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Cited by 14Cellular transport and secretionProtein Kinase Regulation and GTPase SignalingExtracellular vesicles in disease

Regulation of cargo exocytosis by a Reps1-Ralbp1-RalA module

Open access
Wang, Shifeng, Chen, Xu, Crisman, Lauren, Dou, Ximing, Winborn, Christina S., Wan, Chun, Puscher, Harrison, Yin, Qian +2 more·2023·Science Advances
DOI 10.1126/sciadv.ade2540PMC PMC9946360
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Abstract

Surface levels of membrane proteins are determined by a dynamic balance between exocytosis-mediated surface delivery and endocytosis-dependent retrieval from the cell surface. Imbalances in surface protein levels perturb surface protein homeostasis and cause major forms of human disease such as type 2 diabetes and neurological disorders. Here, we found a Reps1-Ralbp1-RalA module in the exocytic pathway broadly regulating surface protein levels. Reps1 and Ralbp1 form a binary complex that recognizes RalA, a vesicle-bound small guanosine triphosphatases (GTPase) promoting exocytosis through interacting with the exocyst complex. RalA binding results in Reps1 release and formation of a Ralbp1-RalA binary complex. Ralbp1 selectively recognizes GTP-bound RalA but is not a RalA effector. Instead, Ralbp1 binding maintains RalA in an active GTP-bound state. These studies uncovered a segment in the exocytic pathway and, more broadly, revealed a previously unrecognized regulatory mechanism for small GTPases, GTP state stabilization.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper identifies a Reps1-Ralbp1-RalA module that promotes cargo exocytosis and surface protein homeostasis. Reps1 and Ralbp1 form a binary complex that hands off RalA, with Ralbp1 stabilizing RalA in its active GTP-bound state rather than acting as a RalA effector.

Key findings

  • A genome-wide CRISPR screen in mouse preadipocyte fibroblasts expressing a GLUT4-based surface reporter (GLUT-SPR) identified Reps1 and Ralbp1 as regulators of surface protein levels; Reps1 KO reduced surface GLUT-SPR while total levels were unchanged, and Reps1 KO cells showed reduced exocytosis rates and fewer plasma membrane-fusing vesicles.
  • Reps1 and Ralbp1 form a binary complex through their C-terminal domains; deletion of either protein diminished the other, and Ralbp1 KO similarly reduced GLUT-SPR exocytosis.
  • Active GTP-bound RalA was reduced to near-background levels in Reps1 KO cells, while total RalA was unchanged; expression of constitutively active RalA Q72L fully rescued exocytosis in Reps1 KO and Ralbp1 KO cells.
  • In neurons, Reps1 KO reduced neurite number and length in CAD cells and primary rat hippocampal neurons; in CAD cells, 80 WT, 93 Reps1 KO, and 95 rescue cells were analyzed, and in primary neurons 23 WT and 21 Reps1 KO neurons were analyzed, with dendritic spines largely eliminated in Reps1 KO neurons.

Methods

  • Genome-wide CRISPR screen: mouse preadipocyte fibroblasts expressing HA-GLUT4-GFP were mutagenized with GeCKO v2, sorted three times for reduced surface but normal total reporter, and analyzed with MAGeCK.
  • Cell and neuronal assays: CRISPR-Cas9 KOs in adipocytes, HeLa, CAD neurons, and primary rat hippocampal neurons; surface and total protein levels measured by flow cytometry, confocal/TIRFM/SIM/EM, and mass spectrometry-based surface and whole-cell proteomics.
  • Biochemical and functional assays: co-immunoprecipitation, GST pull-down, cleavable ATTO-565 endocytosis assays, immunoblotting, and rescue experiments with Reps1 mutants, Ralbp1 mutants, and RalA Q72L; statistical comparisons used t tests or ANOVA.

Limitations

  • The paper states that the reason many surface proteins were up-regulated in Reps1 KO cells is unclear, and it posits that inhibited exocytic delivery of retrieval factors may be responsible.
  • The paper states that Reps1 and Ralbp1 may have additional roles beyond exocytosis, that Ralbp1 could act as a RalA effector in another pathway, and that the Reps1-AP2 interaction may regulate only restricted cargoes or be insignificant in the examined cell types.
  • The paper states that further studies are needed to determine how disease-causing REPS1 mutations affect exocytosis kinetics and neuronal functions using animal models and human pluripotent stem cell-derived neurons or organoids; evident from the text, several mechanistic conclusions rely on KO/rescue and binding assays rather than structural determination of the proposed conformational changes.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1126/sciadv.ade2540

Cites

75 of 75 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

62 of 79 cited sources checked · 25 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Ralbp1 binds to RalA.

    “Ralbp1 binds to RalA, a small GTPase regulating vesicle tethering and fusion through interacting with exocyst (11, 20, 41–44).”
    Quote in the paperSubstantial claim5 of 6 sources checked
    • cites [11] R. Shirakawa, H. Horiuchi, Ral GTPases: Crucial mediators of exocytosis and tumourigenesis. J. Biochem. 157, 285– 299 (2015). 25796063 source
    • cites [20] Y. Skorobogatko, M. Dragan, C. Cordon, S. M. Reilly, C. W. Hung, W. Xia, P. Zhao, M. Wallace, D. E. Lackey, X. W. Chen, O. Osborn, J. G. Bogner-Strauss, D. Theodorescu, C. M. Metallo, J. M. Olefsky, A. R. Saltiel, RalA controls glucose homeostasis by regulating glucose uptake in brown fat. Proc. Natl. Acad. Sci. U.S.A. 115, 7819– 7824 (2018). 29915037 PMC6065037 source

      The paper’s section on Glut4 vesicle dynamics says RalA interacts with the exocyst proteins Sec5 and Exo84. Its discussion of RalGAPB concerns regulation of RalA activity, not Ralbp1. Searches for Ralbp1 and its alternative names found no passage in our copy stating that Ralbp1 binds RalA; RalGAPB should not be treated as Ralbp1. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [41] P. Chardin, A. Tavitian, The ral gene: A new ras related gene isolated by the use of a synthetic probe. EMBO J. 5, 2203– 2208 (1986). 3023062 PMC1167101 source

