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.

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 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 StART-like domains have identified a hydrophobic cavity that can bind sterol (Gatta et al., 2018; Horenkamp et al., 2018; Jentsch et al., 2018; Sandhu et al., 2018; Tong et al., 2018). The StART-like domains of GRAMD1s bind and transport sterols in vitro (Horenkamp et al., 2018; 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 Ca²⁺ homeostasis (Besprozvannaya et al., 2018), whereas GRAMD1b facilitates the transport of HDL-derived cholesterol to the ER in the adrenal glands of mice (Sandhu et al., 2018). 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 sphingomyelin-sequestered 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.

We have demonstrated that the evolutionarily conserved family of ER-anchored GRAMD1s 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 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 non-vesicular 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.

Figure 8

Download asset Open asset

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 (He et al., 2018; 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)P₂ in addition to cholesterol (Horenkamp et al., 2018). 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.

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 3, 4, 5, 6, 7, 3-S-1, 3-S-2, 4-S-2, 4-S-3, 5-S-1, 5-S-2, 6-S-1, 6-S-2, 7-S-1, and 7-S-2.

1. 1. Fairall L
   2. Gurnett JE
   3. Vashi D
   4. Sandhu J
   5. Tontonoz P
   6. Schwabe JWR

   (2018) Protein Data Bank

   ID 6GQF. The structure of mouse AsterA (GramD1a) with 25-hydroxy cholesterol.

   https://www.rcsb.org/structure/6GQF

1. 1. Acton S
   2. Rigotti A
   3. Landschulz KT
   4. Xu S
   5. Hobbs HH
   6. Krieger M

   (1996) Identification of scavenger receptor SR-BI as a high density lipoprotein receptor

   Science 271:518–520.

   https://doi.org/10.1126/science.271.5248.518

   • PubMed
   • Google Scholar
2. 1. AhYoung AP
   2. Jiang J
   3. Zhang J
   4. Khoi Dang X
   5. Loo JA
   6. Zhou ZH
   7. Egea PF

   (2015) Conserved SMP domains of the ERMES complex bind phospholipids and mediate tether assembly

   PNAS 112:E3179–E3188.

   https://doi.org/10.1073/pnas.1422363112

   • Google Scholar
3. 1. Alpy F
   2. Rousseau A
   3. Schwab Y
   4. Legueux F
   5. Stoll I
   6. Wendling C
   7. Spiegelhalter C
   8. Kessler P
   9. Mathelin C
   10. Rio MC
   11. Levine TP
   12. Tomasetto C

   (2013) STARD3 or STARD3NL and VAP form a novel molecular tether between late endosomes and the ER

   Journal of Cell Science 126:5500–5512.

   https://doi.org/10.1242/jcs.139295

   • PubMed
   • Google Scholar
4. 1. Alpy F
   2. Tomasetto C

   (2014) START ships lipids across interorganelle space

   Biochimie 96:85–95.

   https://doi.org/10.1016/j.biochi.2013.09.015

   • Google Scholar
5. 1. Antonny B
   2. Bigay J
   3. Mesmin B

   (2018) The Oxysterol-Binding protein cycle: burning off PI(4)P to transport cholesterol

   Annual Review of Biochemistry 87:809–837.

   https://doi.org/10.1146/annurev-biochem-061516-044924

   • PubMed
   • Google Scholar
6. 1. Baumann NA
   2. Sullivan DP
   3. Ohvo-Rekilä H
   4. Simonot C
   5. Pottekat A
   6. Klaassen Z
   7. Beh CT
   8. Menon AK

   (2005) Transport of newly synthesized sterol to the sterol-enriched plasma membrane occurs via nonvesicular equilibration

   Biochemistry 44:5816–5826.

   https://doi.org/10.1021/bi048296z

   • PubMed
   • Google Scholar
7. 1. Begley MJ
   2. Taylor GS
   3. Kim SA
   4. Veine DM
   5. Dixon JE
   6. Stuckey JA

   (2003) Crystal structure of a phosphoinositide phosphatase, MTMR2: insights into myotubular myopathy and Charcot-Marie-Tooth syndrome

   Molecular Cell 12:1391–1402.

   https://doi.org/10.1016/s1097-2765(03)00486-6

   • PubMed
   • Google Scholar
8. 1. Benedetti L
   2. Barentine AES
   3. Messa M
   4. Wheeler H
   5. Bewersdorf J
   6. De Camilli P

   (2018) Light-activated protein interaction with high spatial subcellular confinement

   PNAS 115:E2238–E2245.

   https://doi.org/10.1073/pnas.1713845115

   • PubMed
   • Google Scholar
9. 1. Besprozvannaya M
   2. Dickson E
   3. Li H
   4. Ginburg KS
   5. Bers DM
   6. Auwerx J
   7. Nunnari J

   (2018) GRAM domain proteins specialize functionally distinct ER-PM contact sites in human cells

   eLife 7:e31019.

