, preventing GRAMD1s from accumulating at ER-PM contacts at rest.
(3) We have deciphered the novel cellular mechanisms by which the accessibility of PM cholesterol is monitored by an LTP at ER-PM contacts. We found that GRAMD1s sense a transient expansion of the accessible pool of PM cholesterol through their GRAM domain and facilitate its transport through their StART-like domain. Disruption of their functions leads to less efficient transport of accessible PM cholesterol to the ER and reduced suppression of SREBP-2 cleavage. Importantly, we showed that the formation of GRAMD1 protein complexes, as well as the StART-like and GRAM domains of GRAMD1 proteins, is critical for the cellular functions of GRAMD1s in vivo.
(4) Our results demonstrate that removal of accessible PM cholesterol occurs within~1 hr, when re-expressed GRAMD1 proteins are artificially recruited to ER-PM contact sites to facilitate transport of accessible cholesterol from the PM to the ER in GRAMD1 TKO cells. These experiments utilized a drug-induced dimerization approach. In addition, this GRAMD1 function requires the sterol-transporting StART-like domain. Previous studies of yeast mutants lacking Lam/Ltc proteins relied on genetic approaches (over a time scale of~20 hr), making it difficult to interpret the significance of their lipid-transfer functions in vivo.
Using the CRISPR/Cas9 gene-editing system, we demonstrated that GRAMD1s are not essential for cell viability. Although cells that lack all three GRAMD1s grow more slowly than wild-type cells, they do not exhibit major abnormalities. Overlapping functions between mammalian GRAMD1s and other sterol-binding STARD proteins may explain the lack of major defects in these cells. Accordingly, PM lipidomics did not reveal major differences in PM cholesterol levels between GRAMD1 TKO cells and wild-type cells. Our analyses suggest, however, that GRAMD1s facilitate the transport of accessible cholesterol from the PM to the ER in response to a transient expansion of the accessible pool of PM cholesterol, thereby contributing to PM cholesterol homeostasis. First, GRAMD1 TKO cells exhibited an exaggerated accumulation of the accessible pool of PM cholesterol in response to acute hydrolysis of sphingomyelin, which liberates the sphingomyelin-sequestered pool of cholesterol into the 'accessible pool'. This was revealed by sustained binding of the PFO D4 probe to the PM and by enhanced PM recruitment of the GRAM domain of GRAMD1b, a novel biosensor for detecting acute expansion of the accessible pool of PM cholesterol that we identified in this study, in GRAMD1 TKO cells. Second, GRAMD1 TKO cells showed reduced suppression of Video 5. Rapamycin-induced acute recruitment of a mutant version of GRAMD1b (5P) to the PM in GRAMD1 triple knockout (TKO) cells. GRAMD1 TKO HeLa cells expressing PM-FRB-mCherry and miRFP-FKBP-GRAMD1b (5P) were imaged under TIRF microscopy. Images were taken every 20 s, and 200 nM rapamycin was added at the 5 min time point. Note the rapamycin-induced recruitment of miRFP-FKBP-GRAMD1b (5P) to the PM. Image size, 66.1 mm x 66.1 mm. https://elifesciences.org/articles/51401#video5 SREBP-2 cleavage upon hydrolysis of sphingomyelin, reflecting less efficient PM to ER transport of the newly expanded pool of accessible cholesterol in these cells. Third, GRAMD1 TKO cells exhibited a chronic expansion of the accessible pool of PM cholesterol, as detected by the D4H probe. All of these phenotypes are consistent with defects in efficient PM to ER transport of the accessible pool of cholesterol. Importantly, re-expression of GRAMD1b rescued these phenotypes, with rescue depending on the sterol-binding property of GRAMD1b's StART-like domain, the GRAM domain, and protein complex formation.
Although the transition of cholesterol between 'inaccessible' and 'accessible' pools in the PM plays crucial roles in controlling cellular cholesterol homeostasis (Das et al., 2014;Endapally et al., 2019), the molecular mechanisms by which these transitions are monitored, and the intracellular transport machinery responsible for the PM to ER transport of the accessible pool of cholesterol, have both remained elusive. Our results suggest that these two mechanisms can be coupled by nonvesicular cholesterol transport mediated by an LTP at ER-PM contact sites. We found that GRAMD1s sense a transient expansion of the accessible pool of PM cholesterol and facilitate its transport to the ER at ER-PM contact sites ( Figure 8A-C). We found that interactions between the purified GRAM domains of GRAMD1a/b and artificial membranes that contain phosphatidylserine, which is a major acidic phospholipid in the PM, are dramatically enhanced by the presence of unsequestered/ accessible cholesterol that exceeds a certain threshold. Furthermore, such interactions are modulated by sphingomyelin and phospholipids, which sequester cholesterol by forming dynamic complexes. Thus, the GRAM domain allows GRAMD1s to sense an increase in accessible PM cholesterol and facilitates the accumulation of GRAMD1s at ER-PM contacts only when the accessible pool of cholesterol transiently expands in this bilayer ( Figure 8B). Importantly, such regulation prevents GRAMD1s from depleting PM cholesterol at steady state. Loss of GRAMD1 functions, however, leads to chronic and acute expansion of the accessible pool of PM cholesterol ( Figure 8B,C). It has been known that accessible cholesterol, upon reaching a certain threshold, is extracted for transport to the ER and regulates cholesterol biosynthesis to maintain homeostasis. How the intracellular transport machinery senses such a sharp threshold has been unknown. The ability of the GRAMD1s to accumulate at ER-PM contacts in a switch-like fashion, by sensing a transient expansion of the accessible pool of PM cholesterol through their GRAM domain, provides a conceptual framework for this process. Importantly, GRAMD1s themselves directly facilitate the transport of the expanded pool of accessible PM cholesterol from the PM to the ER, thereby contributing to PM cholesterol homeostasis as a critical homeostatic regulator.