      The Discussion suggests that Ral may interact with other proteins but says its precise interacting proteins remain to be characterized. A search of our copy for Ralbp1, RLIP76, and binding to RalA found no passage identifying Ralbp1 or establishing the claimed interaction. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [42] L. Wang, G. Li, S. Sugita, RalA-exocyst interaction mediates GTP-dependent exocytosis. J. Biol. Chem. 279, 19875– 19881 (2004). 14978027 source

      The Results section describes RalA binding to Sec5, an exocyst component, but does not identify Sec5 as Ralbp1. Searches for RalBP1, Ral binding protein 1, and RLIP76 found no passage in our copy addressing whether Ralbp1 binds RalA; no page number was available. (LLM, conf=0.73)

      Experimental — accuracy not yet measured
    • cites [43] X. W. Chen, D. Leto, J. Xiao, J. Goss, Q. Wang, J. A. Shavit, T. Xiong, G. Yu, D. Ginsburg, D. Toomre, Z. Xu, A. R. Saltiel, Exocyst function regulated by effector phosphorylation. Nat. Cell Biol. 13, 580– 588 (2011). 21516108 PMC3904505 source
    • cites [44] M. Kawato, R. Shirakawa, H. Kondo, T. Higashi, T. Ikeda, K. Okawa, S. Fukai, O. Nureki, T. Kita, H. Horiuchi, Regulation of platelet dense granule secretion by the Ral GTPase-exocyst pathway. J. Biol. Chem. 283, 166– 174 (2008). 17938170 source
  • Interpretationreasoning

    Ralbp1 and Reps1 are involved in the same biological pathway.

    “Ralbp1 was also identified as a significant hit in our CRISPR screen (Fig. 1D) and is an interacting factor of Reps1 (Figs. 3B and 5B) (35), suggesting that Ralbp1 and Reps1 are involved in the same biological pathway.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [35] A. Yamaguchi, T. Urano, T. Goi, L. A. Feig, An Eps homology (EH) domain protein that binds to the Ral-GTPase target, RalBP1. J. Biol. Chem. 272, 31230– 31234 (1997). 9395447 source
  • Premisecitation

    GLUT4 depends on general exocytic and endocytic pathways to regulate its surface levels.

    “The reporter is based on the glucose transporter GLUT4 (Fig. 1A), which depends on general exocytic and endocytic pathways to regulate its surface levels (3, 19–21).”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [3] N. J. Bryant, R. Govers, D. E. James, Regulated transport of the glucose transporter GLUT4. Nat. Rev. Mol. Cell Biol. 3, 267– 277 (2002). 11994746 source
    • cites [19] D. R. Gulbranson, E. M. Davis, B. A. Demmitt, Y. Ouyang, Y. Ye, H. Yu, J. Shen, RABIF/MSS4 is a Rab-stabilizing holdase chaperone required for GLUT4 exocytosis. Proc. Natl. Acad. Sci. U.S.A. 114, E8224– E8233 (2017). 28894007 PMC5625932 source
    • cites [20] Y. Skorobogatko, M. Dragan, C. Cordon, S. M. Reilly, C. W. Hung, W. Xia, P. Zhao, M. Wallace, D. E. Lackey, X. W. Chen, O. Osborn, J. G. Bogner-Strauss, D. Theodorescu, C. M. Metallo, J. M. Olefsky, A. R. Saltiel, RalA controls glucose homeostasis by regulating glucose uptake in brown fat. Proc. Natl. Acad. Sci. U.S.A. 115, 7819– 7824 (2018). 29915037 PMC6065037 source

      The paper’s discussion of glucose uptake in brown fat says insulin regulates GLUT4 trafficking to the plasma membrane through vesicle tethering and fusion, supporting the exocytic part of the claim. The section “RalA Activation Increases Plasma Membrane Glut4 in Brown Fat” mentions GLUT4 recycling, but our copy does not establish that general endocytic pathways regulate its surface levels. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [21] D. R. Gulbranson, L. Crisman, M. S. Lee, Y. Ouyang, B. L. Menasche, B. A. Demmitt, C. Wan, T. Nomura, Y. Ye, H. Yu, J. Shen, AAGAB controls AP2 adaptor assembly in clathrin-mediated endocytosis. Dev. Cell 50, 436– 446.e5 (2019). 31353312 PMC6702059 source

      The Results section (‘Identification of CME regulators in a genome-wide genetic screen’; page not provided) says that the GLUT4 reporter, like native GLUT4, is continuously internalized by clathrin-mediated endocytosis, and describes measuring its surface levels. The passages found do not establish the claim’s exocytic-pathway component; GLUT4 exocytosis appears in a cited work’s title in the References, not as a finding stated here. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    RalA is a vesicle-bound small GTPase promoting exocytosis through interacting with the exocyst complex.

    “Reps1 and Ralbp1 form a binary complex that recognizes RalA, a vesicle-bound small guanosine triphosphatases (GTPase) promoting exocytosis through interacting with the exocyst complex.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [7] B. Wu, W. Guo, The exocyst at a glance. J. Cell Sci. 128, 2957– 2964 (2015). 26240175 PMC4541039 source

      The Introduction (page not provided) identifies RalA as a Ral GTPase that interacts with the exocyst subunits Sec5 and Exo84, and links RalA–Sec5 engagement to exocytic trafficking. The passages do not establish that RalA is vesicle-bound. The separate statement about Reps1 and Ralbp1 is not the part attributed to this source. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [8] D. M. Lepore, L. Martinez-Nunez, M. Munson, Exposing the elusive exocyst structure. Trends Biochem. Sci. 43, 714– 725 (2018). 30055895 PMC6108956 source

      The sections “What have we learned about the exocyst architecture and function?” and “Protein-protein interactions” identify RalA as a GTPase that interacts with exocyst subunits Exo84 and Sec5. They do not identify RalA as vesicle-bound; the vesicle-associated GTPase they name is Rab. The paper also says Ral–exocyst interactions may help control exocyst function, rather than establishing that RalA promotes exocytosis through that interaction. Page numbers were not available in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [9] R. Jin, J. R. Junutula, H. T. Matern, K. E. Ervin, R. H. Scheller, A. T. Brunger, Exo84 and Sec5 are competitive regulatory Sec6/8 effectors to the RalA GTPase. EMBO J. 24, 2064– 2074 (2005). 15920473 PMC1150893 source
  • Premisecitation

    The activity of the RhoGAP domain is abolished by mutations of a conserved arginine finger (R232) and a stabilizing lysine residue (K268).