   https://doi.org/10.7554/eLife.31019

   • PubMed
   • Google Scholar
10. 1. Bligh EG
    2. Dyer WJ

    (1959) A rapid method of total lipid extraction and purification

    Canadian Journal of Biochemistry and Physiology 37:911–917.

    https://doi.org/10.1139/y59-099

    • PubMed
    • Google Scholar
11. 1. Brown AJ
    2. Sun L
    3. Feramisco JD
    4. Brown MS
    5. Goldstein JL

    (2002) Cholesterol addition to ER membranes alters conformation of SCAP, the SREBP escort protein that regulates cholesterol metabolism

    Molecular Cell 10:237–245.

    https://doi.org/10.1016/S1097-2765(02)00591-9

    • PubMed
    • Google Scholar
12. 1. Brown MS
    2. Radhakrishnan A
    3. Goldstein JL

    (2018) Retrospective on cholesterol homeostasis: the central role of scap

    Annual Review of Biochemistry 87:783–807.

    https://doi.org/10.1146/annurev-biochem-062917-011852

    • PubMed
    • Google Scholar
13. 1. Chakrabarti RS
    2. Ingham SA
    3. Kozlitina J
    4. Gay A
    5. Cohen JC
    6. Radhakrishnan A
    7. Hobbs HH

    (2017) Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin

    eLife 6:e23355.

    https://doi.org/10.7554/eLife.23355

    • Google Scholar
14. 1. Charman M
    2. Colbourne TR
    3. Pietrangelo A
    4. Kreplak L
    5. Ridgway ND

    (2014) Oxysterol-binding protein (OSBP)-related protein 4 (ORP4) is essential for cell proliferation and survival

    Journal of Biological Chemistry 289:15705–15717.

    https://doi.org/10.1074/jbc.M114.571216

    • PubMed
    • Google Scholar
15. 1. Chung J
    2. Torta F
    3. Masai K
    4. Lucast L
    5. Czapla H
    6. Tanner LB
    7. Narayanaswamy P
    8. Wenk MR
    9. Nakatsu F
    10. De Camilli P

    (2015) INTRACELLULAR TRANSPORT. PI4P/phosphatidylserine countertransport at ORP5- and ORP8-mediated ER-plasma membrane contacts

    Science 349:428–432.

    https://doi.org/10.1126/science.aab1370

    • PubMed
    • Google Scholar
16. 1. Cohen CM
    2. Kalish DI
    3. Jacobson BS
    4. Branton D

    (1977) Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells

    The Journal of Cell Biology 75:119–134.

    https://doi.org/10.1083/jcb.75.1.119

    • PubMed
    • Google Scholar
17. 1. Das A
    2. Goldstein JL
    3. Anderson DD
    4. Brown MS
    5. Radhakrishnan A

    (2013) Use of mutant 125I-perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells

    PNAS 110:10580–10585.

    https://doi.org/10.1073/pnas.1309273110

    • PubMed
    • Google Scholar
18. 1. Das A
    2. Brown MS
    3. Anderson DD
    4. Goldstein JL
    5. Radhakrishnan A

    (2014) Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis

    eLife 3:e02882.

    https://doi.org/10.7554/eLife.02882

    • Google Scholar
19. 1. de Duve C

    (1971) Tissue fraction-past and present

    The Journal of Cell Biology 50:20–55.

    https://doi.org/10.1083/jcb.50.1.20d

    • PubMed
    • Google Scholar
20. 1. Drin G

    (2014) Topological regulation of lipid balance in cells

    Annual Review of Biochemistry 83:51–77.

    https://doi.org/10.1146/annurev-biochem-060713-035307

    • PubMed
    • Google Scholar
21. 1. Elbaz Y
    2. Schuldiner M

    (2011) Staying in touch: the molecular era of organelle contact sites

    Trends in Biochemical Sciences 36:616–623.

    https://doi.org/10.1016/j.tibs.2011.08.004

    • PubMed
    • Google Scholar
22. 1. Endapally S
    2. Frias D
    3. Grzemska M
    4. Gay A
    5. Tomchick DR
    6. Radhakrishnan A

    (2019) Molecular discrimination between two conformations of sphingomyelin in plasma membranes

    Cell 176:1040–1053.

    https://doi.org/10.1016/j.cell.2018.12.042

    • Google Scholar
23. 1. Finean JB

    (1953) Phospholipid-cholesterol complex in the structure of myelin

    Experientia 9:17–19.

    https://doi.org/10.1007/BF02147697

    • PubMed
    • Google Scholar
24. 1. Gatta AT
    2. Wong LH
    3. Sere YY
    4. Calderón-Noreña DM
    5. Cockcroft S
    6. Menon AK
    7. Levine TP

    (2015) A new family of StART domain proteins at membrane contact sites has a role in ER-PM sterol transport

    eLife 4:e07253.

