Molecular basis of accessible plasma membrane cholesterol recognition by the GRAM domain of GRAMD1b

WT control GRAM1b bound strongly to liposomes containing 40% or 50% cholesterol.

“WT control GRAM1b bound strongly to liposomes that contained 40% or 50% cholesterol, consistent with our previous results (Naito et al, 2019) (Fig 2D).”

• cites Naito et al, 2019 sourcePartly supported

  The Results section (page unavailable) reports strong binding of the GRAMD1b GRAM domain to liposomes containing 50% cholesterol and 20% phosphatidylserine. It does not explicitly report strong binding at 40% cholesterol, and the strong binding at 50% depended on the presence of phosphatidylserine. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

GRAM1b binds weakly to liposomes containing only cholesterol, with approximately 15-20% binding when liposomes contained 60% cholesterol.

“GRAM1b binds weakly to liposomes that contain only cholesterol (~15–20% binding when liposomes contained 60% cholesterol) (Naito et al, 2019) (Fig 2C) (see also Fig 3A and B).”

• cites Naito et al, 2019 sourcePartly supported

  The Results section reports weak binding of the GRAMD1b GRAM domain to liposomes containing 60% cholesterol, but gives the bound fraction as approximately 25%, not 15–20%. The qualitative finding and cholesterol concentration are supported; the quoted binding percentage is not. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

The GRAM domain facilitates cholesterol-dependent recruitment of GRAMD1s to ER-PM contact sites when the accessible pool of plasma membrane cholesterol transiently expands beyond a threshold.

“Further, the GRAM domain facilitates the cholesterol‐dependent recruitment of GRAMD1s to ER‐PM contact sites when the accessible pool of PM cholesterol transiently expands beyond a certain threshold (Naito et al, 2019).”

• cites Naito et al, 2019 sourceSupported

Unsequestered cholesterol can spontaneously flip-flop between the outer and inner leaflets of the PM bilayers.

“Unsequestered cholesterol can spontaneously flip‐flop between the outer and inner leaflets of the PM bilayers (Leventis & Silvius, 2001; Steck & Lange, 2018).”

• cites Leventis & Silvius, 2001 sourceNot checked — no full text of the source
• cites Steck & Lange, 2018 sourceSupported

Increases in plasma membrane cholesterol beyond a threshold, or liberation of plasma membrane cholesterol from sequestration, result in transient expansions in the accessible pool of plasma membrane cholesterol.

“Increases in the level of PM cholesterol beyond a certain threshold (or liberation of PM cholesterol from sequestration) result in transient expansions in the accessible pool of PM cholesterol.”

• cites Slotte & Bierman, 1988 sourcePartly supported

  The Discussion (page unavailable) reports that depleting sphingomyelin reduces the plasma membrane’s capacity to retain cholesterol and rapidly moves cholesterol to intracellular pools. It also describes fibroblast plasma membranes as saturated with respect to cholesterol solubilization. The paper does not show that adding cholesterol beyond a threshold, or releasing sequestered cholesterol, transiently expands an accessible plasma-membrane pool. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites Lange & Steck, 1997 sourcePartly supported

  The passage labeled Abstract (page unavailable) says that large changes in the ER-associated run-off pool with little change in total cell cholesterol support a threshold mechanism; it also reports that sphingomyelinase treatment increased cholesterol esterification. The paper does not establish that either treatment transiently expands an accessible pool in the plasma membrane: the measured pool is associated with the ER, not identified as a plasma-membrane pool. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Scheek et al, 1997 sourcePartly supported

  The Results and Discussion suggest that sphingomyelin can sequester cholesterol, and the Abstract reports that sphingomyelinase treatment moves cholesterol from the plasma membrane to the ER. Neither passage establishes a plasma-membrane cholesterol threshold or a transient expansion of an accessible plasma-membrane pool; the expanded pool described is in the ER. No page numbers are available in our copy. (LLM, conf=0.80)

  Experimental — accuracy not yet measured

In the absence of GRAMD1s, accessible cholesterol is inefficiently transported from the plasma membrane to the endoplasmic reticulum, resulting in chronic expansion of accessible plasma membrane cholesterol.

“In the absence of GRAMD1s, accessible cholesterol is inefficiently transported from the PM to the ER, resulting in a chronic expansion of accessible PM cholesterol (Naito et al, 2019; Ferrari et al, 2020).”

• cites Naito et al, 2019 sourceSupported
• cites Ferrari et al, 2020 sourcePartly supported

  The Abstract reports that cells lacking Aster expression had an expanded accessible plasma-membrane cholesterol pool accompanied by reduced movement of cholesterol to the ER. The Discussion also reports expansion after Aster knockdown or gene deletion. These passages support the transport defect and expansion, but do not establish that the expansion is chronic. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

The ability of GRAM1b to detect both anionic lipids and accessible cholesterol as a coincidence detector suggests that at least some cholesterol codistributes with phosphatidylserine without being sequestered in the membrane.

“However, the ability of the GRAM domain of GRAMD1b (GRAM1b) to detect the presence of both anionic lipids, including PS (a major acidic phospholipid enriched in the inner leaflet of the PM), and accessible cholesterol as a coincidence detector (Naito et al, 2019) suggests that at least some cholesterol codistributes with PS without being sequestered in the membrane.”

• cites Naito et al, 2019 sourceSupported

Recruitment of GRAM1b to the PM following sphingomyelinase treatment is significantly enhanced in GRAMD1 TKO HeLa cells compared to WT HeLa cells due to exaggerated accumulation of accessible PM cholesterol.

“The recruitment of GRAM1b to the PM following sphingomyelinase treatment was significantly enhanced in HeLa cells that lacked all three GRAMD1s (GRAMD1 TKO cells) compared to WT HeLa cells due to exaggerated accumulation of accessible PM cholesterol in these cells (Naito et al, 2019).”

• cites Naito et al, 2019 sourceSupported

Phospholipid acyl chain saturation has profound effects on the accessibility of cholesterol in membranes.

“In addition to sphingomyelin, phospholipid acyl chain saturation has profound effects on the accessibility of cholesterol in membranes (Radhakrishnan & McConnell, 2000; Sokolov & Radhakrishnan, 2010; Lange et al, 2013; Gay et al, 2015; Chakrabarti et al, 2017).”

• cites Radhakrishnan & McConnell, 2000 sourceNot checked — no full text of the source
• cites Sokolov & Radhakrishnan, 2010 sourceSupported
• cites Lange et al, 2013 sourceSupported

Levels of SREBP-2 cleavage are higher in GRAMD1 TKO cells than control cells following sphingomyelinase treatment.

“Using SREBP‐2 cleavage to estimate the efficiency of accessible cholesterol transport from the PM to the ER, we previously showed that levels of SREBP‐2 cleavage are higher in GRAMD1 TKO cells than control cells following sphingomyelinase treatment (Naito et al, 2019).”

• cites Naito et al, 2019 sourceSupported

mCherry-D4H strongly bound to the PM of control GRAMD1 TKO cells stably expressing EGFP, consistent with accumulation of accessible cholesterol in the PM in these cells.

“mCherry‐D4H strongly bound to the PM of control GRAMD1 TKO cells that stably expressed EGFP, consistent with accumulation of accessible cholesterol in the PM in these cells (Naito et al, 2019) (Fig 7D and E).”