Levels of PM cholesterol are maintained at an equilibrium by balancing the efflux and influx of cholesterol out of and into the PM, respectively. Thus, inhibition of the efflux pathway only (e.g., by loss of GRAMD1s) disrupts this equilibrium, potentially increasing total levels of PM cholesterol. However, levels of PM cholesterol are unchanged in GRAMD1 TKO cells (Figure 4-figure supplement 3C,D). Thus, alternative backup systems may support the efflux of PM cholesterol (or reduce the influx of cholesterol to the PM) to maintain total cholesterol levels in the PM in the absence of GRAMD1s. For example, recent studies found that macrophages are able to dispose of accessible cholesterol by releasing PM-derived particles that are rich in cholesterol Hu et al., 2019). It is also important to note that suppression of SREBP-2 cleavage upon sphingomyelinase treatment (an estimate of the efficiency of PM to ER transport of accessible cholesterol) is reduced but not eliminated in the absence of GRAMD1s ( Figure 6B,C). Thus, other intracellular cholesterol transport machineries might also participate in the transport of accessible cholesterol from the PM to the ER. Such robust parallel mechanisms may be partially mediated by other non-vesicular sterol transport systems, such as those mediated by STARDs or ORPs; vesicular transport may also be involved in maintaining total levels of PM cholesterol. Further studies are needed to better understand the interplay between GRAMD1s and other sterol efflux/transfer systems in the regulation of cellular cholesterol homeostasis.
GRAMD1s interact with each other through their transmembrane domains and predicted luminal amphipathic helices (e.g., GRAMD1b homomeric and GRAMD1b/GRAMD1a heteromeric interactions). Mutant GRAMD1b proteins that lack these regions fail to interact with one another and are diffusely distributed throughout the tubular ER. Other lipid transfer proteins that are known to localize to membrane contact sites also form complexes or oligomers. These proteins include E-Syts (Giordano et al., 2013;Saheki et al., 2016), ORP2 (Wang et al., 2019), and ORP5/8 (Chung et al., 2015), as well as the yeast ERMES complex (AhYoung et al., 2015). ORP2 oligomerization and ERMES assembly enhance the ability of these proteins to transfer lipids (Kawano et al., 2018;Wang et al., 2019). Accordingly, mutant GRAMD1b proteins that lack the ability to form protein complexes accumulate less robustly at ER-PM contacts, and less effectively rescue phenotypes associated with GRAMD1 TKO cells. Thus, the formation of GRAMD1 complexes plays important roles in regulating GRAMD1 localization and function. A recent study by Sandhu et al. (2018) reported that GRAMD1s facilitate the transport of PM cholesterol that is additionally loaded from external sources. In particular, they showed that GRAMD1b mediates PM to ER transport of HDL-derived cholesterol that is taken up by adrenal glands via the scavenger receptor SR-B1 in mice. On the basis of these results, they proposed that GRAMD1s might also play more general roles in intracellular cholesterol transport by sensing accessible cholesterol in the PM. Our findings support this notion and further demonstrate that GRAMD1s contribute to PM cholesterol homeostasis and cholesterol metabolism by sensing a transient expansion of the accessible pool of PM cholesterol by their GRAM domain, and facilitate the transport of this cholesterol to the ER by their StART-like domain. Interestingly, GRAMD1a is broadly expressed in many tissues with particular enrichment in the brain. Future studies will be needed to determine the physiological functions of GRAMD1s in other tissues in mammals.
In summary, our study has demonstrated that ER-localized GRAMD1s help to maintain PM cholesterol homeostasis in trans via their ability to transport accessible cholesterol from the PM to the ER at ER-PM contact sites. Our results show that GRAMD1s sense a transient expansion of the pool of accessible PM cholesterol and facilitate its transport to the ER through non-vesicular transport. These proteins probably perform additional functions, as yeast mutants that lack Lam/Ltc proteins show pleiotropic defects in cell signaling, including altered mTOR kinase signaling (Murley et al., 2017). Furthermore, the StART-like domain of GRAMD1b has been shown to transport PI(4,5)P 2 in addition to cholesterol . Thus, potential changes in the properties of other organelles and the PM in GRAMD1 TKO cells deserve future investigations. Further elucidating the physiological functions of these proteins, as well as other sterol-transfer proteins, at membrane contact sites will be important to gain insight into how cellular cholesterol homeostasis is regulated.