    https://doi.org/10.7554/eLife.07253

    • Google Scholar
25. 1. Gatta AT
    2. Sauerwein AC
    3. Zhuravleva A
    4. Levine TP
    5. Matthews S

    (2018) Structural insights into a StART-like domain in Lam4 and its interaction with sterol ligands

    Biochemical and Biophysical Research Communications 495:2270–2274.

    https://doi.org/10.1016/j.bbrc.2017.12.109

    • PubMed
    • Google Scholar
26. 1. Gautier R
    2. Douguet D
    3. Antonny B
    4. Drin G

    (2008) HELIQUEST: a web server to screen sequences with specific alpha-helical properties

    Bioinformatics 24:2101–2102.

    https://doi.org/10.1093/bioinformatics/btn392

    • PubMed
    • Google Scholar
27. 1. Gay A
    2. Rye D
    3. Radhakrishnan A

    (2015) Switch-like responses of two cholesterol sensors do not require protein oligomerization in membranes

    Biophysical Journal 108:1459–1469.

    https://doi.org/10.1016/j.bpj.2015.02.008

    • PubMed
    • Google Scholar
28. 1. Giordano F
    2. Saheki Y
    3. Idevall-Hagren O
    4. Colombo SF
    5. Pirruccello M
    6. Milosevic I
    7. Gracheva EO
    8. Bagriantsev SN
    9. Borgese N
    10. De Camilli P

    (2013) PI(4,5)P(2)-dependent and ca(2+)-regulated ER-PM interactions mediated by the extended synaptotagmins

    Cell 153:1494–1509.

    https://doi.org/10.1016/j.cell.2013.05.026

    • PubMed
    • Google Scholar
29. 1. Goldstein JL
    2. Brown MS

    (1990) Regulation of the mevalonate pathway

    Nature 343:425–430.

    https://doi.org/10.1038/343425a0

    • PubMed
    • Google Scholar
30. 1. Goldstein JL
    2. Brown MS

    (2015) A century of cholesterol and coronaries: from plaques to genes to statins

    Cell 161:161–172.

    https://doi.org/10.1016/j.cell.2015.01.036

    • PubMed
    • Google Scholar
31. 1. Guan Y
    2. Zhu Q
    3. Huang D
    4. Zhao S
    5. Jan Lo L
    6. Peng J

    (2015) An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide

    Scientific Reports 5:13370.

    https://doi.org/10.1038/srep13370

    • PubMed
    • Google Scholar
32. 1. Hao M
    2. Lin SX
    3. Karylowski OJ
    4. Wüstner D
    5. McGraw TE
    6. Maxfield FR

    (2002) Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle

    The Journal of Biological Chemistry 277:609–617.

    https://doi.org/10.1074/jbc.M108861200

    • PubMed
    • Google Scholar
33. 1. He C
    2. Hu X
    3. Weston TA
    4. Jung RS
    5. Sandhu J
    6. Huang S
    7. Heizer P
    8. Kim J
    9. Ellison R
    10. Xu J
    11. Kilburn M
    12. Bensinger SJ
    13. Riezman H
    14. Tontonoz P
    15. Fong LG
    16. Jiang H
    17. Young SG

    (2018) Macrophages release plasma membrane-derived particles rich in accessible cholesterol

    PNAS 115:E8499–E8508.

    https://doi.org/10.1073/pnas.1810724115

    • PubMed
    • Google Scholar
34. 1. Heino S
    2. Lusa S
    3. Somerharju P
    4. Ehnholm C
    5. Olkkonen VM
    6. Ikonen E

    (2000) Dissecting the role of the golgi complex and lipid rafts in Biosynthetic transport of cholesterol to the cell surface

    PNAS 97:8375–8380.

    https://doi.org/10.1073/pnas.140218797

    • PubMed
    • Google Scholar
35. 1. Holthuis JC
    2. Menon AK

    (2014) Lipid landscapes and pipelines in membrane homeostasis

    Nature 510:48–57.

    https://doi.org/10.1038/nature13474

    • PubMed
    • Google Scholar
36. 1. Horenkamp FA
    2. Valverde DP
    3. Nunnari J
    4. Reinisch KM

    (2018) Molecular basis for sterol transport by StART-like lipid transfer domains

    The EMBO Journal 37:e98002.

    https://doi.org/10.15252/embj.201798002

    • PubMed
    • Google Scholar
37. 1. Hoyer MJ
    2. Chitwood PJ
    3. Ebmeier CC
    4. Striepen JF
    5. Qi RZ
    6. Old WM
    7. Voeltz GK

    (2018) A novel class of ER membrane proteins regulates ER-Associated endosome fission

    Cell 175:254–265.