• cites Naito et al, 2019 sourcePartly supported

  The Results section (“Chronic expansion of the accessible pool of PM cholesterol,” Figure 7A–B) reports stronger D4H binding at the plasma membrane of GRAMD1 TKO cells and interprets it as an expanded accessible cholesterol pool. It used recombinant EGFP–D4H, however, and does not report the stated mCherry–D4H observation in TKO cells stably expressing EGFP. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Sphingomyelinase treatment greatly expands the accessible pool of PM cholesterol in GRAMD1 TKO cells due to inefficient transport of newly liberated accessible cholesterol from the PM to the ER.

“Sphingomyelinase treatment greatly expands the accessible pool of PM cholesterol in GRAMD1 TKO cells due to inefficient transport of newly liberated accessible cholesterol from the PM to the ER (Naito et al, 2019).”

• cites Naito et al, 2019 sourceSupported

Yeast mutants that lack GRAMD1 homologs (Lam/Ltc proteins) are highly susceptible to treatment with the polyene antibiotic Amphotericin B compared to WT cells.

“Yeast mutants that lack GRAMD1 homologs (Lam/Ltc proteins) are highly susceptible to treatment with the polyene antibiotic, Amphotericin B, compared to WT cells (Gatta et al, 2015; Marek et al, 2020).”

• cites Gatta et al, 2015 sourcePartly supported

  The Results section (‘Ysp2p function correlates with sterol binding to its StART-like domain’; Figure 4A) reports that Δysp1 and Δsip3 were highly sensitive to amphotericin B compared with wild type. But Δysp2 was only moderately sensitive, while Δlam4, Δlam5 and Δlam6 behaved like wild type, so the claim is too broad for mutants lacking Lam/Ltc proteins generally. (LLM, conf=0.91)

  Experimental — accuracy not yet measured
• cites Marek et al, 2020 sourceSupported

GRAMD1b has a direct role in extracting and transporting accessible cholesterol from the PM to the ER.

“consistent with the direct role of GRAMD1b in extracting and transporting accessible cholesterol from the PM to the ER (Naito et al, 2019; Ferrari et al, 2020)”

• cites Naito et al, 2019 sourceSupported
• cites Ferrari et al, 2020 sourceSupported

Sphingomyelin can effectively sequester cholesterol.

“consistent with the ability of sphingomyelin to effectively sequester cholesterol (Endapally et al, 2019).”

• cites Endapally et al, 2019 sourceSupported

Structural modeling revealed that residue R189 is located in the β7-α2 loop of GRAM1b, near amino acid residues that contribute to PS/anionic lipid-sensing (K161, R191).

“Structural modeling revealed that residue R189 is located in the β7‐α2 loop of GRAM1b, near amino acid residues that contribute to PS/anionic lipid‐sensing (K161, R191) (Fig 3C).”

• AssumptionRests on an assumption: The structural model of GRAM1b based on the Lam6/Ltc1 GRAM domain accurately represents the structure of GRAM1b.

Anionic lipid-sensing defective EGFP-GRAM1b mutants that exhibit some anionic lipid binding (K161A and R191A) were partially recruited to the PM following sphingomyelinase treatment.

“Anionic lipid‐sensing defective EGFP‐GRAM1b mutants that exhibit some anionic lipid binding (K161A and R191A) were partially recruited to the PM following sphingomyelinase treatment.”

At steady state, all EGFP-GRAM1b variants were distributed throughout the cytosol.

“At steady state, all EGFP‐GRAM1b variants were distributed throughout the cytosol (Appendix Fig S1B).”

The codistribution of accessible cholesterol and phosphatidylserine can be modulated by the additional presence of sphingomyelin.

“Importantly, these results also demonstrate that such codistribution can be modulated by the additional presence of sphingomyelin, which normally sequesters cholesterol into inaccessible pools in the PM.”

GRAM1b shows no plasma membrane recruitment in cells cultured with lipoprotein-deficient serum and mevastatin.

“Note the absence of PM recruitment.”

The presence of phosphatidylserine does not affect the binding of ECFP-D4H to cholesterol-containing liposomes.

“Note that the presence of PS does not affect the binding of ECFP‐D4H to cholesterol‐containing liposomes”

A single glycine residue at position 187 (G187) is critical for the sensitivity of the GRAM domain to accessible plasma membrane cholesterol.

“Finally, we performed a mini‐mutagenesis screen and found that a single glycine residue at the position 187 (G187) is critical for the sensitivity of the GRAM domain to accessible PM cholesterol.”

The GRAM domain of GRAMD1b possesses distinct but synergistic sites for sensing accessible cholesterol and anionic lipids.

“We show that the GRAM domain of GRAMD1b, a coincidence detector for anionic lipids, including phosphatidylserine (PS), and cholesterol, possesses distinct but synergistic sites for sensing accessible cholesterol and anionic lipids.”

• Also stated in: § Introduction · boosted

  “Our results show that the GRAM domain of GRAMD1b possesses distinct but physically proximal lipid‐sensing sites and that one is dedicated to cholesterol and one is dedicated to anionic lipids.”

GRAM1b-R189W/R191A bound slightly less efficiently to liposomes containing cholesterol and PS than GRAM1b-R189W, with approximately 17% binding when liposomes contained 20% PS and 50% cholesterol.

“Purified GRAM1b carrying the R189W/R191A double mutation (GRAM1b–R189W/R191A) bound slightly less efficiently to liposomes containing cholesterol and PS than GRAM1b–R189W (~17% binding when liposomes contained 20% PS and 50% cholesterol)”

GRAMD1b-WT and GRAMD1b-G187L proteins mediate concentration-dependent DHE transfer from LPM to LER.

“Note the concentration‐dependent DHE transfer mediated by GRAMD1b–WT and GRAMD1b–G187L proteins.”

The R189W mutation dramatically impairs the ability of GRAMD1b to sense transient expansions in the accessible pool of plasma membrane cholesterol.

“dramatically impairing the ability of GRAMD1b to sense transient expansions in the accessible pool of PM cholesterol.”

Binding of GRAM1b-R189W/R191A to liposomes containing only PS was significantly reduced compared to GRAM1b-R189W or GRAM1b.

“Binding of GRAM1b–R189W/R191A to liposomes containing only PS was significantly reduced compared to GRAM1b–R189W or GRAM1b, as predicted based on the importance of R191 in anionic lipid recognition.”

Control and mutant GRAM1b bound similarly to liposomes containing PS.

“Control and mutant GRAM1b bound similarly to liposomes containing PS (Fig 3E).”

GRAM1b bound equally robustly to liposomes containing 20% PS and either 60% desmosterol or 60% cholesterol, with approximately 95% binding.

“GRAM1b bound equally robustly to liposomes containing 20% PS and either 60% desmosterol or 60% cholesterol (~95% binding) (Appendix Fig S1A).”

Treating GRAMD1 TKO cells with sphingomyelinase for 1 h led to the recruitment of EGFP-GRAM1b to the PM due to the expansion of the accessible pool of PM cholesterol.

“Treating GRAMD1 TKO cells with sphingomyelinase for 1 h led to the recruitment of EGFP‐GRAM1b to the PM due to the expansion of the accessible pool of PM cholesterol (Fig 3F and G).”

PM recruitment of GRAM1b was completely abolished by pre-incubation with purified ECFP-D4H proteins.