Antibodies and chemicals
Primary and secondary antibodies, chemicals, lipids and other reagents used in this study are listed in Supplementary file 1.

DNA plasmids
DNA plasmids used in this study are listed in Supplementary file 1; the sequences of oligos and primers used are listed in Supplementary file 2.
The amino-acid residues present in the StART-like domain that were predicted to be critical for binding cholesterol on the basis of our structural modeling (Y430 and V445) were mutated to alanine using site-directed mutagenesis in mRuby-GRAMD1b, with the primer set, GRAMD1b_Y430A_-V445A_F and GRAMD1b_Y430A_V445A_R, to generate mRuby-GRAMD1b (Y430A, V445A).
Cloning of EGFP-GRAMD1b (TM swap) and mRuby-GRAMD1b (TM swap) cDNAs corresponding to the residues 404-621 of GRAMD1b and 67-96 of Sec61b were individually amplified by PCR with the following primer sets: 5'_GRAMD1b_iEcoRV_Fw_HiFi and 3'_GRAMD1b_621_Rv_HiFi for GRAMD1b; and 5'_GRAMD1b-Sec61b_TM_Fw_HiFi and 3'_pEGFP_Sec61b_TM_Rv_HiFI for Sec61b. The two PCR products were simultaneously ligated at the EcoRV and KpnI sites of mRuby-GRAMD1b by DNA HiFi assembly kit (NEB) to generate mRuby-GRAMD1b (TM swap). cDNA corresponding to GRAMD1b (TM swap) was excised and ligated into pEGFP-C1 vector at the XhoI and KpnI sites to generate EGFP-GRAMD1b (TM swap).
A clone of PM-FRB-mCherry cDNA corresponding to the PM targeting signal (the residues 1-20 of mouse GAP43) and the FRB module was digested from PM-FRB-CFP (a gift from the De Camilli Lab) and ligated into the NheI and AgeI sites of the pmCherry-N1 vector.

Cell culture and transfection
HeLa and COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% or 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37˚C and 5% CO 2 . Transfection of plasmids was carried out with Lipofectamine 2000 (Thermo Fisher Scientific). Both wild-type and genome-edited HeLa cell lines were routinely verified as free of mycoplasma contamination at least every two months, using MycoGuard Mycoplasma PCR Detection Kit (Genecopoeia). No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample.