    https://doi.org/10.1016/j.cell.2018.08.030

    • PubMed
    • Google Scholar
38. 1. Hsu PD
    2. Scott DA
    3. Weinstein JA
    4. Ran FA
    5. Konermann S
    6. Agarwala V
    7. Li Y
    8. Fine EJ
    9. Wu X
    10. Shalem O
    11. Cradick TJ
    12. Marraffini LA
    13. Bao G
    14. Zhang F

    (2013) DNA targeting specificity of RNA-guided Cas9 nucleases

    Nature Biotechnology 31:827–832.

    https://doi.org/10.1038/nbt.2647

    • PubMed
    • Google Scholar
39. 1. Hu X
    2. Weston TA
    3. He C
    4. Jung RS
    5. Heizer PJ
    6. Young BD
    7. Tu Y
    8. Tontonoz P
    9. Wohlschlegel JA
    10. Jiang H
    11. Young SG
    12. Fong LG

    (2019) Release of cholesterol-rich particles from the macrophage plasma membrane during movement of filopodia and lamellipodia

    eLife 8:e50231.

    https://doi.org/10.7554/eLife.50231

    • PubMed
    • Google Scholar
40. 1. Iaea DB
    2. Mao S
    3. Lund FW
    4. Maxfield FR

    (2017) Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane

    Molecular Biology of the Cell 28:1111–1122.

    https://doi.org/10.1091/mbc.e16-07-0499

    • PubMed
    • Google Scholar
41. 1. Ikonen E

    (2008) Cellular cholesterol trafficking and compartmentalization

    Nature Reviews Molecular Cell Biology 9:125–138.

    https://doi.org/10.1038/nrm2336

    • PubMed
    • Google Scholar
42. 1. Infante RE
    2. Radhakrishnan A

    (2017) Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol

    eLife 6:e25466.

    https://doi.org/10.7554/eLife.25466

    • PubMed
    • Google Scholar
43. 1. Jentsch JA
    2. Kiburu I
    3. Pandey K
    4. Timme M
    5. Ramlall T
    6. Levkau B
    7. Wu J
    8. Eliezer D
    9. Boudker O
    10. Menon AK

    (2018) Structural basis of sterol binding and transport by a yeast StARkin domain

    Journal of Biological Chemistry 293:5522–5531.

    https://doi.org/10.1074/jbc.RA118.001881

    • PubMed
    • Google Scholar
44. 1. Jeyasimman D
    2. Saheki Y

    (2019) SMP domain proteins in membrane lipid dynamics

    Biochimica Et Biophysica Acta. Molecular and Cell Biology of Lipids 1865:S1388-1981.

    https://doi.org/10.1016/j.bbalip.2019.04.007

    • Google Scholar
45. 1. Johnson BB
    2. Moe PC
    3. Wang D
    4. Rossi K
    5. Trigatti BL
    6. Heuck AP

    (2012) Modifications in perfringolysin O domain 4 alter the cholesterol concentration threshold required for binding

    Biochemistry 51:3373–3382.

    https://doi.org/10.1021/bi3003132

    • PubMed
    • Google Scholar
46. 1. Johnson KA
    2. Endapally S
    3. Vazquez DC
    4. Infante RE
    5. Radhakrishnan A

    (2019) Ostreolysin A and anthrolysin O use different mechanisms to control movement of cholesterol from the plasma membrane to the endoplasmic reticulum

    Journal of Biological Chemistry jbc.RA119.010393.

    https://doi.org/10.1074/jbc.RA119.010393

    • PubMed
    • Google Scholar
47. 1. Kawano S
    2. Tamura Y
    3. Kojima R
    4. Bala S
    5. Asai E
    6. Michel AH
    7. Kornmann B
    8. Riezman I
    9. Riezman H
    10. Sakae Y
    11. Okamoto Y
    12. Endo T

    (2018) Structure-function insights into direct lipid transfer between membranes by Mmm1-Mdm12 of ERMES

    The Journal of Cell Biology 217:959–974.

    https://doi.org/10.1083/jcb.201704119

    • PubMed
    • Google Scholar
48. 1. Kelley LA
    2. Mezulis S
    3. Yates CM
    4. Wass MN
    5. Sternberg MJ

    (2015) The Phyre2 web portal for protein modeling, prediction and analysis

    Nature Protocols 10:845–858.

    https://doi.org/10.1038/nprot.2015.053

    • PubMed
    • Google Scholar
49. 1. Kumar N
    2. Leonzino M
    3. Hancock-Cerutti W
    4. Horenkamp FA
    5. Li P
    6. Lees JA
    7. Wheeler H
    8. Reinisch KM
    9. De Camilli P

    (2018) VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites

    The Journal of Cell Biology 217:3625–3639.

    https://doi.org/10.1083/jcb.201807019

    • PubMed
    • Google Scholar
50. 1. Lahiri S
    2. Toulmay A
    3. Prinz WA

    (2015) Membrane contact sites, gateways for lipid homeostasis

    Current Opinion in Cell Biology 33:82–87.

    https://doi.org/10.1016/j.ceb.2014.12.004

    • PubMed
    • Google Scholar
51. 1. Lange Y
    2. Swaisgood MH
    3. Ramos BV
    4. Steck TL

    (1989)

    Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts

    The Journal of Biological Chemistry 264:3786–3793.