“However, PM recruitment of GRAM1b (mCherry‐GRAM1b or EGFP‐GRAM1b) was completely abolished by pre‐incubation with purified ECFP‐D4H proteins (Fig 1D)”

The K161A/R191A, R189W, and R189W/R191A mutants failed to localize to the PM, even after 60 min.

“However, the K161A/R191A, R189W, and R189W/R191A mutants failed to localize to the PM, even after 60 min (Fig 3H, Appendix Fig S1C, Movie EV1).”

PM recruitment of GRAM1b was significantly attenuated by mCherry-Lact-C2 overexpression.

“and significantly attenuated by mCherry‐Lact‐C2 overexpression (Fig 1E)”

GRAM1b plasma membrane recruitment after sphingomyelinase treatment is stronger in GRAMD1 triple knockout cells than in control HeLa cells.

“Note the very weak PM recruitment of mCherry‐GRAM1b in HeLa cells compared to the strong PM recruitment of mCherry‐GRAM1b in GRAMD1 TKO cells.”

GRAMD1 triple knockout HeLa cells stably expressing EGFP-GRAMD1b-G187L show strong resistance against Amphotericin B.

“Note the strong resistance against Amphotericin B of GRAMD1 TKO HeLa cells that stably expressed EGFP‐GRAMD1b–G187L.”

WT and mutant GRAM1b bound to liposomes containing only cholesterol (60%) to the same extent, approximately 15% binding.

“In contrast to PS detection, WT and mutant GRAM1b bound to liposomes that contained only cholesterol (60%) to the same extent (~15% binding) (Fig 2C).”

GRAMD1b-R189W/R191A proteins show no DHE transfer at any concentration tested.

“Note also the absence of DHE transfer by GRAMD1b‐R189W/R191A proteins at any concentration tested”

The presence of cholesterol does not affect the binding of mVenus-Lact-C2 to phosphatidylserine-containing liposomes.

“and that the presence of cholesterol does not affect the binding of mVenus‐Lact‐C2 to PS containing liposomes.”

FRET is decreased by addition of either untagged Lact-C2 or untagged D4H in a concentration-dependent manner.

“FRET was decreased by addition of either untagged Lact‐C2 or untagged D4H in a concentration‐dependent manner (Fig EV1, EV2, EV3, EV4, EV5).”

GRAMD1 triple knockout cells expressing EGFP-GRAMD1b-G187L are more resistant to Amphotericin B than those expressing EGFP or EGFP-GRAMD1b-WT.

“Note the resistance of GRAMD1 TKO cells that stably expressed EGFP‐GRAMD1b–G187L compared to GRAMD1 TKO cells that stably expressed either EGFP or EGFP‐GRAMD1b–WT”

GRAM1b-R189W bound much less efficiently to liposomes compared to control GRAM1b, with approximately 22% binding compared to approximately 90% binding for WT when liposomes contained 20% PS and 50% cholesterol.

“Strikingly, GRAM1b–R189W bound much less efficiently to liposomes compared to control GRAM1b (~22% binding compared to ~90% binding for the WT control when liposomes contained 20% PS and 50% cholesterol).”

R189 is a residue that is evolutionarily conserved from yeast to humans.

“Interestingly, R189 is a residue that is evolutionarily conserved from yeast to humans (Fig EV2, EV3, EV4, EV5).”

Strong FRET is observed when both cholesterol (50%) and phosphatidylserine (20%) are simultaneously incorporated into liposomes.

“Strikingly, strong FRET was observed when both cholesterol (50%) and PS (20%) were simultaneously incorporated into liposomes”

• Also stated in: § The GRAM domain of GRAMD1b detects the codistribution of accessible cholesterol and PS, which is regulated by sphingomyelin · plain

  “Note that FRET signal was increased in the presence of both cholesterol (50%) and phosphatidylserine (DOPS) (20%)”

GRAM1b-R189W bound less efficiently to liposomes containing 60% cholesterol and no PS, with approximately 4% binding compared to approximately 15% for WT controls.

“However, GRAM1b–R189W bound less efficiently to liposomes containing 60% cholesterol and no PS (~4% binding compared to ~15% for WT controls) (Fig 3E).”

Mutant versions of GRAM1b carrying K161A, R191A, or K161A/R191A bound to liposomes less efficiently than WT controls.

“However, mutant versions of GRAM1b that carried the K161A, R191A, or K161A/R191A mutation bound to liposomes less efficiently than WT controls.”

Within 30 min of sphingomyelinase treatment, robust PM recruitment of EGFP-GRAM1b was observed.

“Within 30 min of sphingomyelinase treatment, robust PM recruitment of EGFP‐GRAM1b was observed.”

No further reduction in binding to liposomes containing only cholesterol was observed when the R191A mutation was added.

“No further reduction in binding to liposomes containing only cholesterol was observed when the R191A mutation was added (Fig 3E).”

The K161A and R191A mutants were still recruited to the PM, albeit less efficiently compared to control EGFP-GRAM1b.

“The K161A and R191A mutants were still recruited to the PM, albeit less efficiently compared to control EGFP‐GRAM1b (Fig 3H, Appendix Fig S1C).”

Sphingomyelinase treatment resulted in strong recruitment of GRAM1b to the PM in GRAMD1 TKO cells in control conditions.

“In both cases, sphingomyelinase treatment resulted in strong recruitment of GRAM1b (mCherry‐GRAM1b or EGFP‐GRAM1b) to the PM in GRAMD1 TKO cells in control conditions (Fig 1D and E).”

Mutant GRAM1b (K161A, R191A, or K161A/R191A) bound significantly less to liposomes compared to WT GRAM1b.

“Mutant GRAM1b (K161A, R191A, or K161A/R191A) bound significantly less to liposomes compared to WT GRAM1b.”

WT control GRAM1b bound to liposomes containing PS alone (0-80%) in a concentration-dependent manner.

“WT control GRAM1b bound to liposomes that contained PS alone (0‐80%) in a concentration‐dependent manner, as we previously reported (Naito et al, 2019) (Fig 2C).”

• cites Naito et al, 2019 sourceNot checked yet

GRAMD1 TKO cells pre-treated with sphingomyelinase for 3 h were more efficiently killed by Amphotericin B than WT cells subjected to the same treatment.

“GRAMD1 TKO cells pre‐treated with sphingomyelinase for 3 h were more efficiently killed by Amphotericin B compared to WT cells subjected to the same treatment”

The binding of mCherry-D4H to the PM was further reduced by expressing EGFP-GRAMD1b-G187L compared to EGFP-GRAMD1b-WT.

“Remarkably, the binding of mCherry‐D4H to the PM was further reduced by expressing EGFP‐GRAMD1b–G187L compared to EGFP‐GRAMD1b–WT (Fig 7D and E).”

Following sphingomyelinase treatment, mCherry-GRAM1b was more strongly recruited to the PM in GRAMD1 TKO cells compared to control HeLa cells.

“mCherry‐GRAM1b was more strongly recruited to the PM in GRAMD1 TKO cells compared to control HeLa cells (Fig EV3B).”

The addition of near full-length GRAMD1b protein (GRAMD1b-WT) resulted in an increase in optical turbidity over time, consistent with the progressive tethering of PM-like and ER-like liposomes.

“The addition of near full‐length GRAMD1b protein (GRAMD1b–WT) resulted in an increase in optical turbidity over time, consistent with the progressive tethering of PM‐like and ER‐like liposomes (Fig 4B).”