Fluorescence microscopy
For imaging experiments, cells were plated onto 35 mm glass bottom dishes at low density (MatTek Corporation). All live-cell imaging was carried out one day after transfection.
Spinning disc confocal (SDC) microscopy ( Figures 3A, 4D were performed on a setup built around a Nikon Ti2 inverted microscope equipped with a Yokogawa CSU-W1 confocal spinning head, a Plan-Apo objective (100 Â 1.45 NA), a back-illuminated sCMOS camera (Prime 95B; Photometrics), and a super-resolution module (Live-SR; Gataca Systems) that was based on structured illumination with optical reassignment and image processing (Roth and Heintzmann, 2016). The method, known as multifocal structured illumination microscopy (York et al., 2012), makes it possible to double the resolution and the optical sectioning capability of confocal microscopy simultaneously. The maximum resolution is 128 nm with a pixel size in superresolution mode of 64 nm. Excitation light was provided by 488 nm/150 mW (Coherent) (for GFP), 561 nm/100 mW (Coherent) (for mCherry/mRFP/mRuby) and 642 nm/110 mW (Vortran) (for iRFP/ miRFP) (power measured at optical fiber end) DPSS laser combiner (iLAS system; Gataca systems). All image acquisition and processing was controlled by MetaMorph (Molecular Device) software. Images were acquired with exposure times in the 400-500 msec range.
Cells were washed twice and incubated with Ca 2+ containing buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, 10 mM glucose, and 2 mM CaCl 2 [pH 7.4]) before imaging with either an SDC microscope or a TIRF microscope. All types of microscopy were carried out at 37˚C except for the experiments with recombinant EGFP-D4H proteins, which were performed at room temperature via SDC microscopy.

Drug stimulation for time-lapse TIRF imaging
For all time-lapse TIRF imaging experiments with drug stimulation, drugs were added to the cells 5 min after the initiation of the imaging, except for methyl-b-cyclodextrin (MCD) treatment, where 10 mM MCD (Sigma-Aldrich/Merck) was added to the cells as indicated in a figure (Figure 4-figure  supplement 2B). Other drugs were used at the following concentrations: 200 mM cholesterol/MCD complex generated as described previously (Brown et al., 2002); 100 mU/ml sphingomyelinase (SMase) (Sigma-Aldrich/Merck); and 200 nM rapamycin (Sigma-Aldrich/Merck).

Assays with recombinant EGFP-D4H proteins
For the assays with recombinant EGFP-D4H proteins ( Figure 7A For the experiments with MCD treatment (Figure 7-figure supplement 1A,B), GRAMD1 TKO cells were transfected with iRFP-PH-PLCd. On the following day, cells were washed with Ca 2+-containing buffer and incubated for 30 min at room temperature with the same buffer containing 10 mM MCD (Sigma-Aldrich/Merck) before staining with recombinant EGFP-D4H proteins for SDC microscopy.
For rescue experiments with mRuby-GRAMD1 constructs (Figure 7-figure supplement 2A,B,E, F,G,H), GRAMD1 TKO cells were transfected with iRFP-PH-PLCd together with the indicated mRuby-GRAMD1 constructs. On the following day, cells were stained with recombinant EGFP-D4H proteins and imaged under SDC microscopy.
For rapamycin-induced dimerization experiments ( Figure 7E,F), GRAMD1 TKO cells were transfected with PM-FRB-mCherry and one of the three miRFP-FKBP-GRAMD1b constructs (WT, 5P, or T469D). On the following day, 200 nM rapamycin (Sigma-Aldrich/Merck) was added to the culture media, which was further incubated for either 30 min or 60 min at 37˚C before staining with recombinant EGFP-D4H proteins for SDC microscopy.

Image analysis
All images were analyzed off-line using Fiji (http://fiji.sc/wiki/index.php/Fiji). Quantification of fluorescence signals was performed using Excel (Microsoft) and Prism 7 or 8 (GraphPad Software). All data are presented as mean ± SEM. In dot plots, each dot represents the value from a single cell with the black bar as the mean.
For time-lapse imaging via TIRF microscopy, changes in PM fluorescence over time were analyzed by manually selecting regions of interest covering the largest possible area of the cell foot-print. Mean fluorescence intensity values of the selected regions were obtained and normalized to the average fluorescence intensity before stimulation after background subtraction.
For analysis of the binding of recombinant EGFP-D4H proteins to the PM via SDC microscopy, line scan analysis was performed. A line of 5 mm in length was manually drawn around the PM (see dashed white lines in Figure 7A and Figure 7-figure supplement 1A), and EGFP fluorescence intensity along the manually drawn line was measured. The minimum fluorescence intensity along the line was subtracted from the maximum fluorescence intensity (corresponding to the PM-bound EGFP-D4H fluorescence) and plotted for quantification.