    • PubMed
    • Google Scholar
52. 1. Lange Y
    2. Ye J
    3. Steck TL

    (2004) How cholesterol homeostasis is regulated by plasma membrane cholesterol in excess of phospholipids

    PNAS 101:11664–11667.

    https://doi.org/10.1073/pnas.0404766101

    • PubMed
    • Google Scholar
53. 1. Lange Y
    2. Tabei SM
    3. Ye J
    4. Steck TL

    (2013) Stability and stoichiometry of bilayer phospholipid-cholesterol complexes: relationship to cellular sterol distribution and homeostasis

    Biochemistry 52:6950–6959.

    https://doi.org/10.1021/bi400862q

    • PubMed
    • Google Scholar
54. 1. Lange Y
    2. Ye J
    3. Steck TL

    (2014) Essentially all excess fibroblast cholesterol moves from plasma membranes to intracellular compartments

    PLOS ONE 9:e98482.

    https://doi.org/10.1371/journal.pone.0098482

    • Google Scholar
55. 1. Lange Y
    2. Steck TL

    (1997) Quantitation of the pool of cholesterol associated with acyl-CoA:cholesterol acyltransferase in human fibroblasts

    Journal of Biological Chemistry 272:13103–13108.

    https://doi.org/10.1074/jbc.272.20.13103

    • PubMed
    • Google Scholar
56. 1. Laraia L
    2. Friese A
    3. Corkery DP
    4. Konstantinidis G
    5. Erwin N
    6. Hofer W
    7. Karatas H
    8. Klewer L
    9. Brockmeyer A
    10. Metz M
    11. Schölermann B
    12. Dwivedi M
    13. Li L
    14. Rios-Munoz P
    15. Köhn M
    16. Winter R
    17. Vetter IR
    18. Ziegler S
    19. Janning P
    20. Wu YW
    21. Waldmann H

    (2019) The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis

    Nature Chemical Biology 15:710–720.

    https://doi.org/10.1038/s41589-019-0307-5

    • PubMed
    • Google Scholar
57. 1. Lev S

    (2012) Nonvesicular lipid transfer from the endoplasmic reticulum

    Cold Spring Harbor Perspectives in Biology 4:a013300.

    https://doi.org/10.1101/cshperspect.a013300

    • PubMed
    • Google Scholar
58. 1. Leventis R
    2. Silvius JR

    (2001) Use of cyclodextrins to monitor transbilayer movement and differential lipid affinities of cholesterol

    Biophysical Journal 81:2257–2267.

    https://doi.org/10.1016/S0006-3495(01)75873-0

    • PubMed
    • Google Scholar
59. 1. Lin D
    2. Sugawara T
    3. Strauss JF
    4. Clark BJ
    5. Stocco DM
    6. Saenger P
    7. Rogol A
    8. Miller WL

    (1995) Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis

    Science 267:1828–1831.

    https://doi.org/10.1126/science.7892608

    • PubMed
    • Google Scholar
60. 1. Luo J
    2. Jiang L-Y
    3. Yang H
    4. Song B-L

    (2019) Intracellular cholesterol transport by sterol transfer proteins at membrane contact sites

    Trends in Biochemical Sciences 44:273–292.

    https://doi.org/10.1016/j.tibs.2018.10.001

    • Google Scholar
61. 1. Lyons ET
    2. Hemken RD
    3. Button FS

    (1981)

    Overwintering of larvae of the cattle lungworm (Dictyocaulus viviparus) on pasture in Kentucky

    Journal of the American Veterinary Medical Association 179:456–457.

    • PubMed
    • Google Scholar
62. 1. Maekawa M
    2. Fairn GD

    (2015) Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol

    Journal of Cell Science 128:1422–1433.

    https://doi.org/10.1242/jcs.164715

    • PubMed
    • Google Scholar
63. 1. McConnell HM
    2. Radhakrishnan A

    (2003) Condensed complexes of cholesterol and phospholipids

    Biochimica Et Biophysica Acta (BBA) - Biomembranes 1610:159–173.

    https://doi.org/10.1016/S0005-2736(03)00015-4

    • Google Scholar
64. 1. Mesmin B
    2. Pipalia NH
    3. Lund FW
    4. Ramlall TF
    5. Sokolov A
    6. Eliezer D
    7. Maxfield FR

    (2011) STARD4 abundance regulates sterol transport and sensing

    Molecular Biology of the Cell 22:4004–4015.

    https://doi.org/10.1091/mbc.e11-04-0372

    • PubMed
    • Google Scholar
65. 1. Murley A
    2. Sarsam RD
    3. Toulmay A
    4. Yamada J
    5. Prinz WA
    6. Nunnari J

