Fig 2: SCRN1 does not exhibit proteolytic activity and its C‐terminal is recruited to VAP MSP domain Schematic illustration of autolytic protease activation of C69 family members.Sequence alignment of predicted proteolytic sites of SCRN family members according to the MEROPS database.Western blot of lysates from HEK293T cells expressing BioGFP‐SCRN1‐WT, BioGFP‐SCRN1‐C9A, BioGFP‐SCRN2‐WT, BioGFP‐SCRN2‐C12A, BioGFP‐SCRN3‐WT, or BioGFP‐SCRN3‐C6A. Arrowheads represent (1) full‐length BioGFP‐SCRN proteins and (2) N‐terminal cleaved BioGFP‐SCRN2 and BioGFP‐SCRN3.Pull‐down assay of HEK293T cells co‐expressing Myc‐VAPA with BioGFP, BioGFP‐SCRN1‐WT, BioGFP‐SCRN1‐C9A, BioGFP‐SCRN1‐N, or BioGFP‐SCRN1‐C.Pull‐down assay of HEK293T cells co‐expressing Myc‐VAPB with BioGFP, BioGFP‐SCRN1‐WT, BioGFP‐SCRN1‐C9A, BioGFP‐SCRN1‐N, or BioGFP‐SCRN1‐C.Hippocampal neurons (DIV16) co‐expressing HA‐VAPB with GFP‐SCRN1‐N or GFP‐SCRN1‐C. Scale bars: 10 μm (full size) and 2 μm (zoom).COS7 cells co‐expressing GFP‐SCRN1‐C with HA‐VAPA or HA‐VAPB. Scale bars: 10 μm (full size) and 5 μm (zoom).COS7 cells co‐expressing HA‐SCRN1 with GFP‐VAPB‐TM, GFP‐VAPB‐MSP‐CC, or GFP‐VAPB‐MSP. Scale bars: 10 μm (full size) and 5 μm (zoom).COS7 cells co‐expressing GFP‐SCRN1‐N with HA‐VAPA or HA‐VAPB. Scale bars: 10 μm (full size) and 5 μm (zoom). Source data are available online for this figure.

Fig 3: VAP and SCRN1 control ER continuity and remodeling Summary of observed phenotypes on SCRN1 recruitment and ER morphology in cells co‐expressing indicated SCRN1 and VAP constructs (asterisks represent mutations).Live hippocampal neurons (DIV17–18) co‐expressing TagRFP‐ER with GFP, GFP‐SCRN1, GFP‐SCRN1‐F402A, SCRN1 shRNA, or VAPA/B shRNAs. Scale bars: 5 μm (full size) and 2 μm (zoom).Time‐lapse of TagRFP‐ER dynamics in hippocampal neurons (DIV16–18) co‐expressing GFP, GFP‐SCRN1, GFP‐SCRN1‐F402A, SCRN1 shRNA, or VAPA/B shRNAs. Intact and stable ER structures (dark arrowheads), intact and dynamic ER structures (light arrowheads), and impaired non‐dynamic ER structures (arrows) are indicated. Scale bars: 5 μm (full size) and 2 μm (zoom).Schematic illustration of the effect of VAP‐SCRN1 interactions on ER structure and dynamics.ER nanostructures visualized with GFP‐Sec61β in axons of hippocampal neurons (DIV18) immunostained for α‐tubulin and co‐expressed with pSuper empty vector, SCRN1 shRNA, or VAPA/B shRNAs, and subjected to expansion microscopy. Scale bars: 2 μm (full size) and 500 nm (zoom).FRAP experiment of TagRFP‐ER in hippocampal neurons (DIV17–18) co‐expressing GFP, GFP‐SCRN1, and GFP‐SCRN1‐F402A. Scale bar: 2 μm.Average normalized fluorescent TagRFP‐ER recovery after photo‐bleaching in hippocampal neurons (DIV17–18) co‐expressing GFP, GFP‐SCRN1, and GFP‐SCRN1‐F402A. N = 2, n = 9 neurons.Normalized fluorescent TagRFP‐ER recovery after photo‐bleaching at T = 60 s in hippocampal neurons (DIV17–18) co‐expressing GFP, GFP‐SCRN1, and GFP‐SCRN1‐F402A. N = 2, n = 9 neurons.Average normalized fluorescent TagRFP‐ER recovery after photo‐bleaching in hippocampal neurons (DIV18) co‐expressing pSuper empty vector, SCRN1 shRNA, and VAPA/B shRNAs. N = 4, n = 6–12 neurons.Normalized fluorescent TagRFP‐ER recovery after photo‐bleaching at T = 60 s in hippocampal neurons (DIV18) co‐expressing pSuper empty vector, SCRN1 shRNA, and VAPA/B shRNAs. N = 4, n = 6–12 neurons.Data information: Data represent mean ± SEM; n.s.: not significant; *P < 0.05; **P < 0.01, by Mann–Whitney U‐test.