Generation of GRAMD1 knockout HeLa cell lines
The GRAMD1B, GRAMD1A and GRAMD1C genes were sequentially targeted to generate GRAMD1 triple knockout cells. The sequences of oligos and primers used are listed in Supplementary file 2.
For the generation of HeLa cells lacking GRAMD1b, control wild-type HeLa cells were transfected with a plasmid encoding spCas9 and the GRAMD1b-targeting guide RNA ( Figure 4A), followed by isolation of individual clones by dilution cloning. Two clones (#10 and #17) were further characterized by sequencing and immunoblotting (i.e. western blotting). These analyses revealed deletions and insertions within the guide RNA-binding sites, frame-shift and early termination in the open-reading frame of GRAMD1B gene, and the loss of GRAMD1b protein expression (Figure 4-figure supplement 1A,C). To generate GRAMD1a/1b double knockout (DKO) cell lines, a subclone of the GRAMD1b KO cell line #10 was transfected with a plasmid encoding spCas9 and the GRAMD1a-targeting guide RNA with ssDNA oligos containing stop codons and homology-arms ( Figure 4A). These cells were subjected to single cell sorting, and individually isolated clones [lines #38 and #40 (hereafter GRAMD1a/1b DKO #38 and GRAMD1a/1b DKO #40)] showed insertion of ssDNA within the guide RNA-targeted locus, resulting in the lack of GRAMD1a protein expression ( Figure 4B and To generate GRAMD1 triple knockout (TKO) cell lines, the GRAMD1a/1b DKO #40 cell line was transfected with two plasmids encoding spCas9 and each one of the two GRAMD1b-targeting guide RNAs ( Figure 4A). Two clones (GRAMD1a/1b/1c TKO #1 and GRAMD1a/1b/1c TKO #15) that showed large deletions in the exon 11 of GRAMD1C, as assessed by genomic PCR (Figure 4-figure supplement 1E), were isolated and knock-outs were confirmed by direct sequencing ( Figure 4C). In all figures and texts, TKO denotes GRAMD1a/1b/1c TKO #15 unless stated.

GRAMD1b knockout
The genomic sequence surrounding the exon 13, which encodes the amino-acid stretch in the StART-like domain of human GRAMD1b, was analyzed for potential CRISPR/Cas9 targets in silico using the Cas9 design target tool (http://crispr.mit.edu) . The GRAMD1B genomic sequence targeted by the predicted CRISPR gRNA is: TCGCTACACGCTCACCCGTGTGG (GRAMD1b-sgRNA).
HeLa cells were transiently transfected with the PX459-GRAMD1B_Back plasmid. 24 hr after transfection, cells were supplemented with growth medium containing puromycin (1.5 mg/mL) and incubated for 72 hr. Cells that were resistant to puromycin selection were then incubated with puromycin-free medium for 24 hr before harvesting for dilution cloning, and then assessed by genotyping PCR using the primer set, Genotyping_1B_F2 and Genotyping_1B_R1, to obtain GRAMD1b knockout cells.