• AssumptionRests on an assumption: The PM-like and ER-like liposomes used in the in vitro transport assay mimic the lipid composition and properties of the plasma membrane and endoplasmic reticulum.

GRAM1b-G187L bound to liposomes containing only cholesterol more strongly than GRAM1b.

“However, GRAM1b–G187L bound to liposomes containing only cholesterol more strongly than GRAM1b (Fig 6I).”

Suppression of SREBP-2 cleavage could be reliably detected by immunoblotting only after approximately 90 min.

“Suppression of SREBP‐2 cleavage could be reliably detected by immunoblotting only after ~90 min”

In cells, masking/trapping accessible PM cholesterol via purified accessible cholesterol-binding proteins (D4H) or masking the head group of PS within the inner leaflet of the PM via overexpressed PS-selective C2 domain (Lact-C2) led to reduced PM recruitment of GRAM1b even when the accessible pool of PM cholesterol was expanded by hydrolysis of sphingomyelin.

“In cells, masking/trapping accessible PM cholesterol via purified accessible cholesterol‐binding proteins (D4H) or masking the head group of PS within the inner leaflet of the PM via overexpressed PS‐selective C2 domain (Lact‐C2) led to reduced PM recruitment of GRAM1b even when the accessible pool of PM cholesterol was expanded by hydrolysis of sphingomyelin.”

GRAM1b-G187L was recruited to the PM even at steady state.

“GRAM1b–G187L was recruited to the PM even at steady state.”

Both GRAM1b-G187L and GRAM1b bound to liposomes containing only PS at similar levels.

“Both proteins bound to liposomes containing only PS at similar levels (Fig 6I).”

EGFP-GRAM1b-G187L was recruited to the PM even in control HeLa cells, despite limited PM cholesterol accessibility at rest.

“Notably, EGFP‐GRAM1b–G187L was recruited to the PM even in control HeLa cells, despite limited PM cholesterol accessibility at rest.”

• AssumptionRests on an assumption: The modeled structure of the GRAM domain of GRAMD1b accurately represents the three-dimensional arrangement of the residues.

Sphingomyelinase treatment after pre-depletion resulted in a more rapid PM recruitment of EGFP-GRAM1b-G187L compared to EGFP-GRAM1b.

“Sphingomyelinase treatment after pre‐depletion resulted in a more rapid PM recruitment of EGFP‐GRAM1b–G187L compared to EGFP‐GRAM1b, further indicating that the G187L mutation increases the sensitivity of EGFP‐GRAM1b to accessible PM cholesterol (Fig 6G, Movie EV3).”

A fraction of accessible cholesterol codistributes with PS in artificial membranes.

“We found that a fraction of accessible cholesterol codistributes with PS in artificial membranes.”

Stably expressed EGFP-GRAMD1b proteins localized to the ER as discrete patches, similar to previously reported localization of transiently transfected and functional EGFP-GRAMD1b.

“Stably expressed EGFP‐GRAMD1b proteins localized to the ER as discrete patches, similar to previously reported localization of transiently transfected and functional EGFP‐GRAMD1b (Naito et al, 2019)”

• cites Naito et al, 2019 sourceNot checked yet

For liposomes that contained 60% cholesterol, ~30% of GRAM1b-G187L bound to liposomes, whereas only ~15% of GRAM1b were bound.

“For liposomes that contained 60% cholesterol, ~30% of GRAM1b–G187L bound to liposomes, whereas only ~15% of GRAM1b were bound (Fig 6I).”

Expression of EGFP-GRAMD1b-R189W or EGFP-GRAMD1b-R189W/R191A in GRAMD1 TKO cells failed to rescue the SREBP-2 cleavage phenotype.

“However, the expression of EGFP‐GRAMD1b–R189W or EGFP‐GRAMD1b–R189W/R191A in TKO cells failed to rescue the phenotype (Fig 5E).”

The addition of GRAMD1b-R189W or GRAMD1b-R189W/R191A failed to increase optical turbidity despite their equally robust interaction with ER-like liposomes compared to GRAMD1b-WT.

“In contrast, the addition of GRAMD1b–R189W (defective in sensing cholesterol) or GRAMD1b–R189W/R191A (defective in sensing both cholesterol and anionic lipids) failed to increase optical turbidity despite their equally robust interaction with ER‐like liposomes compared to GRAMD1b–WT (Appendix Fig S3E)”

• AssumptionRests on an assumption: The PM-like and ER-like liposomes used in the in vitro transport assay mimic the lipid composition and properties of the plasma membrane and endoplasmic reticulum.

Further reduction in mCherry-D4H binding to the PM was observed in GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-G187L compared to GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-WT.

“Further reduction in mCherry‐D4H binding to the PM was observed in GRAMD1 TKO HeLa cells that stably expressed EGFP‐GRAMD1b–G187L compared to GRAMD1 TKO HeLa cells that stably expressed EGFP‐GRAMD1b–WT.”

• AssumptionRests on an assumption: The mCherry-D4H protein specifically binds accessible PM cholesterol.

Re-expression of EGFP-GRAMD1b in GRAMD1 TKO cells robustly suppressed SREBP-2 cleavage, rescuing the phenotype.

“Re‐expression of EGFP‐GRAMD1b in TKO cells robustly suppressed SREBP‐2 cleavage, thereby rescuing the phenotype (Fig 5E).”

Treatment of GRAMD1 TKO cells with methyl-β-cyclodextrin resulted in acute loss of EGFP-GRAM1b-G187L from the PM within 2 min.

“Importantly, the additional treatment of GRAMD1 TKO cells with methyl‐β‐cyclodextrin, which extracts cholesterol from cellular membranes, resulted in acute loss of EGFP‐GRAM1b–G187L from the PM within 2 min (Fig EV4B)”

The G187L mutant retained its selectivity for accessible cholesterol.

“Importantly, the G187L mutant retained its selectivity for accessible cholesterol.”

Suppression of SREBP-2 cleavage was observed in control cell lysates, but this suppression was significantly attenuated (though not completely eliminated) in GRAMD1 TKO cells.

“Suppression of SREBP‐2 cleavage was observed in control cell lysates; however, this suppression was significantly attenuated (but not completely eliminated) in GRAMD1 TKO cells.”

PM recruitment of EGFP-GRAM1b-G187L was further enhanced in both control and GRAMD1 TKO HeLa cells after sphingomyelinase treatment.

“PM recruitment of EGFP‐GRAM1b–G187L was further enhanced in both control and GRAMD1 TKO HeLa cells after sphingomyelinase treatment (Fig 6D and E).”

• AssumptionRests on an assumption: The modeled structure of the GRAM domain of GRAMD1b accurately represents the three-dimensional arrangement of the residues.

Mutant versions of EGFP-GRAMD1b (R189W and R189W/R191A) were recruited less efficiently to the PM.

“In contrast, mutant versions of EGFP‐GRAMD1b (R189W and R189W/R191A) were recruited less efficiently to the PM”

EGFP-GRAMD1b-G187L was more strongly recruited to ER-PM contacts compared to EGFP-GRAMD1b-WT following cholesterol loading.

“EGFP‐GRAMD1b–G187L was more strongly recruited to ER‐PM contacts compared to EGFP‐GRAMD1b–WT following cholesterol loading, consistent with the critical role of G187 in determining sensitivity of the GRAM domain to accessible PM cholesterol and in promoting tethering of the ER to the PM (Fig EV5G, Movie EV4).”

