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

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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.

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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

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