    “The activity of the RhoGAP domain is abolished by mutations of a conserved arginine finger (R232) and a stabilizing lysine residue (K268) (50).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [50] N. F. Neel, K. L. Rossman, T. D. Martin, T. K. Hayes, J. J. Yeh, C. J. Der, The RalB small GTPase mediates formation of invadopodia through a GTPase-activating protein-independent function of the RalBP1/RLIP76 effector. Mol. Cell. Biol. 32, 1374– 1386 (2012). 22331470 PMC3318593 source

      The Results (page not provided) identify R232 as the arginine finger and K268 as a conserved lysine in the RalBP1 RhoGAP domain. They report that the R232A/K268A double mutant failed to enhance Cdc42 GTP hydrolysis, unlike the wild-type domain. Our copy does not describe K268 as stabilizing or establish that either mutation alone abolishes activity. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 28
  • May combine two claims. 26
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mutations in RALBP1 and RALA are also linked to neurodegeneration.

    “Mutations in RALBP1 and RALA are also linked to neurodegeneration (66, 67).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [66] A. Hindle, S. P. Singh, J. A. Pradeepkiran, C. Bose, M. Vijayan, S. Kshirsagar, N. A. Sawant, P. H. Reddy, Rlip76: An unexplored player in neurodegeneration and Alzheimer’s disease? Int. J. Mol. Sci. 23, 6098 (2022). 35682775 PMC9181721 source

      Section 10, “Rlip Gene Polymorphisms and Mechanistic Links” (page unavailable), says that any contribution of Rlip changes to neurodegenerative disorders would likely involve altered regulation or mutations in interaction partners, rather than coding mutations in RALBP1 itself. The cited passages also do not establish a link between RALA mutations and neurodegeneration. (LLM, conf=0.82)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [67] M. Wagner, Y. Skorobogatko, B. Pode-Shakked, C. M. Powell, B. Alhaddad, A. Seibt, O. Barel, G. Heimer, C. Hoffmann, L. A. Demmer, Y. Perilla-Young, M. Remke, D. Wieczorek, T. Navaratnarajah, P. Lichtner, D. Klee, H. E. Shamseldin, F. al Mutairi, E. Mayatepek, T. Strom, T. Meitinger, F. S. Alkuraya, Y. Anikster, A. R. Saltiel, F. Distelmaier, Bi-allelic variants in RALGAPA1 cause profound neurodevelopmental disability, muscular hypotonia, infantile spasms, and feeding abnormalities. Am. J. Hum. Genet. 106, 246– 255 (2020). 32004447 PMC7010976 source

      The supplied passages contain a garbled title and a list of links, but no substantive text about RALBP1, RALA, or neurodegeneration. Searches of the indexed copy for those terms returned no relevant passage, so the claim cannot be verified from our copy. (LLM, conf=0.70)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Neurons are highly polarized and compartmentalized cells.

    “Neurons are highly polarized and compartmentalized cells (51).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [51] M. J. Kennedy, C. Hanus, Architecture and dynamics of the neuronal secretory network. Annu. Rev. Cell Dev. Biol. 35, 543– 566 (2019). 31283381 PMC6935261 source

  • Classified as probably not a substantial claim.

    Premisecitation

    RalA regulates vesicle tethering and fusion through interacting with exocyst.

    “Ralbp1 binds to RalA, a small GTPase regulating vesicle tethering and fusion through interacting with exocyst (11, 20, 41–44).”
    Quote in the paperSubstantial claim5 of 6 sources checked
    • cites [11] R. Shirakawa, H. Horiuchi, Ral GTPases: Crucial mediators of exocytosis and tumourigenesis. J. Biochem. 157, 285– 299 (2015). 25796063 source
    • cites [20] Y. Skorobogatko, M. Dragan, C. Cordon, S. M. Reilly, C. W. Hung, W. Xia, P. Zhao, M. Wallace, D. E. Lackey, X. W. Chen, O. Osborn, J. G. Bogner-Strauss, D. Theodorescu, C. M. Metallo, J. M. Olefsky, A. R. Saltiel, RalA controls glucose homeostasis by regulating glucose uptake in brown fat. Proc. Natl. Acad. Sci. U.S.A. 115, 7819– 7824 (2018). 29915037 PMC6065037 source
    • cites [41] P. Chardin, A. Tavitian, The ral gene: A new ras related gene isolated by the use of a synthetic probe. EMBO J. 5, 2203– 2208 (1986). 3023062 PMC1167101 source

      The Discussion describes ral as a ras-related protein and says its conserved GTP-binding regions strongly suggest it binds GTP. It does not identify an exocyst interaction or a role in vesicle tethering and fusion; instead, it says ral’s precise interacting proteins remained to be characterized. The cited paper therefore supports only the small-GTPase characterization, and even that was an inference rather than a demonstrated result. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [42] L. Wang, G. Li, S. Sugita, RalA-exocyst interaction mediates GTP-dependent exocytosis. J. Biol. Chem. 279, 19875– 19881 (2004). 14978027 source