    (2015) Ltc1 is an ER-localized sterol transporter and a component of ER-mitochondria and ER-vacuole contacts

    The Journal of Cell Biology 209:539–548.

    https://doi.org/10.1083/jcb.201502033

    • PubMed
    • Google Scholar
66. 1. Murley A
    2. Yamada J
    3. Niles BJ
    4. Toulmay A
    5. Prinz WA
    6. Powers T
    7. Nunnari J

    (2017) Sterol transporters at membrane contact sites regulate TORC1 and TORC2 signaling

    The Journal of Cell Biology jcb.201610032.

    https://doi.org/10.1083/jcb.201610032

    • Google Scholar
67. 1. Muthuswamy SK
    2. Gilman M
    3. Brugge JS

    (1999) Controlled dimerization of ErbB receptors provides evidence for differential signaling by Homo- and heterodimers

    Molecular and Cellular Biology 19:6845–6857.

    https://doi.org/10.1128/MCB.19.10.6845

    • PubMed
    • Google Scholar
68. 1. Ngo M
    2. Ridgway ND

    (2009) Oxysterol binding protein-related protein 9 (ORP9) is a cholesterol transfer protein that regulates golgi structure and function

    Molecular Biology of the Cell 20:1388–1399.

    https://doi.org/10.1091/mbc.e08-09-0905

    • PubMed
    • Google Scholar
69. 1. Nishimura T
    2. Stefan CJ

    (2019) Specialized ER membrane domains for lipid metabolism and transport

    Biochimica Et Biophysica Acta. Molecular and Cell Biology of Lipids 1865:S1388-1981.

    https://doi.org/10.1016/j.bbalip.2019.07.001

    • Google Scholar
70. 1. Ohvo-Rekilä H
    2. Ramstedt B
    3. Leppimäki P
    4. Slotte JP

    (2002) Cholesterol interactions with phospholipids in membranes

    Progress in Lipid Research 41:66–97.

    https://doi.org/10.1016/S0163-7827(01)00020-0

    • PubMed
    • Google Scholar
71. 1. Petrungaro C
    2. Kornmann B

    (2019) Lipid exchange at ER-mitochondria contact sites: a puzzle falling into place with quite a few pieces missing

    Current Opinion in Cell Biology 57:71–76.

    https://doi.org/10.1016/j.ceb.2018.11.005

    • PubMed
    • Google Scholar
72. 1. Phillips MC

    (2014) Molecular mechanisms of cellular cholesterol efflux

    Journal of Biological Chemistry 289:24020–24029.

    https://doi.org/10.1074/jbc.R114.583658

    • Google Scholar
73. 1. Phillips MJ
    2. Voeltz GK

    (2016) Structure and function of ER membrane contact sites with other organelles

    Nature Reviews Molecular Cell Biology 17:69–82.

    https://doi.org/10.1038/nrm.2015.8

    • PubMed
    • Google Scholar
74. 1. Radhakrishnan A
    2. McConnell HM

    (2000) Chemical activity of cholesterol in membranes

    Biochemistry 39:8119–8124.

    https://doi.org/10.1021/bi0005097

    • PubMed
    • Google Scholar
75. 1. Ran FA
    2. Hsu PD
    3. Wright J
    4. Agarwala V
    5. Scott DA
    6. Zhang F

    (2013) Genome engineering using the CRISPR-Cas9 system

    Nature Protocols 8:2281–2308.

    https://doi.org/10.1038/nprot.2013.143

    • Google Scholar
76. 1. Ray TK
    2. Skipski VP
    3. Barclay M
    4. Essner E
    5. Archibald FM

    (1969)

    Lipid composition of rat liver plasma membranes

    The Journal of Biological Chemistry 244:5528–5536.

    • PubMed
    • Google Scholar
77. 1. Roth S
    2. Heintzmann R

    (2016) Optical photon reassignment with increased axial resolution by structured illumination

    Methods and Applications in Fluorescence 4:045005.

    https://doi.org/10.1088/2050-6120/4/4/045005

    • PubMed
    • Google Scholar
78. 1. Saheki Y
    2. Bian X
    3. Schauder CM
    4. Sawaki Y
    5. Surma MA
    6. Klose C
    7. Pincet F
    8. Reinisch KM
    9. De Camilli P

    (2016) Control of plasma membrane lipid homeostasis by the extended synaptotagmins

    Nature Cell Biology 18:504–515.

    https://doi.org/10.1038/ncb3339

    • PubMed
    • Google Scholar
79. 1. Saheki Y
    2. De Camilli P

    (2017a) Endoplasmic Reticulum-Plasma membrane contact sites

    Annual Review of Biochemistry 86:659–684.

    https://doi.org/10.1146/annurev-biochem-061516-044932

    • PubMed
    • Google Scholar
80. 1. Saheki Y
    2. De Camilli P

    (2017b) The Extended-Synaptotagmins

    Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1864:1490–1493.