EGFP-GRAM1b-G187L exhibited enhanced PM recruitment compared to EGFP-GRAM1b in GRAMD1 TKO cells.

“Strikingly, EGFP‐GRAM1b–G187L exhibited enhanced PM recruitment compared to EGFP‐GRAM1b in GRAMD1 TKO cells (Fig 6B–D).”

• AssumptionRests on an assumption: The modeled structure of the GRAM domain of GRAMD1b accurately represents the three-dimensional arrangement of the residues.

The binding curve of GRAM1b-G187L was shifted toward lower cholesterol concentrations compared to that of GRAM1b in both 10% PS and 20% PS conditions.

“Strikingly, the binding curve of GRAM1b–G187L was shifted toward lower cholesterol concentrations compared to that of GRAM1b in both conditions (Fig 6H).”

The binding of both GRAM1b-G187L and GRAM1b was significantly increased as the ordering tendency of PC was lowered (i.e., as the cholesterol sequestration effect was reduced).

“The binding of both GRAM1b–G187L and GRAM1b was significantly increased as the ordering tendency of PC was lowered (i.e., as the cholesterol sequestration effect was reduced) (Figs 6H and EV4F and G).”

GRAMD1b-R191A and GRAMD1b-K161A/R191A interacted equally robustly to ER-like liposomes compared to GRAMD1b-WT.

“Both of them interacted equally robustly to ER‐like liposomes compared to GRAMD1b–WT (Appendix Fig S3E).”

• AssumptionRests on an assumption: The PM-like and ER-like liposomes used in the in vitro transport assay mimic the lipid composition and properties of the plasma membrane and endoplasmic reticulum.

GRAMD1 TKO cells pre-treated with sphingomyelinase for 3 h were more efficiently killed by Amphotericin B compared to wild-type cells subjected to the same treatment.

“We found that GRAMD1 TKO cells pre‐treated with sphingomyelinase for 3 h were more efficiently killed by Amphotericin B compared to WT cells subjected to the same treatment (Fig 5F).”

Expression of EGFP-GRAMD1b-R189W failed to reduce the binding of mCherry-D4H to the PM.

“In contrast, expression of EGFP‐GRAMD1b–R189W failed to reduce the binding of mCherry‐D4H to the PM (Fig 7D and E).”

Depletion of accessible cholesterol using LPDS and mevastatin for 16 h resulted in almost complete loss of EGFP-GRAM1b-G187L from the PM.

“This resulted in an almost complete loss of EGFP‐GRAM1b–G187L from the PM, confirming the specificity of GRAM1b–G187L to accessible cholesterol (Figs 6F and EV4C).”

The codistribution of accessible cholesterol and PS is less pronounced in the presence of sphingomyelin.

“Importantly, this codistribution is less pronounced in the presence of sphingomyelin”

The addition of GRAMD1b-WT, but not GRAMD1b-R189W or GRAMD1b-R189W/R191A, resulted in robust DHE transfer from PM-like to ER-like liposomes over time in a concentration-dependent manner, approximately 1.2 DHE molecules per min.

“Consistent with the progressive tethering of PM‐like and ER‐like liposomes, the addition of GRAMD1b–WT, but not GRAMD1b–R189W or GRAMD1b–R189W/R191A, resulted in robust DHE transfer from PM‐like to ER‐like liposomes over time in a concentration‐dependent manner (~1.2 DHE molecules per min) (Fig 4C and Appendix Fig S2E and F).”

• AssumptionRests on an assumption: The PM-like and ER-like liposomes used in the in vitro transport assay mimic the lipid composition and properties of the plasma membrane and endoplasmic reticulum.

Addition of GRAMD1b-G187L resulted in much stronger tethering, as judged by optical turbidity, compared to the addition of near full-length GRAMD1b-WT.

“Addition of GRAMD1b–G187L resulted in much stronger tethering, as judged by optical turbidity, compared to the addition of near full‐length GRAMD1b–WT (Fig 7B).”

Efficient PM recruitment of GRAM1b requires the presence of both PS and accessible cholesterol.

“Efficient PM recruitment of GRAM1b requires the presence of both PS and accessible cholesterol”

Wild-type GRAM domains are recruited to the PM only after transient expansion of the accessible pool of PM cholesterol induced by sphingomyelin hydrolysis.

“While wild‐type GRAM domains are recruited to the PM only after transient expansion of the accessible pool of PM cholesterol (induced by sphingomyelin hydrolysis)”

GRAM1b-R189W interacts much less with the PM even after expansion of the accessible pool of cholesterol in the bilayer.

“Further, GRAM1b–R189W interacts much less with the PM, even after expansion of the accessible pool of cholesterol in this bilayer.”

Addition of GRAMD1b-G187L resulted in more efficient DHE transfer from PM-like to ER-like liposomes over time in a concentration-dependent manner (~3.6 DHE molecules per min by GRAMD1b-G187L versus ~1.2 DHE molecules per min by GRAMD1b versus ~0 DHE molecules per min by GRAMD1b-R189W/R191A).

“Consistent with the enhanced tethering, addition of GRAMD1b–G187L resulted in more efficient DHE transfer from PM‐like to ER‐like liposomes over time in a concentration‐dependent manner (~3.6 DHE molecules per min by GRAMD1b–G187L versus ~1.2 DHE molecules per min by GRAMD1b versus ~0 DHE molecules per min by GRAMD1b–R189W/R191A) (Figs 7C and EV5C and D).”

• AssumptionRests on an assumption: The rate of DHE transfer between liposomes reflects the rate of cholesterol transfer by GRAMD1b.

mCherry-GRAM1b was less recruited to the PM in GRAMD1 TKO cells stably expressing EGFP-GRAMD1b-G187L than in those expressing EGFP-GRAMD1b-WT after 1 h of sphingomyelinase treatment.

“mCherry‐GRAM1b was less recruited to the PM in GRAMD1 TKO cells that stably expressed EGFP‐GRAMD1b–G187L compared to GRAMD1 TKO cells that stably expressed EGFP‐GRAMD1b–WT (Appendix Fig S5B)”

Robust PM recruitment of EGFP-GRAMD1b was observed within 20 min of cholesterol loading.

“Robust PM recruitment of EGFP‐GRAMD1b was observed within 20 min of cholesterol loading (Fig EV3A, Movie EV2).”

GRAM1b possesses distinct but closely apposed sites that synergistically sense anionic lipids, including PS, and accessible cholesterol.

“Using a mutagenesis approach, we found that GRAM1b possesses distinct, but closely apposed sites that synergistically sense anionic lipids, including PS, and accessible cholesterol.”

More EGFP-GRAM1b-G187L was bound to the PM at rest compared to EGFP-GRAM1b, as assessed by TIRF microscopy.

“Consistent with the results from SDC microscopy, more EGFP‐GRAM1b–G187L was bound to the PM at rest compared to EGFP‐GRAM1b (Fig EV4B; compare with Fig 6D and E).”

PM recruitment was mostly enhanced when G187 was replaced by more hydrophobic residues.

“PM recruitment was mostly enhanced when G187 was replaced by more hydrophobic residues.”

• AssumptionRests on an assumption: The modeled structure of the GRAM domain of GRAMD1b accurately represents the three-dimensional arrangement of the residues.