GRAMD1a knockout
The genomic sequence surrounding the exon 13, which encodes the amino-acid stretch in the StART-like domain of human GRAMD1a, was analyzed in silico using the Cas9 design target tool (http://crispr.mit.edu) . The GRAMD1A genomic sequence targeted by the predicted CRISPR gRNA is: GGACTCCGAGGTGCTGACGCAGGG (GRAMD1a-sgRNA).
To knock-in the sequence with stop codons, ssDNA containing stop codons and homology-arms surrounding the guide RNA targeting site was designed. The ssDNA of the reverse complementary sequence was synthesized by IDT and used for the transfection with the PX459-GRAMD1A_V2_Front plasmid. The sequence of ssDNA was: GTGGGCAGTGTAGAAGTAGTCCTGGTAGGGGATGCCC TGCGGATCCCGGGCCCGCGGTACCGAATTCGAAGCTTGAGCTCGAGATCTActagttaatcaGTCAG-CACCTCGGAGTCCACCACACACCCGCCGGCCTGGG, where homology arms are indicated by the underline (ssDNA_StopKI_HR_GRAMD1a_V2).
Control HeLa cells and GRAMD1b knockout cell line #10 were transiently transfected with the PX459-GRAMD1A_V2_Front plasmid with ssDNA. 24 hr after transfection, cells were supplemented with growth medium containing puromycin (1.5 mg/mL) and incubated for 72 hr. Cells that were resistant to puromycin selection were then incubated with puromycin-free medium for 24 hr before harvesting for single-cell sorting, and individually isolated cell clones were assessed by genotyping PCR using the primer set, GRAMD1a_Genotyping_V2V3_F2 and GRAMD1a_Genoty-ping_V2V3_R2, to obtain GRAMD1a knockout and GRAMD1a/1b DKO cell lines.
GRAMD1a/1b DKO cell line #40 was transiently transfected with the two GRAMD1c CRISPR/Cas9 plasmids, PX459-GRAMD1c_sgRNA_#1 and PX459-GRAMD1c_sgRNA_#2. 24 hr after transfection, cells were supplemented with growth medium containing puromycin (1.5 mg/mL) and incubated for 72 hr. Cells that were resistant to puromycin selection were then incubated with puromycin-free medium for 24 hr before harvesting for single-cell sorting, and individually isolated clones were assessed by genotyping PCR using the primer set, GRAMD1c_Genotyping_F1 and GRAMD1c_Ge-notyping_R1, to obtain GRAMD1a/1b/1c triple knockout cell lines.

Sequencing of mutant alleles
For GRAMD1a and GRAMD1b knockout cells, sequencing of mutated alleles was carried out by cloning PCR products into the pCR4 Blunt-TOPO vector using the Zero Blunt TOPO PCR Cloning Kit for sequencing (Thermo Fisher Scientific). Biallelic insertions/deletions were confirmed by sequencing at least 10 individual colonies. The same primers were used as genotyping primers. For GRAMD1c knockout cells, sequencing of mutated alleles was carried out by direct-sequencing of the genomic PCR products. The same primers were used as genotyping primers.

Biochemical analyses Plasma membrane isolation and protein extraction
The procedure was modified from Cohen et al. (1977) and Saheki et al. (2016). Briefly, 2 g of Cytodex three microcarrier beads (Sigma-Aldrich/Merck) were reconstituted in 100 ml phosphate-buffered saline (PBS), autoclaved and coated by incubation with a poly-D-lysine solution overnight at 37C . Cells were added to the reconstituted beads in sterile PETG flasks (Thermo Fisher Scientific), allowed to attach to the beads for 4 hr with a gentle stirring every 30 min, and then further incubated overnight with continuous stirring on a rotating incubator at 37˚C with 5% CO 2 . Beads were subsequently collected by spontaneous sedimentation and incubated with 220 mM sucrose and 40 mM sodium acetate (pH 5.0) for 5 min at room temperature (Cohen et al., 1977). After the acid treatment, beads were collected, incubated with a hypotonic solution [10 mM Tris-HCl (pH 8.0)] and vortexed for 10 s. A 10 s sonication pulse was then applied to the beads with Vibra Cell VCX130 (Sonics and Materials, Inc) in the same solution. Beads were finally washed three times with the same solution and once with PBS for lipidomic analysis and for protein extraction with SDS lysis buffer [10 mM Tris-HCl, 150 mM NaCl, 2% SDS (pH 8.0)]. assays are shown in Supplementary file 3. The dried lipid films were hydrated with HK buffer [50 mM HEPES, 120 mM potassium acetate (pH 7.5)]. Liposomes were then formed by five freeze-thaw cycles (liquid N 2 and 37˚C water bath) followed by extrusion using Nanosizer with a pore size of 100 nm (T and T Scientific Corporation). All liposomes except those shown in Figure 3-figure supplement 1D were subjected to extrusion.