    https://doi.org/10.1016/j.bbamcr.2017.03.013

    • Google Scholar
81. 1. Sales S
    2. Knittelfelder O
    3. Shevchenko A

    (2017) Lipidomics of human blood plasma by High-Resolution shotgun mass spectrometry

    Methods in Molecular Biology 1619:203–212.

    https://doi.org/10.1007/978-1-4939-7057-5_16

    • PubMed
    • Google Scholar
82. 1. Sandhu J
    2. Li S
    3. Fairall L
    4. Pfisterer SG
    5. Gurnett JE
    6. Xiao X
    7. Weston TA
    8. Vashi D
    9. Ferrari A
    10. Orozco JL
    11. Hartman CL
    12. Strugatsky D
    13. Lee SD
    14. He C
    15. Hong C
    16. Jiang H
    17. Bentolila LA
    18. Gatta AT
    19. Levine TP
    20. Ferng A
    21. Lee R
    22. Ford DA
    23. Young SG
    24. Ikonen E
    25. Schwabe JWR
    26. Tontonoz P

    (2018) Aster proteins facilitate nonvesicular plasma membrane to ER cholesterol transport in mammalian cells

    Cell 175:514–529.

    https://doi.org/10.1016/j.cell.2018.08.033

    • Google Scholar
83. 1. Scheek S
    2. Brown MS
    3. Goldstein JL

    (1997) Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1

    PNAS 94:11179–11183.

    https://doi.org/10.1073/pnas.94.21.11179

    • PubMed
    • Google Scholar
84. 1. Segrest JP
    2. De Loof H
    3. Dohlman JG
    4. Brouillette CG
    5. Anantharamaiah GM

    (1990) Amphipathic Helix motif: classes and properties

    Proteins: Structure, Function, and Genetics 8:103–117.

    https://doi.org/10.1002/prot.340080202

    • PubMed
    • Google Scholar
85. 1. Shimada Y
    2. Maruya M
    3. Iwashita S
    4. Ohno-Iwashita Y

    (2002) The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains

    European Journal of Biochemistry 269:6195–6203.

    https://doi.org/10.1046/j.1432-1033.2002.03338.x

    • PubMed
    • Google Scholar
86. 1. Slotte JP

    (1992) Enzyme-catalyzed oxidation of cholesterol in mixed phospholipid monolayers reveals the stoichiometry at which free cholesterol clusters disappear

    Biochemistry 31:5472–5477.

    https://doi.org/10.1021/bi00139a008

    • PubMed
    • Google Scholar
87. 1. Slotte JP
    2. Bierman EL

    (1988) Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts

    Biochemical Journal 250:653–658.

    https://doi.org/10.1042/bj2500653

    • PubMed
    • Google Scholar
88. 1. Soccio RE
    2. Adams RM
    3. Romanowski MJ
    4. Sehayek E
    5. Burley SK
    6. Breslow JL

    (2002) The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transfer protein with two closely related homologues, StarD5 and StarD6

    PNAS 99:6943–6948.

    https://doi.org/10.1073/pnas.052143799

    • PubMed
    • Google Scholar
89. 1. Sokolov A
    2. Radhakrishnan A

    (2010) Accessibility of cholesterol in endoplasmic reticulum membranes and activation of SREBP-2 switch abruptly at a common cholesterol threshold

    Journal of Biological Chemistry 285:29480–29490.

    https://doi.org/10.1074/jbc.M110.148254

    • PubMed
    • Google Scholar
90. 1. Steck TL
    2. Lange Y

    (2018) Transverse distribution of plasma membrane bilayer cholesterol: picking sides

    Traffic 19:750–760.

    https://doi.org/10.1111/tra.12586

    • PubMed
    • Google Scholar
91. 1. Stocco DM

    (2001) StAR protein and the regulation of steroid hormone biosynthesis

    Annual Review of Physiology 63:193–213.

    https://doi.org/10.1146/annurev.physiol.63.1.193

    • PubMed
    • Google Scholar
92. 1. Surma MA
    2. Herzog R
    3. Vasilj A
    4. Klose C
    5. Christinat N
    6. Morin-Rivron D
    7. Simons K
    8. Masoodi M
    9. Sampaio JL

    (2015) An automated shotgun lipidomics platform for high throughput, comprehensive, and quantitative analysis of blood plasma intact lipids

    European Journal of Lipid Science and Technology 117:1540–1549.

    https://doi.org/10.1002/ejlt.201500145

    • PubMed
    • Google Scholar
93. 1. Tong J
    2. Manik MK
    3. Im YJ

    (2018) Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites

    PNAS 115:E856–E865.

    https://doi.org/10.1073/pnas.1719709115

    • PubMed
    • Google Scholar
94. 1. Urbani L
    2. Simoni RD

    (1990)

    Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane

    The Journal of Biological Chemistry 265:1919–1923.