Sphingomyelinase treatment induced even stronger recruitment of EGFP-GRAM1b-G187L to the PM over the entire 1-h imaging session compared to EGFP-GRAM1b.

“Sphingomyelinase treatment induced even stronger recruitment of EGFP‐GRAM1b–G187L to the PM over the entire 1‐h imaging session compared to EGFP‐GRAM1b (Fig EV4B).”

Addition of sphingomyelin into the membranes downregulated the codistribution of accessible cholesterol and PS.

“Addition of sphingomyelin into the membranes downregulated the codistribution of accessible cholesterol and PS”

Converting a single amino acid within GRAM1b (G187) to another amino acid either increased or decreased its sensitivity for accessible PM cholesterol, depending on its hydrophobicity.

“Converting a single amino acid within GRAM1b (G187) to another amino acid either increased or decreased its sensitivity for accessible PM cholesterol, depending on its hydrophobicity (Fig 7A).”

mCherry-D4H bound significantly less to the PM when EGFP-GRAMD1b-WT was expressed.

“mCherry‐D4H bound significantly less to the PM by expressing EGFP‐GRAMD1b–WT”

Expression of EGFP-GRAMD1b-WT, but not EGFP-GRAMD1b-R189W, reduced binding of mCherry-D4H to the PM in GRAMD1 TKO HeLa cells.

“Note that expression of EGFP‐GRAMD1b–WT, but not EGFP‐GRAMD1b–R189W, reduced binding of mCherry‐D4H to the PM, in GRAMD1 TKO HeLa cells.”

• AssumptionRests on an assumption: The mCherry-D4H protein specifically binds accessible PM cholesterol.

In GRAMD1 TKO cells stably expressing EGFP-GRAMD1b-WT or EGFP-GRAMD1b-G187L, sphingomyelinase treatment robustly suppressed SREBP-2 cleavage over time.

“Robust suppression of SREBP‐2 cleavage over time was observed in both conditions (Appendix Fig S5A).”

GRAM1b bound 0%, 0%, and ~60% to POPC-containing, DOPC-containing, and DPhyPC-containing liposomes, respectively, when they contained 30% cholesterol.

“GRAM1b bound 0, 0, and ~60% to POPC‐containing, DOPC‐containing, and DPhyPC‐containing liposomes, respectively, when they contained 30% cholesterol.”

Both GRAMD1b-WT and GRAMD1b-G187L bound equally robustly to ER-like liposomes.

“Both GRAMD1b‐WT and GRAMD1b‐G187L bound equally robustly to ER‐like liposomes (Fig EV5E).”

A version of GRAMD1b with the R189W mutation was unable to rescue cholesterol transport defects in cells lacking GRAMD1s.

“Accordingly, a version of GRAMD1b with this mutation was unable to rescue cholesterol transport defects in cells lacking GRAMD1s.”

The ability of GRAM1b to bind accessible cholesterol fine-tuned both membrane tethering and cholesterol transport functions of GRAMD1b.

“Using a cell‐free reconstitution approach, we showed that the ability of GRAM1b to bind accessible cholesterol fine‐tuned both membrane tethering and cholesterol transport functions of GRAMD1b.”

GRAM1b has distinct recognition sites for anionic lipids and cholesterol.

“Taken together, our results reveal the presence of distinct recognition sites for anionic lipids and cholesterol within GRAM1b.”

Recent human genetic studies identified several mutations in GRAMD1b that are associated with intellectual disability.

“Recent human genetic studies identified several mutations in GRAMD1b that are associated with intellectual disability (Reuter et al, 2017; Santos‐Cortez et al, 2018).”

• cites Reuter et al, 2017 sourceNot checked — no full text of the source
• cites Santos‐Cortez et al, 2018 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Cells detect the codistribution of accessible cholesterol and anionic lipids in the plasma membrane and fine-tune the non-vesicular transport of plasma membrane cholesterol to the endoplasmic reticulum via GRAMD1s.

“Thus, cells detect the codistribution of accessible cholesterol and anionic lipids in the PM and fine‐tune the non‐vesicular transport of PM cholesterol to the ER via GRAMD1s.”

• Also stated in: § Introduction · boosted

  “Collectively, our results demonstrate that cells monitor the extent to which accessible cholesterol and anionic lipids are codistributed within the inner leaflet of the PM via the GRAM domain of GRAMD1s and use this information to fine‐tune the extraction and transport of accessible PM cholesterol to the ER via GRAMD1s.”

The GRAM domain of GRAMD1s acts as a coincidence detector for accessible cholesterol and anionic lipids.

“Importantly, the GRAM domain of GRAMD1s acts as a unique coincidence detector, detecting the presence of both accessible cholesterol and anionic lipids (Naito et al, 2019)”

• cites Naito et al, 2019 sourceNot checked yet

The FRET results are consistent with codistribution of accessible cholesterol and phosphatidylserine in membranes.

“These results are consistent with the codistribution of accessible cholesterol and PS in membranes.”

The R189 residue has a critical role in the coincidence detection of cholesterol and anionic lipids, including PS, in membranes.

“This indicates a critical role of the R189 residue in the coincidence detection of cholesterol and anionic lipids, including PS, in membranes (Fig 3D).”

The basic patch has a general role in anionic lipid sensing primarily through electrostatic interaction.

“These results support a general role for the basic patch in anionic lipid sensing primarily through electrostatic interaction (Fig 2E).”

The R189W mutation in the GRAM domain of GRAMD1b is associated with intellectual disability in humans.

“Notably, a missense mutation in the GRAM domain of GRAMD1b (R189W) was identified in a consanguineous family with moderate intellectual disability, indicating the importance of the GRAM domain in GRAMD1b function (Reuter et al, 2017).”

• Also stated in: § Abstract · plain

  “a mutation within the GRAM domain of GRAMD1b that is associated with intellectual disability in humans”

• cites Reuter et al, 2017 sourceNot checked — no full text of the source

GRAM1b is recruited to the plasma membrane when cholesterol is liberated from sphingomyelin-sequestered pools of PM cholesterol via hydrolysis of sphingomyelin by sphingomyelinase treatment.

“We previously showed that GRAM1b is recruited to the PM when cholesterol is liberated from sphingomyelin‐sequestered pools of PM cholesterol via the hydrolysis of sphingomyelin by sphingomyelinase treatment (Naito et al, 2019).”

• cites Naito et al, 2019 sourceNot checked yet

The basic patch of GRAM1b plays an important role in detecting the codistribution of PS and cholesterol within membranes.

“This demonstrates the important role of the basic patch of GRAM1b in detecting the codistribution of PS and cholesterol within membranes.”

The R189W mutation was identified in a consanguineous family with intellectual disability.

“One of these mutations is R189W, which was identified in a consanguineous family with intellectual disability (Reuter et al, 2017).”

• cites Reuter et al, 2017 sourceNot checked — no full text of the source

A small fraction of plasma membrane cholesterol (~15% of plasma membrane lipids) remains unsequestered and accessible for extraction and transport.

“However, a small fraction of PM cholesterol (~15% of PM lipids) remains unsequestered and accessible for extraction and transport (also known as “chemically active”) (Das et al, 2014).”

• cites Das et al, 2014Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Despite being enriched in the outer leaflet of the PM, sphingomyelin contributes significantly to suppressing the accessibility of cholesterol in the inner leaflet of the PM at steady state.