      The Abstract reports that RalA interacts with the exocyst and that this interaction is essential for GTP-dependent exocytosis in PC12 cells. It does not establish that RalA regulates vesicle tethering specifically, and it says how the interaction communicates with SNAREs to trigger membrane fusion remains to be determined. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [43] X. W. Chen, D. Leto, J. Xiao, J. Goss, Q. Wang, J. A. Shavit, T. Xiong, G. Yu, D. Ginsburg, D. Toomre, Z. Xu, A. R. Saltiel, Exocyst function regulated by effector phosphorylation. Nat. Cell Biol. 13, 580– 588 (2011). 21516108 PMC3904505 source
    • cites [44] M. Kawato, R. Shirakawa, H. Kondo, T. Higashi, T. Ikeda, K. Okawa, S. Fukai, O. Nureki, T. Kita, H. Horiuchi, Regulation of platelet dense granule secretion by the Ral GTPase-exocyst pathway. J. Biol. Chem. 283, 166– 174 (2008). 17938170 source

      The Results (page unavailable) show that the exocyst interacts with active RalA in vitro and that disrupting the Ral–exocyst interaction inhibits platelet dense-granule secretion. The Discussion (page unavailable) says the experiments could not distinguish RalA’s role from RalB’s; the paper also does not establish that RalA specifically regulates the tethering and fusion steps. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CME requires the AP2 complex to recruit cargo proteins to endocytic vesicles.

    “To recruit cargo proteins to endocytic vesicles, CME requires the heterotetrameric adaptor protein complex AP2 complex that acts as an adaptor between cargo proteins and clathrin coats (5, 15–18).”
    Quote in the paperNot a substantial claim4 of 5 sources checked
    • cites [5] K. Briant, L. Redlingshofer, F. M. Brodsky, Clathrin’s life beyond 40: Connecting biochemistry with physiology and disease. Curr. Opin. Cell Biol. 65, 141– 149 (2020). 32836101 source

      In “Phylogenetic and population diversity” (page unavailable), the review identifies AP2 as a tetrameric adaptor associated with clathrin at the plasma membrane and describes cargo binding to AP2. Its “Cargo transport pathways” section also describes ankyrin B as an adaptor for GLUT4 uptake by CME. The source therefore supports AP2’s adaptor role, but not the unqualified claim that CME requires AP2 to recruit cargo. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [15] B. M. Pearse, M. S. Robinson, Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with clathrin. EMBO J. 3, 1951– 1957 (1984). 6149117 PMC557627 source

      The Discussion (page unavailable) says the 100-kd proteins promote clathrin assembly and may interact with receptor tails to help select molecules for coated pits. That supports a possible adaptor role, but the paper does not establish that cargo recruitment requires AP2 or identify the studied complexes as AP2. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [16] S. D. Conner, S. L. Schmid, Differential requirements for AP-2 in clathrin-mediated endocytosis. J. Cell Biol. 162, 773– 780 (2003). 12952931 PMC2172816 source

      The Introduction describes AP-2 as a heterotetramer that facilitates coupling clathrin to cargo. But the Results and discussion conclude that AP-2 has a cargo-selective requirement in CME and that other adaptors can package cargo independently of it. The claim overstates AP-2 as universally required for cargo recruitment. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [17] B. M. Collins, A. J. McCoy, H. M. Kent, P. R. Evans, D. J. Owen, Molecular architecture and functional model of the endocytic AP2 complex. Cell 109, 523– 535 (2002). 12086608 source

      The Introduction (page unavailable) identifies AP2 as the predominant adaptor in clathrin-coated vesicles mediating CME and says it binds protein cargo. The Abstract (page unavailable) describes AP2 as a heterotetrameric clathrin adaptor. These passages support its adaptor role, but do not establish the stronger claim that CME requires AP2 to recruit cargo. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [18] M. Kaksonen, A. Roux, Mechanisms of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 19, 313– 326 (2018). 29410531 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    REPS1 mutations in humans cause neurodegeneration with brain iron accumulation (NBIA).

    “REPS1 mutations in humans cause neurodegeneration with brain iron accumulation (NBIA), a progressive neurodegenerative disease (24–26).”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [24] A. Drecourt, J. Babdor, M. Dussiot, F. Petit, N. Goudin, M. Garfa-Traoré, F. Habarou, C. Bole-Feysot, P. Nitschké, C. Ottolenghi, M. D. Metodiev, V. Serre, I. Desguerre, N. Boddaert, O. Hermine, A. Munnich, A. Rötig, Impaired transferrin receptor palmitoylation and recycling in neurodegeneration with brain iron accumulation. Am. J. Hum. Genet. 102, 266– 277 (2018). 29395073 PMC5985451 source
    • cites [25] S. Levi, V. Tiranti, Neurodegeneration with brain iron accumulation disorders: Valuable models aimed at understanding the pathogenesis of iron deposition. Pharmaceuticals (Basel) 12, 27 (2019). 30744104 PMC6469182 source
    • cites [26] S. Levi, D. Finazzi, Neurodegeneration with brain iron accumulation: Update on pathogenic mechanisms. Front. Pharmacol. 5, 99 (2014). 24847269 PMC4019866 source

      The Introduction describes NBIA as a group of degenerative genetic disorders, and the paper discusses mutations in several associated genes. Neither the supplied passages nor searches for REPS1 and its proposed link to NBIA found a passage addressing REPS1 mutations, so our copy cannot verify the claimed causal link. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Reps1 has two EH domains and an evolutionarily conserved C-terminal domain (CTD).

    “Reps1 has two EH (Eps15 homology) domains, protein modules involved in protein-protein interactions (40), and an evolutionarily conserved C-terminal domain (CTD) with no sequence homology with other proteins (Fig. 5I).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [40] A. E. Salcini, S. Confalonieri, M. Doria, E. Santolini, E. Tassi, O. Minenkova, G. Cesareni, P. G. Pelicci, P. P. di Fiore, Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module. Genes Dev. 11, 2239– 2249 (1997). 9303539 PMC275390 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ral and Rab GTPases cycle between GDP-bound inactive and GTP-bound active states.