    • PubMed
    • Google Scholar
95. 1. van Meer G
    2. Voelker DR
    3. Feigenson GW

    (2008) Membrane lipids: where they are and how they behave

    Nature Reviews Molecular Cell Biology 9:112–124.

    https://doi.org/10.1038/nrm2330

    • PubMed
    • Google Scholar
96. 1. Vance JE

    (2015) Phospholipid synthesis and transport in mammalian cells

    Traffic 16:1–18.

    https://doi.org/10.1111/tra.12230

    • PubMed
    • Google Scholar
97. 1. Wang H
    2. Ma Q
    3. Qi Y
    4. Dong J
    5. Du X
    6. Rae J
    7. Wang J
    8. Wu WF
    9. Brown AJ
    10. Parton RG
    11. Wu JW
    12. Yang H

    (2019) ORP₂ delivers cholesterol to the plasma membrane in exchange for phosphatidylinositol 4, 5-Bisphosphate (PI(4,5)P₂)

    Molecular Cell 73:458–473.

    https://doi.org/10.1016/j.molcel.2018.11.014

    • PubMed
    • Google Scholar
98. 1. Wilhelm LP
    2. Wendling C
    3. Védie B
    4. Kobayashi T
    5. Chenard MP
    6. Tomasetto C
    7. Drin G
    8. Alpy F

    (2017) STARD3 mediates endoplasmic reticulum-to-endosome cholesterol transport at membrane contact sites

    The EMBO Journal 36:1412–1433.

    https://doi.org/10.15252/embj.201695917

    • PubMed
    • Google Scholar
99. 1. Wong LH
    2. Gatta AT
    3. Levine TP

    (2018) Lipid transfer proteins: the lipid commute via shuttles, bridges and tubes

    Nature Reviews. Molecular Cell Biology 20:85-101.

    https://doi.org/10.1038/s41580-018-0071-5

    • Google Scholar
100. 1. Wu H
     2. Carvalho P
     3. Voeltz GK

     (2018) Here, there, and everywhere: the importance of ER membrane contact sites

     Science 361:eaan5835.

     https://doi.org/10.1126/science.aan5835

     • PubMed
     • Google Scholar
101. 1. Yeung T
     2. Gilbert GE
     3. Shi J
     4. Silvius J
     5. Kapus A
     6. Grinstein S

     (2008) Membrane phosphatidylserine regulates surface charge and protein localization

     Science 319:210–213.

     https://doi.org/10.1126/science.1152066

     • PubMed
     • Google Scholar
102. 1. York AG
     2. Parekh SH
     3. Dalle Nogare D
     4. Fischer RS
     5. Temprine K
     6. Mione M
     7. Chitnis AB
     8. Combs CA
     9. Shroff H

     (2012) Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy

     Nature Methods 9:749–754.

     https://doi.org/10.1038/nmeth.2025

     • PubMed
     • Google Scholar

Author details

1. Tomoki Naito

   Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing—review and editing, Designed and performed all aspects of genetic manipulations, Designed and performed all aspects of imaging and biochemical studies, Performed the isolation of plasma membrane sheets

   Competing interests

   No competing interests declared

2. Bilge Ercan

   Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing—review and editing, Designed and performed all aspects of lipid transfer assays, Designed and performed all aspects of liposome sedimentation assays, Performed all the structural modeling and designed StART-like domain mutations, Designed and performed protein purification work

   Competing interests

   No competing interests declared

3. Logesvaran Krshnan

   Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

   Present address

   Sir William Dunn School of Pathology, University of Oxford, Oxford, United kingdom

   Contribution

   Conceptualization, Validation, Investigation, Methodology, Designed and performed genetic manipulations, Designed imaging and biochemical studies

   Competing interests

   No competing interests declared

4. Alexander Triebl

   Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

   Contribution

   Formal analysis, Investigation, Methodology, Performed lipidomics analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8423-8224

5. Dylan Hong Zheng Koh

   Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

   Contribution

   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

   Competing interests

   No competing interests declared

6. Fan-Yan Wei

   Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan

   Contribution

   Resources, Writing—review and editing

   Competing interests

   No competing interests declared

7. Kazuhito Tomizawa

   Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan

   Contribution

   Resources, Supervision

   Competing interests

   No competing interests declared

8. Federico Tesio Torta

   Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

   Contribution

   Formal analysis, Supervision, Investigation, Methodology, Performed lipidomics analysis

   Competing interests

   No competing interests declared

9. Markus R Wenk

   Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

   Contribution

   Resources, Supervision, Funding acquisition, Methodology, Performed lipidomics analysis

   Competing interests

   No competing interests declared

10. Yasunori Saheki

    1. Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
    2. Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan

    Contribution

    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

    For correspondence

    yasunori.saheki@ntu.edu.sg

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-1229-6668

• 10,482

  views

• 1,166

  downloads

• 121

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.