“Thus, despite being enriched in the outer leaflet of the PM, sphingomyelin contributes significantly to suppressing the accessibility of cholesterol in the inner leaflet of the PM at steady state.”

Efficient binding of GRAM1b to the PM requires both accessible PM cholesterol and PS as coligands.

“indicating that the efficient binding of GRAM1b to the PM requires both accessible PM cholesterol and PS as coligands.”

Binding of GRAM1b to the PM may additionally depend on other anionic lipids within the bilayer.

“The moderate effect of mCherry‐Lact‐C2 overexpression suggests that the binding of GRAM1b to the PM may additionally depend on other anionic lipids within this bilayer.”

There is another dedicated site for detecting accessible cholesterol that may act independently from the basic patch.

“Thus, these results indicate the presence of another dedicated site for detecting accessible cholesterol that may act independently from the basic patch.”

GRAM1b likely possesses distinct sites for recognizing anionic lipids and cholesterol.

“As major defects in the ability of mutant GRAM1b (e.g. basic patch mutants) to sense anionic lipids did not alter their ability to sense cholesterol, GRAM1b likely possesses distinct sites for recognizing anionic lipids and cholesterol.”

The GRAM domain plays a critical role in regulating GRAMD1-mediated transport of accessible cholesterol.

“and suggest that the GRAM domain plays a critical role in regulating GRAMD1‐mediated transport of accessible cholesterol.”

Both cholesterol-sensing and anionic lipid-sensing properties are important in the ability of GRAM1b to detect expansions of the accessible pool of PM cholesterol.

“These results demonstrate the importance of both cholesterol‐sensing and anionic lipid‐sensing properties in the ability of GRAM1b to detect expansions of the accessible pool of PM cholesterol”

Cholesterol-sensing and anionic lipid-sensing properties of the GRAM domain act independently.

“thereby suggesting that cholesterol‐sensing and anionic lipid‐sensing properties of the GRAM domain act independently (Fig 3D and E).”

GRAM1b possesses motifs for sensing anionic lipids and/or accessible cholesterol to detect their codistribution.

“These data further indicate that GRAM1b possesses motifs for sensing anionic lipids and/or accessible cholesterol to detect their codistribution.”

These sites act synergistically to detect the codistribution of cholesterol and anionic lipids in membranes.

“Further, these sites act synergistically to detect the codistribution of cholesterol and anionic lipids in membranes.”

Anionic lipid and cholesterol recognition are mediated by independent sites.

“These results are consistent with the notion that anionic lipid and cholesterol recognition are mediated by independent sites.”

GRAM1b-G187L has increased sensitivity to membranes that contain both PS and cholesterol.

“These results are consistent with an increased sensitivity of GRAM1b–G187L to membranes that contain both PS and cholesterol.”

GRAMD1 is important for the transport of accessible cholesterol from the PM to the ER in response to acute expansion of the accessible pool of PM cholesterol, thereby preventing overaccumulation of cholesterol in the PM.

“Taken together, these data confirm the importance of GRAMD1 in the transport of accessible cholesterol from the PM to the ER in response to acute expansion of the accessible pool of PM cholesterol, thereby preventing the overaccumulation of cholesterol in the PM.”

The R189W mutation affects the ability of GRAMD1b to transfer cholesterol.

“Thus, the R189W mutation, which specifically impairs the cholesterol‐sensing property of the GRAM domain and has been associated with intellectual disability in humans, affects the ability of GRAMD1b to transfer cholesterol.”

The accessible pool of PM cholesterol is exaggeratedly expanded in GRAMD1 TKO cells following sphingomyelinase treatment.

“further supporting exaggerated expansions in the accessible pool of PM cholesterol in GRAMD1 TKO cells following sphingomyelinase treatment.”

The GRAM domain is essential for GRAMD1-dependent cholesterol transport at ER-PM contact sites.

“Our results demonstrate that the GRAM domain is essential for GRAMD1‐dependent cholesterol transport at ER‐PM contact sites.”

The G187 position is critically important in modulating the sensitivity of GRAM1b to accessible PM cholesterol.

“These results support the critical importance of the G187 position in modulating the sensitivity of GRAM1b to accessible PM cholesterol.”

• AssumptionRests on an assumption: The modeled structure of the GRAM domain of GRAMD1b accurately represents the three-dimensional arrangement of the residues.

A hydrophobic amino acid is preferred at position G187 for the effective recognition of accessible cholesterol.

“Further, a hydrophobic amino acid is preferred in this position for the effective recognition of accessible cholesterol.”

• AssumptionRests on an assumption: The modeled structure of the GRAM domain of GRAMD1b accurately represents the three-dimensional arrangement of the residues.

Some fraction of cholesterol can remain closely associated with PS (and possibly other anionic lipids) without being sequestered by this lipid.

“This suggests that some fraction of cholesterol can remain closely associated with PS (and possibly other anionic lipids) without being sequestered by this lipid.”

The similar levels of expression allowed comparison of the activities of various versions of EGFP-GRAMD1b proteins in GRAMD1 TKO cells.

“allowing us to compare the activities of various versions of EGFP‐GRAMD1b proteins at similar levels of expression in GRAMD1 TKO cells (Fig 5B).”

EGFP-GRAMD1b-G187L was expressed at levels similar to EGFP-GRAMD1b in another stable cell line.

“Immunoblot analysis of total cell lysates confirmed that EGFP‐GRAMD1b–G187L was expressed at levels similar to EGFP‐GRAMD1b in another stable cell line (Fig EV5F; compare with Fig 5A).”

GRAM1b contributes to the regulation of GRAMD1-mediated PM to ER cholesterol transport at ER-PM contact sites.

“We have also demonstrated that GRAM1b contributes to the regulation of GRAMD1‐mediated PM to ER cholesterol transport at ER‐PM contact sites.”

The G187L mutation enhances the ability of GRAM1b to sense accessible PM cholesterol.

“These results suggest that the newly identified GRAM1b mutation, G187L, enhances the ability of GRAM1b to sense accessible PM cholesterol.”

• AssumptionRests on an assumption: The modeled structure of the GRAM domain of GRAMD1b accurately represents the three-dimensional arrangement of the residues.

The GRAM domain detects the codistribution of anionic lipids, including PS, and accessible cholesterol in the inner leaflet of the PM when it becomes prominent.

“strongly indicating that the GRAM domain detects the codistribution of anionic lipids, including PS, and accessible cholesterol in the inner leaflet of the PM when it becomes prominent.”

The cholesterol-sensing property of the GRAM domain plays a critical role in StART-like domain-dependent cholesterol transport via facilitation of membrane tethering.

“These results demonstrate that the cholesterol‐sensing property of the GRAM domain plays a critical role in StART‐like domain‐dependent cholesterol transport via facilitation of membrane tethering.”

• AssumptionRests on an assumption: The PM-like and ER-like liposomes used in the in vitro transport assay mimic the lipid composition and properties of the plasma membrane and endoplasmic reticulum.

Proper sensitivity of the GRAM domain to accessible PM cholesterol is critical for regulation of GRAMD1-dependent cholesterol extraction and transport to the ER, thereby maintaining the size of the accessible pool of cholesterol in the PM.

“These results reveal the critical role of the proper sensitivity of the GRAM domain to accessible PM cholesterol in the regulation of GRAMD1‐dependent cholesterol extraction and transport to the ER, thereby maintaining the size of the accessible pool of cholesterol in the PM.”