    “Similar to other small GTPases, Ral and Rab cycle between a guanosine diphosphate (GDP)–bound inactive state and a GTP-bound active state (10–12).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [10] C. Yan, D. Theodorescu, RAL GTPases: Biology and potential as therapeutic targets in cancer. Pharmacol. Rev. 70, 1– 11 (2018). 29196555 PMC5712631 source
    • cites [11] R. Shirakawa, H. Horiuchi, Ral GTPases: Crucial mediators of exocytosis and tumourigenesis. J. Biochem. 157, 285– 299 (2015). 25796063 source
    • cites [12] J. Cherfils, M. Zeghouf, Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol. Rev. 93, 269– 309 (2013). 23303910 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    During neuronal morphogenesis, neuronal progenitor cells send out processes that become neurites including a single axon and multiple dendrites.

    “During neuronal morphogenesis, neuronal progenitor cells send out processes that become neurites including a single axon and multiple dendrites (52).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [52] F. L. Urbina, S. L. Gupton, SNARE-mediated exocytosis in neuronal development. Front. Mol. Neurosci. 13, 133 (2020). 32848598 PMC7427632 source

      The Development of Neurons section says immature neurons extend multiple neurites, one of which becomes an axon while the others acquire dendritic components. It distinguishes those neurons from the neural progenitor cells they were born from; it does not say progenitor cells extend the neurites. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Ralbp1 has a putative RhoGAP domain, although its catalytic activity is very low.

    “In addition, Ralbp1 has a putative RhoGAP domain, although its catalytic activity is very low (48, 49).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [48] S. H. Park, R. A. Weinberg, A putative effector of Ral has homology to Rho/Rac GTPase activating proteins. Oncogene 11, 2349– 2355 (1995). 8570186
    • cites [49] K. V. Rajasekar, L. J. Campbell, D. Nietlispach, D. Owen, H. R. Mott, The Structure of the RLIP76 RhoGAP-Ral binding domain dyad: Fixed position of the domains leads to dual engagement of small G proteins at the membrane. Structure 21, 2131– 2142 (2013). 24207123 PMC3852207 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Surface levels of membrane proteins are determined by the dynamic balance of exocytosis-mediated delivery and endocytosis-dependent retrieval.

    “The surface level of a protein is determined by the dynamic balance of its delivery to the plasma membrane through exocytic pathways (including recycling exocytosis) and removal from the plasma membrane by endocytic pathways (4–6).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [4] S. Fukai, H. T. Matern, J. R. Jagath, R. H. Scheller, A. T. Brunger, Structural basis of the interaction between RalA and Sec5, a subunit of the sec6/8 complex. EMBO J. 22, 3267– 3278 (2003). 12839989 PMC165653 source

      The Introduction describes the exocyst as a tethering complex for exocytosis at the plasma membrane and discusses protein transport to that membrane, supporting the delivery side of the claim. Our copy does not address endocytic removal, recycling exocytosis, or the assertion that their balance determines surface protein levels. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [5] K. Briant, L. Redlingshofer, F. M. Brodsky, Clathrin’s life beyond 40: Connecting biochemistry with physiology and disease. Curr. Opin. Cell Biol. 65, 141– 149 (2020). 32836101 source
    • cites [6] M. Mettlen, P. H. Chen, S. Srinivasan, G. Danuser, S. L. Schmid, Regulation of clathrin-mediated endocytosis. Annu. Rev. Biochem. 87, 871– 896 (2018). 29661000 PMC6383209 source

      The Introduction (page not available) says clathrin-mediated endocytosis takes up surface receptors and regulates the surface abundance of transmembrane transporters. It does not address exocytic delivery, including recycling exocytosis, or state that surface levels reflect a balance between delivery and removal. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Neurite outgrowth depends on exocytosis to deliver membrane materials to specific surface regions, a process requiring RalA and exocyst.

    “Neurite outgrowth depends on exocytosis to deliver membrane materials to specific surface regions, a process requiring RalA and exocyst (53–55).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [53] G. Lalli, RalA and the exocyst complex influence neuronal polarity through PAR-3 and aPKC. J. Cell Sci. 122, 1499– 1506 (2009). 19383721 source
    • cites [54] G. Lalli, A. Hall, Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex. J. Cell Biol. 171, 857– 869 (2005). 16330713 PMC2171284 source

      The Discussion (page unavailable) reports that disrupting the RalA–exocyst interaction impaired RalA-promoted neurite branching and proposes that the exocyst may coordinate membrane delivery during branching. It does not establish that neurite outgrowth generally requires RalA-dependent exocytosis to deliver membrane materials to specific surface regions; that extends a proposed branching mechanism into a broader requirement. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [55] R. O. Teodoro, G. Pekkurnaz, A. Nasser, M. E. Higashi-Kovtun, M. Balakireva, I. G. McLachlan, J. Camonis, T. L. Schwarz, Ral mediates activity-dependent growth of postsynaptic membranes via recruitment of the exocyst. EMBO J. 32, 2039– 2055 (2013). 23812009 PMC3715865 source

      The Introduction states that membrane addition permits neurite outgrowth and that the exocyst directs membrane delivery to specific surface regions and is required for neurite outgrowth. The Discussion says Ral promotes neurite branching via the exocyst, but also says some exocyst functions do not require Ral. It does not establish that RalA is required for neurite outgrowth. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Reps1, Ralbp1, and RalA are all implicated in a range of physiological and pathological processes.