Fig 4: VAP and VAP‐associated protein SCRN1 modulate SV cycling Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons (DIV16) expressing GFP‐Sec61β. Zooms represent (1) an axonal structure with presynaptic boutons (arrowheads), and (2) a dendritic structure. Scale bars: 10 μm (full size) and 5 μm (zoom).Schematic illustration of the Syt antibody uptake assay: live neurons were stimulated with bicuculline and incubated with primary Syt antibodies, and next neurons were fixed and stained with secondary antibodies.Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co‐expressing RFP and pSuper empty vector or VAPA/B shRNAs. Yellow and gray arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 μm (full size) and 2 μm (zoom).Quantifications of fluorescence intensity of internalized endogenous Syt at single presynaptic boutons of hippocampal neurons (DIV18) co‐expressing RFP and pSuper empty vector or VAPA/B shRNAs. N = 2, n = 288–541 boutons.Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non‐neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal.Pull‐down assay of HEK293T cells co‐expressing Myc‐VAPA with BioGFP or BioGFP‐SCRN1.Pull‐down assay of HEK293T cells co‐expressing Myc‐VAPB with BioGFP or BioGFP‐SCRN1.Scaled representation of SCRN1‐associated proteins identified with pull‐down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP‐SCRN1 from HEK293T cell lysates with adult rat brain extracts. All candidates showed > 10 enrichment of PSM compared to control.Endogenous localization of SCRN1 and Syt in cortical neurons (DIV18) expressing GFP. Zoom represents an axon structure with presynaptic boutons (arrowheads). Scale bars: 10 μm (full size) and 5 μm (zoom).Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co‐expressing RFP and pSuper empty vector, SCRN1 shRNA, or SCRN1 shRNA with GFP‐SCRN1. Yellow and gray arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 μm (full size) and 2 μm (zoom).Quantifications of fluorescence intensity of internalized endogenous Syt at single presynaptic boutons of hippocampal neurons (DIV18) co‐expressing RFP and pSuper empty vector, SCRN1 shRNA alone, or SCRN1 shRNA with GFP‐SCRN1. N = 2, n = 201–300 boutons.Data information: Data represent mean ± SEM; ***P < 0.001, by Mann–Whitney U‐test.Source data are available online for this figure.

Fig 5: VAP‐SCRN1 interaction at the ER is mediated by a single FFAT‐like motif Schematic overview of SCRN1 and VAP constructs (asterisks represent mutations). Indicated is if expressed VAP/SCRN1 proteins co‐localize in both hippocampal neurons and COS cells, and co‐precipitate in pull‐down assays.COS7 cells co‐expressing GFP‐SCRN1 with HA‐VAPB‐K87D/M89D. Normalized intensity plot represents indicated line (dotted). Scale bars: 10 μm (full size) and 5 μm (zoom).COS7 cells co‐expressing HA‐VAPB with GFP‐SCRN1‐Y40A, GFP‐SCRN1‐F144A, GFP‐SCRN1‐F153A, or GFP‐SCRN1‐F402A. Normalized intensity plot represents indicated line (dotted).Scale bars: 10 μm (full size) and 5 μm (zoom).Hippocampal neurons (DIV16) co‐expressing HA‐VAPB with GFP‐SCRN1‐Y40A, GFP‐SCRN1‐F144A, GFP‐SCRN1‐F153A, or GFP‐SCRN1‐F402A. Scale bars: 10 μm (full size) and 5 μm (zoom).Quantifications of SCRN1 recruitment to VAPB‐positive structures in COS7 cells (%). Left graph: co‐expression of GFP‐SCRN1 with HA‐VAPB or HA‐VAPB‐K87D/M89D (N = 2–3, n = 44–46). Right graph: co‐expression of HA‐VAPB with GFP or GFP‐SCRN1‐FL, GFP‐SCRN1‐N, GFP‐SCRN1‐C, GFP‐SCRN1‐Y40A, GFP‐SCRN1‐F144A, GFP‐SCRN1‐F153A, or GFP‐SCRN1‐F402A (N = 2–3, n = 41–64).Pull‐down assay of HEK293T cells co‐expressing Myc‐VAPB with GFP, GFP‐SCRN1‐WT, GFP‐SCRN1‐Y40A, GFP‐SCRN1‐F144A, GFP‐SCRN1‐F153A, or GFP‐SCRN1‐F402A.Sequence alignment of the C‐terminal human SCRN1, SCRN2, and SCRN3. Amino acid position 3 (asterisk) in FFAT‐like motif (orange) of SCRN1 is not shared in SCRN2 and SCRN3.Data information: ***P < 0.001, by chi‐square test with post hoc analysis including Bonferroni correction.Source data are available online for this figure.