Sphingomyelin forms a complex with cholesterol, thereby reducing cholesterol's accessibility.

“which forms a complex with cholesterol, thereby reducing its accessibility.”

Accessible PM cholesterol was more efficiently extracted by EGFP-GRAMD1b-G187L than by EGFP-GRAMD1b-WT.

“indicating that accessible PM cholesterol was more efficiently extracted by EGFP‐GRAMD1b–G187L compared to EGFP‐GRAMD1b–WT.”

Immunoblot analysis of total cell lysates from the newly established cell lines confirmed similar levels of the transduced proteins.

“Immunoblot analysis of total cell lysates from these newly established cell lines confirmed similar levels of the transduced proteins.”

The molecular basis by which the GRAM domain of GRAMD1b detects accessible cholesterol in the presence of anionic lipids in the PM has been identified.

“We have identified the molecular basis by which the GRAM domain of GRAMD1b (GRAM1b) detects accessible cholesterol in the presence of anionic lipids in the PM.”

• AssumptionRests on an assumption: The molecular basis for the synergistic binding of GRAM domains to membranes containing both cholesterol and anionic lipids was unclear before this study.

Recruitment of EGFP-GRAM1b-G187L to the PM is cholesterol-dependent.

“demonstrating that recruitment of EGFP‐GRAM1b–G187L to the PM is cholesterol‐dependent.”

The enhanced tethering observed with GRAMD1b-G187L was due to stronger binding of GRAMD1b-G187L to PM-like liposomes compared to GRAMD1b-WT.

“Thus, enhanced tethering observed with GRAMD1b‐G187L was due to stronger binding of GRAMD1b‐G187L to PM‐like liposomes compared to GRAMD1b‐WT.”

Failure to increase turbidity indicates failure of liposome tethering.

“indicating the failure of liposome tethering (Fig 4B).”

The G187L mutation enhances GRAMD1b-mediated accessible PM cholesterol extraction.

“These results further support the notion that the G187L mutation enhances GRAMD1b‐mediated accessible PM cholesterol extraction.”

The G187L mutation increases the sensitivity of EGFP-GRAM1b to accessible PM cholesterol.

“further indicating that the G187L mutation increases the sensitivity of EGFP‐GRAM1b to accessible PM cholesterol”

Cholesterol-sensing properties of the GRAM domain are essential for mediating cholesterol-dependent PM recruitment of GRAMD1b.

“consistent with the essential role of cholesterol‐sensing properties of the GRAM domain in mediating cholesterol‐dependent PM recruitment of GRAMD1b (Fig EV3A, Movie EV2).”

The cholesterol-sensing properties of GRAM1b play important roles in regulating GRAMD1b-mediated transport of accessible cholesterol in cells.

“Thus, these results suggest important roles of the cholesterol‐sensing properties of GRAM1b in regulating GRAMD1b‐mediated transport of accessible cholesterol in cells.”

Amphotericin B preferentially targets accessible cholesterol in the PM of mammalian cells.

“These results suggest that Amphotericin B preferentially targets accessible cholesterol in the PM of mammalian cells, further supporting exaggerated expansions in the accessible pool of PM cholesterol in GRAMD1 TKO cells following sphingomyelinase treatment.”

There are other amino acids outside of the GRAM domain that may contribute to the anionic lipid-sensing property of full-length GRAMD1b proteins.

“Thus, the results from the in vitro lipid transport assay suggest that there are other amino acids outside of the GRAM domain that may contribute to the anionic lipid‐sensing property of full‐length GRAMD1b proteins (see Discussion).”

• AssumptionRests on an assumption: The PM-like and ER-like liposomes used in the in vitro transport assay mimic the lipid composition and properties of the plasma membrane and endoplasmic reticulum.

GRAM1b-G187L is specific to accessible cholesterol.

“confirming the specificity of GRAM1b–G187L to accessible cholesterol”

Both accessible cholesterol and anionic lipids are required for the binding of the GRAM domain to the PM as coligands.

“and that 2) both accessible cholesterol and anionic lipids are required for the binding of the GRAM domain to the PM as coligands (Fig 7F).”

There may be other amino acids outside of GRAM1b that contribute to the anionic lipid-sensing property of full-length GRAMD1b proteins.

“Thus, there may be other amino acids outside of the GRAM1b that contribute to anionic lipid‐sensing property of full‐length GRAMD1b proteins.”

Toxin-based biosensors are not suitable for long-term, live cell imaging of mammalian cells.

“Thus, they are not suitable for long‐term, live cell imaging of mammalian cells.”

The switch-like property of the GRAM domain in binding the PM only when necessary prevents GRAMD1s from extracting too much cholesterol.

“The switch‐like property of the GRAM domain in binding the PM only when necessary prevents GRAMD1s from extracting too much cholesterol.”

Non-vesicular lipid transport between cellular membranes is most efficient when donor and target membranes are closely apposed.

“Non‐vesicular lipid transport between cellular membranes is most efficient when donor and target membranes are closely apposed (Lev, 2012; Wong et al, 2019).”

• cites Lev, 2012 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites Wong et al, 2019 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Anionic lipids, including PS, have a critical role as coligands for PM recruitment of GRAMD1b for the PM to ER cholesterol transport.

“This is in good agreement with our data, which revealed a critical role of anionic lipids, including PS, as coligands for PM recruitment of GRAMD1b for the PM to ER cholesterol transport.”

The GRAMD1 GRAM domain is the first example of an intracellular membrane-binding domain in eukaryotes that is specifically tuned to sense levels of accessible PM cholesterol.

“The GRAMD1 GRAM domain is the first example of an intracellular membrane‐binding domain in eukaryotes that is specifically tuned to sense levels of accessible PM cholesterol.”

Coincidence detection of accessible cholesterol and anionic lipids via distinct ligand binding sites would ensure robust recruitment of the GRAM domain to the PM, whose inner leaflet is enriched with PS and other anionic lipids, only when levels of accessible PM cholesterol exceed a certain threshold.

“Coincidence detection of accessible cholesterol and anionic lipids via distinct ligand binding sites would ensure robust recruitment of the GRAM domain to the PM, whose inner leaflet is enriched with PS and other anionic lipids, only when levels of accessible PM cholesterol exceed a certain threshold.”

Cells monitor the codistribution of accessible cholesterol and anionic lipids within the inner leaflet of the PM and fine-tune the rate at which accessible PM cholesterol is extracted and transported to the ER via GRAMD1s.

“In summary, we have shown that cells monitor the codistribution of accessible cholesterol and anionic lipids within the inner leaflet of the PM and fine‐tune the rate at which accessible PM cholesterol is extracted and transported to the ER via GRAMD1s.”

The R189W mutation in the GRAM domain results in severe GRAMD1b loss of function in humans.

“These results demonstrate that the R189W mutation in the GRAM domain results in severe GRAMD1b loss of function in humans.”

• AssumptionRests on an assumption: The R189W mutation in GRAM1b is linked to intellectual disability in humans.

Because GRAMD1s are conserved from yeast to humans, the findings provide important insights into conserved mechanisms by which levels of intracellular sterols are sensed and monitored in all eukaryotes.

“Because GRAMD1s are conserved from yeast to humans, our findings provide important insights into conserved mechanisms by which levels of intracellular sterols are sensed and monitored in all eukaryotes.”

• AssumptionRests on an assumption: GRAMD1s are conserved from yeast to humans.