    “Reps1, Ralbp1, and RalA are all implicated in a range of physiological and pathological processes (10, 61, 62).”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [10] C. Yan, D. Theodorescu, RAL GTPases: Biology and potential as therapeutic targets in cancer. Pharmacol. Rev. 70, 1– 11 (2018). 29196555 PMC5712631 source

      The RAL Binding Protein 1/RLIP76 section describes RALBP1’s roles in endocytosis and cancer-cell migration, and identifies REPS1 as a protein involved in receptor-mediated endocytosis. The RAL in Human Cancer section describes RALA’s role in tumor transformation. Our copy does not establish a range of pathological roles for REPS1, so it supports only part of the claim about all three proteins. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [61] K. H. Lim, K. O’Hayer, S. J. Adam, S. D. S. Kendall, P. M. Campbell, C. J. Der, C. M. Counter, Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells. Curr. Biol. 16, 2385– 2394 (2006). 17174914 source

      The Discussion describes RalA’s role in pancreatic tumor initiation, invasion, and metastasis, supporting its implication in pathological processes. The passages found do not address Reps1 or Ralbp1, so this source does not support the sentence’s claim about all three proteins. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [62] J. Cornish, D. Owen, H. R. Mott, RLIP76: A structural and functional triumvirate. Cancers (Basel) 13, 2206 (2021). 34064388 PMC8124665 source

      The Introduction identifies RalBP1 as RLIP76, links Ral proteins to cell growth and metastasis, and describes RLIP76’s involvement in multiple cancers. Section 2 identifies REPS1 as an RLIP76-interacting protein involved in receptor-mediated endocytosis, but the paper does not establish a range of pathological processes involving REPS1. Thus it supports the broad statement for RalA and RalBP1, but not fully for all three proteins. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    RalA and Ralbp1 have been implicated in mitochondrial dynamics.

    “Moreover, RalA and Ralbp1 have been implicated in mitochondrial dynamics (65).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [65] D. F. Kashatus, K. H. Lim, D. C. Brady, N. L. K. Pershing, A. D. Cox, C. M. Counter, RALA and RALBP1 regulate mitochondrial fission at mitosis. Nat. Cell Biol. 13, 1108– 1115 (2011). 21822277 PMC3167028 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TfR trafficking is slightly altered in fibroblasts bearing NBIA-causing mutations.

    “TfR trafficking is slightly altered in fibroblasts bearing NBIA-causing mutations (24).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [24] A. Drecourt, J. Babdor, M. Dussiot, F. Petit, N. Goudin, M. Garfa-Traoré, F. Habarou, C. Bole-Feysot, P. Nitschké, C. Ottolenghi, M. D. Metodiev, V. Serre, I. Desguerre, N. Boddaert, O. Hermine, A. Munnich, A. Rötig, Impaired transferrin receptor palmitoylation and recycling in neurodegeneration with brain iron accumulation. Am. J. Hum. Genet. 102, 266– 277 (2018). 29395073 PMC5985451 source

      The Results report significantly delayed transferrin recycling and impaired TfR1 trafficking in fibroblasts from NBIA subjects. They do not characterize the alteration as slight; the Discussion instead calls abnormal TfR1 recycling a common feature of NBIA. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    NBIA is a heterogeneous group of rare monogenetic neurodegenerative diseases exhibiting focal accumulation of intracellular iron.

    “NBIA is a heterogenous group of rare monogenetic neurodegenerative diseases exhibiting focal accumulation of intracellular iron (25).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [25] S. Levi, V. Tiranti, Neurodegeneration with brain iron accumulation disorders: Valuable models aimed at understanding the pathogenesis of iron deposition. Pharmaceuticals (Basel) 12, 27 (2019). 30744104 PMC6469182 source

      The Introduction describes NBIA as a rare, genetically heterogeneous group of inherited neurodegenerative diseases with iron deposits concentrated mainly in the basal ganglia. Section 1.1 describes intracellular iron in a particular NBIA disorder, but the paper does not establish intracellular accumulation as a feature of the group as a whole. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Iron accumulation is also observed in other neurodegenerative disorders including Alzheimer's disease and Parkinson's disease.

    “Iron accumulation is also observed in other neurodegenerative disorders including Alzheimer’s disease and Parkinson’s disease (68, 69), although a causal link between iron overload and neurodegeneration has not been established.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [68] R. J. Ward, F. A. Zucca, J. H. Duyn, R. R. Crichton, L. Zecca, The role of iron in brain ageing and neurodegenerative disorders. Lancet Neurol. 13, 1045– 1060 (2014). 25231526 PMC5672917 source
    • cites [69] L. Zecca, M. B. Youdim, P. Riederer, J. R. Connor, R. R. Crichton, Iron, brain ageing and neurodegenerative disorders. Nat. Rev. Neurosci. 5, 863– 873 (2004). 15496864 source

  • May combine two claims.

    Findingown data

    Both the number and size of protrusions were strongly reduced in Reps1 KO cells.

    “Strikingly, both the number and size of these protrusions were strongly reduced in Reps1 KO cells (Fig. 4, F to H), confirming an exocytosis defect in the KO cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ralbp1 contains a Ral-binding domain and an Reps1-binding domain (CTD), both of which are essential to the function of Ralbp1 in exocytosis.

    “Ralbp1 contains a Ral-binding domain and an Reps1-binding domain (CTD) (fig. S4A), both of which are essential to the function of Ralbp1 in exocytosis (Fig. 6, G and H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    KO of Reps1 did not affect the differentiation or insulin signaling of adipocytes.

    “KO of Reps1 did not affect the differentiation or insulin signaling of adipocytes (fig. S2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    RalA binding results in Reps1 release and formation of a Ralbp1-RalA binary complex.

    “RalA binding results in Reps1 release and formation of a Ralbp1-RalA binary complex.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    A Reps1-Ralbp1-RalA module exists in the exocytic pathway, in which Ralbp1 regulates RalA through a previously unrecognized mechanism of GTP state stabilization.

    “Together, these results revealed a Reps1-Ralbp1-RalA module in the exocytic pathway, in which Ralbp1 regulates RalA through a previously unrecognized mechanism of GTP state stabilization.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CME is a major endocytic route driven by clathrin.

    “A major endocytic route is clathrin-mediated endocytosis (CME), a vesicle budding event on the plasma membrane driven by the coat protein clathrin (13, 14).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [13] A. Fotin, Y. Cheng, P. Sliz, N. Grigorieff, S. C. Harrison, T. Kirchhausen, T. Walz, Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573– 579 (2004). 15502812 source
    • cites [14] M. Kaksonen, C. P. Toret, D. G. Drubin, Harnessing actin dynamics for clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 7, 404– 414 (2006). 16723976 source

  • May combine two claims.

    Interpretationown data

    When the Reps1-Ralbp1 binary complex recognizes the vesicle-bound small GTPase RalA, Reps1 dissociates from Ralbp1, allowing for the formation of a new binary complex of Ralbp1 and RalA.

    “When the Reps1-Ralbp1 binary complex recognizes the vesicle-bound small GTPase RalA, Reps1 dissociates from Ralbp1, allowing for the formation of a new binary complex of Ralbp1 and RalA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Ralbp1 binding maintains RalA in its GTP-bound active state, enabling the latter to interact with the Sec5 and Exo84 subunits of the plasma membrane-localized exocyst complex to facilitate vesicle tethering and fusion.

    “Ralbp1 binding maintains RalA in its GTP-bound active state, enabling the latter to interact with the Sec5 and Exo84 subunits of the plasma membrane-localized exocyst complex to facilitate vesicle tethering and fusion.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Similar to RalA, Reps1 and Ralbp1 are required for neurite outgrowth.

    “Thus, similar to RalA, Reps1 and Ralbp1 are required for neurite outgrowth.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    Detection of the Reps1-AP2 interaction suggests that recombinant GST-Reps1 proteins were properly folded.

    “Although the Reps1-AP2 interaction is dispensable for Reps1 function in exocytosis, detection of the interaction further suggests that recombinant GST-Reps1 proteins were properly folded.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationreasoning

    The GSF function represents a previously unrecognized regulatory mechanism that is likely involved in the activation cycles of other small GTPases.

    “The GSF function represents a previously unrecognized regulatory mechanism that is likely involved in the activation cycles of other small GTPases.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GAPs recognize GTP-bound small GTPases and stimulate GTP hydrolysis, inactivating them.

    “GTPase-activating proteins (GAPs), on the other hand, recognize the GTP-bound form of small GTPases and stimulate guanosine 5′-triphosphate (GTP) hydrolysis, inactivating the small GTPases (12).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] J. Cherfils, M. Zeghouf, Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol. Rev. 93, 269– 309 (2013). 23303910 source

  • May combine two claims.

    Interpretationown data

    Reps1 and Ralbp1 form a binary complex and act in concert to promote exocytosis.

    “Together, these data demonstrate that Reps1 and Ralbp1 form a binary complex and act in concert to promote exocytosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Reps1 regulates the subcellular localization but not the overall expression of GLUT-SPR.

    “Thus, Reps1 regulates the subcellular localization but not the overall expression of GLUT-SPR.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GEFs recognize GDP-bound small GTPases and accelerate GDP release, leading to GTP binding and activation.

    “Guanine nucleotide exchange factors (GEFs) recognize the GDP-bound form of small GTPases and accelerate GDP release, leading to GTP binding and activation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] J. Cherfils, M. Zeghouf, Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol. Rev. 93, 269– 309 (2013). 23303910 source

  • May combine two claims.

    Interpretationown data

    Reps1 and Ralbp1 are physically and functionally linked.

    “These biochemical and genetic data suggest that Reps1 and Ralbp1 are physically and functionally linked.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Reps1 is an essential component of the Reps1-Ralbp1-RalA module, although it does not directly associate with RalA.

    “Reps1 is an essential component of the Reps1-Ralbp1-RalA module, although it does not directly associate with RalA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Reps1 not only stabilizes Ralbp1 but also presents binding-competent Ralbp1 to RalA.

    “Our findings suggest that Reps1 not only stabilizes Ralbp1 but also presents binding-competent Ralbp1 to RalA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    While GEFs directly activate RalA through nucleotide exchange, a dedicated GSF is required to maintain the active state of RalA till the arrival of RalA effectors.

    “A key conclusion of these findings is that while GEFs directly activate RalA through nucleotide exchange, a dedicated GSF is required to maintain the active state of RalA till the arrival of RalA effectors.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

    Interpretationreasoning

    REPS1-linked NBIA pathology involves other cargo molecule(s).

    “However, KO of Reps1 or Ralbp1 in CAD cells had little effect on the surface and total levels of TfR or Tf endocytosis (fig. S7), raising the possibility that REPS1-linked NBIA pathology involves other cargo molecule(s).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Most NBIA-causing genes including REPS1 have no obvious connection to iron metabolism or transport.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    False discovery rates were set to 0.01 for both protein and peptide identifications in mass spectrometry.

    “False discovery rates were set to 0.01 for both protein and peptide identifications”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All experiments in the immunoblotting and IP experiments were run in biological triplicates.

    “All experiments were run in biological triplicates.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The lipid droplet staining and analysis experiments were performed in biological triplicates.

    “The experiments were performed in biological triplicates.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

    The Reps1-Ralbp1-RalA module regulates distinct cargo proteins across cell types, but its molecular mechanism of action is the same.

    “The Reps1-Ralbp1-RalA module regulates distinct cargo proteins across cell types, but its molecular mechanism of action is the same.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    NBIA-causing REPS1 mutations are likely hypomorphic in nature with mutant proteins retaining partial functionality, selectively affecting neurite outgrowth at specific brain regions most sensitive to reduction of its function.

    “Thus, NBIA-causing REPS1 mutations are likely hypomorphic in nature with mutant proteins retaining partial functionality, selectively affecting neurite outgrowth at specific brain regions most sensitive to reduction of its function.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The GSF function of Ralbp1 is supported by three lines of evidence: Ralbp1 selectively recognizes the active GTP-bound form of RalA but is not a RalA effector; its binding stabilizes the GTP-bound state of RalA; and its function in exocytosis can be bypassed by artificially locking RalA in a GTP-bound state.

    “The GSF function of Ralbp1 is supported by three lines of evidence: (i) Ralbp1 selectively recognizes the active GTP-bound form of RalA but is not a RalA effector; (ii) its binding stabilizes the GTP-bound state of RalA; and (iii) its function in exocytosis can be bypassed by artificially locking RalA in a GTP-bound state.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    The Reps1-Ralbp1-RalA module constitutes an integral segment of the Ral-dependent exocytic pathway, which operates in parallel with the Rab-dependent pathway in mediating cargo exocytosis and maintaining surface protein homeostasis.

    “The Reps1-Ralbp1-RalA module constitutes an integral segment of the Ral-dependent exocytic pathway, which operates in parallel with the Rab-dependent pathway in mediating cargo exocytosis and maintaining surface protein homeostasis (11, 59).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [11] R. Shirakawa, H. Horiuchi, Ral GTPases: Crucial mediators of exocytosis and tumourigenesis. J. Biochem. 157, 285– 299 (2015). 25796063 source
    • cites [59] D. Leto, A. R. Saltiel, Regulation of glucose transport by insulin: Traffic control of GLUT4. Nat. Rev. Mol. Cell Biol. 13, 383– 396 (2012). 22617471 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The minimum peptide number for mass spectrometry identification was two.

    “a minimum peptide number of two”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The minimum peptide length for mass spectrometry identification was four residues.

    “a minimum peptide length of four residues”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationown data

    Mutations of Reps1 inhibit exocytic delivery of factors involved in retrieval of certain surface proteins, leading to increases in their surface levels.

    “We posit that mutations of Reps1 inhibit exocytic delivery of factors involved in retrieval of certain surface proteins, leading to increases in their surface levels.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

    Study design and conductnone given

    All experiments in the recombinant protein expression and GST pull-down assays were run in biological triplicates.

    “All experiments were run in biological triplicates.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationreasoning

    Since other small GTPases face the same challenges in their activation cycles, they may also require a similar GSF-dependent regulatory mechanism.

    “Since other small GTPases face the same challenges in their activation cycles, they may also require a similar GSF-dependent regulatory mechanism.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    A complete loss of Reps1 abolishes neuronal morphogenesis and is expected to cause early lethality during mammalian development.

    “A complete loss of Reps1 abolishes neuronal morphogenesis and is expected to cause early lethality during mammalian development.”
    Quote in the paperSubstantial claim
49 of 49

  • The paper relies on this without support.

    Reps1 lacks an annotated function.

    “A high-ranking gene isolated in the screen is Reps1 (Ralbp1-associated eps domain containing 1), which encodes a ubiquitously expressed soluble protein lacking an annotated function.”

  • The paper relies on this without support.

    The biological function of Reps1 was unknown.

    “The biological function of Reps1, however, was unknown.”

  • The paper relies on this without support.

    RalA Q72L is constitutively active.

    “Normalized surface levels of GLUT-SPR in WT or Reps1 KO adipocytes stably expressing the constitutively active RalA Q72L mutant.”

  • The paper relies on this without support.

    The roles of Reps1 and Ralbp1 in neuronal morphogenesis remained unknown.

    “However, the roles of Reps1 and Ralbp1 in neuronal morphogenesis remained unknown.”

  • The paper relies on this without support.

    Most NBIA-causing genes including REPS1 have no obvious connection to iron metabolism or transport.

    “Most of NBIA-causing genes including REPS1 have no obvious connection to iron metabolism or transport.”
5 of 5

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Exocytosis time course is cited to Figure 2 instead of Figure 4

    Minor

    The text cites Figure 2C and D for the time course and calculated exocytosis rates. The caption assigns those measurements to Figure 4C and D; Figure 2 has no panel D in its caption.

  • GLUT-SPR endocytosis is cited to exocytosis panels

    Minor

    The text cites Figure 4E and F for the GLUT-SPR endocytosis result, but those panels are described as an exocytosis-rate measurement and images of the reporter. Figure 3E and F are the panels described as measuring GLUT-SPR endocytosis.

2 of 2

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Related

Authors
Wang, S., Chen, X., Crisman, L., Dou, X., Winborn, C. S., Wan, C., Puscher, H., Yin, Q., Kennedy, M. J., & Shen, J.
Affiliations
  • Beijing University of Chinese Medicine (CN)
  • University of Colorado Boulder (US)
  • University of Colorado Anschutz (US)
  • Florida State University (US)
Corresponding author
Shen, Jingshi
ORCID
Venue
Science Advances
Publisher
American Association for the Advancement of Science (AAAS)
Published
2023-02-24
Volume
9
Issue
8
Pages
eade2540-eade2540
ISSN
2375-2548 (ISSN-L)
DOI
10.1126/sciadv.ade2540
PMC
PMC9946360
OpenAlex
W4321481660
Cited by
14
References
76
Citations by year
2026: 1 · 2025: 9 · 2024: 3 · 2023: 1
FWCI
2.02
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Science Advances (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • CU Scholar (University of Colorado Boulder) (submittedVersion, cc-by)
Funders
  • Beijing University of Chinese Medicine
  • National Institutes of Health (1s10od025267-01, GM088759)
  • University of Colorado Boulder
  • National Institute of General Medical Sciences (R01GM138685, R35GM126960)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK124431)
  • National Institute of Neurological Disorders and Stroke (R35NS116879)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
Keywords
RALBP1, RalA, small GTPases, exocyst complex, binary complexes, vesicle trafficking, type 2 diabetes, neurological disorders
MeSH terms
Calcium-Binding Proteins, Diabetes Mellitus, Type 2, Exocytosis, Guanosine Triphosphate, Humans, Membrane Proteins, ATP-Binding Cassette Transporters, GTP Phosphohydrolases, GTPase-Activating Proteins, ral GTP-Binding Proteins

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-02.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-02.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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