FRET-based DHE transfer assays
Buffer of the purified proteins was replaced with HK buffer prior to the FRET-based DHE transfer assay. Reactions were performed in 50 ml volumes. The final lipid concentration in the reaction was 1 mM, with donor and acceptor liposomes added at a 1:1 ratio (only acceptor liposomes contain 2.5% DNS-PE, see Supplementary file 3 for the lipid compositions). Reactions were initiated by the addition of protein to a final concentration of 0.5-2 mM in a 96-well plate (Corning). The fluorescence intensity of DNS-PE (i.e. FRET signals), resulting from FRET between DNS-PE and DHE (excited at 310 nm), was monitored at 525 nm every 15 s over 30 min at room temperature by using a Synergy H1 microplate reader (Biotek). The values of blank solution (buffer only) were subtracted from all the values from each time point, and data were presented by setting the first value to zero at t = 0.
In some experiments, data were expressed as the number of DHE molecules transferred using the calibration curve ( Figure 5-figure supplement 1B-E). For the generation of the calibration curve, FRET signals were measured for the liposomes containing 0%, 5% (1.25 nmole), 10% (2.5 nmole) or 15% (3.75 nmole) DHE and 2.5% DNS-PE (0.5 mM lipids in total: compositions of the liposomes can be found in Supplementary file 3). The mean of FRET signals at t = 0 from three replicates were plotted against the DHE mole number in liposomes ( Figure 5-figure supplement 1A). Then, the mole number of the transferred DHE from the donor to acceptor liposomes in in vitro DHE transfer assay was obtained using the following formula: y = 7649.6667 + 5946.61531x (derived from the linear fit of the calibration curve). To obtain x (the amount of transferred DHE in nmole shown in the y axis of

Liposome sedimentation assays
Heavy liposomes were prepared by hydrating 1.6 mM dried lipid films in HK buffer containing sucrose [50 mM HEPES (pH 7.5), 120 mM K-acetate and 0.75 M sucrose] and subjected to freezethaw cycles five times. Next, 200 ml of heavy liposomes were pelleted and washed with HK buffer without sucrose twice to remove unencapsulated sucrose. Pelleted heavy liposomes were resuspended in 200 ml HK buffer and incubated with 7.5-10 mg of the indicated proteins for 1 hr at room temperature. Unbound proteins (supernatant) were separated from liposome-bound proteins (pellet) by centrifugation at 21,000 x g for 1 hr at 25˚C. After centrifugation, the supernatant was removed, and pellets were re-suspended in 200 ml HK buffer. 20 ml samples were taken from both fractions and run on SDS-PAGE followed by colloidal blue staining. Quantification of the bands was performed using Fiji.

Molecular modeling
The modeled structure of GRAMD1b StART was obtained by submitting the primary sequence (residues 375-545) to the I-TASSER server, using the GRAMD1a StART structure (PDB: 6GQF) as the template.

Statistical analysis
No statistical method was used to predetermine sample size, and the experiments were not randomized for live-cell imaging. Sample size and information about replicates are described in the figure legends. The number of biological replicates for all cell-based experiments and the number of technical replicates for all other biochemical assays are shown as the number of independent experiments within the figure legends for each figure. Comparisons of data were carried out by the twotailed unpaired Student's t-test, Holm-Sidak's t-test or one-way ANOVA, followed by Tukey or Dunnett corrections for multiple comparisons as appropriate with Prism 7 or 8 (GraphPad software). Unless p<0.0001, exact P values are shown within the figure legends for each figure. p>0.05 was considered not significant.
Methodology, Designed and performed genetic manipulations, Designed imaging and biochemical studies; Alexander Triebl, Formal analysis, Investigation, Methodology, Performed lipidomics analysis; Dylan Hong Zheng Koh, Resources, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing-review and editing, Designed and performed genetic manipulations, Designed and performed imaging and biochemical studies, Performed the isolation of plasma membrane sheets; Fan-Yan Wei, Resources, Writing-review and editing; Kazuhito Tomizawa, Resources, Supervision; Federico Tesio Torta, Formal analysis, Supervision, Investigation, Methodology, Performed lipidomics analysis; Markus R Wenk, Resources, Supervision, Funding acquisition, Methodology, Performed lipidomics analysis; Yasunori Saheki, Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing-original draft, Project administration, Writing-review and editing, Designed all experiments, Supervised the project, Performed the isolation of plasma membrane sheets  Table 2. Lipid compositions of liposomes used for lipid transfer assays. Moles % of lipids used for the acceptor and donor liposomes in FRET-based lipid transfer experiments are described.
The following previously published dataset was used: