Rabphilin 3A binds the N-peptide of SNAP-25 to promote SNARE complex assembly in exocytosis
- Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, China
Figures
Figure 1 with 3 supplements
The intramolecular interplay of Rph3A enables strong binding to SN25.
(A) Schematic diagram showing domain organization and variant fragments of Rph3A. The relative SN25 binding affinity (–, +, +++) derived from binding experiments is indicated on the right. (B, C) Binding of Rph3A FL or the fragments to GST-SN25 measured by GST pull-down assay (B) and quantification of the Rph3A binding (C). Asterisks in (B) show bands of bound Rph3A FL or fragment proteins. (D, E) Binding of Rph3A FL or deletion mutations (Δ185–371, Δ1–45 & Δ161–371) to GST-SN25 measured by GST pull-down assay (D) and quantification of the Rph3A binding (E). Asterisks in (D) show bands of bound Rph3A FL or fragment proteins. (F) Structure alignment of Rph3A with RIM-ZF–Munc13-C2A complex (PDB entry: 2CJS). The Rph3A-RBD (PDB entry: 1ZBD) and C2B (PDB entry: 3RPB) domain were aligned and superposed with RIM-ZF, Munc13-C2A, respectively. The G102/L104 site was mapped to Rph3A-RBD structure. (G, H) Binding of His6-tagged Rph3A-C2AB to GST-Rph3A-RBD (40–170) or its G102A/L104A mutant (GLAA) measured by GST pull-down assay (G) and quantification of the bound His6-C2AB (H). The top panel shows a Coomassie blue stained gel of the GST-proteins to illustrate that similar amounts of protein were employed. Bound His6-C2AB proteins were analyzed by immunoblotting with anti-His6 antibody (bottom). (I, J) Binding of Rph3A FL to the GST-SN25 in the presence of Rab3A Q81L measured by GST pull-down assay (I) and quantification of the bound Rph3A (J). Asterisk in (I) shows the band of bound Rab3A Q81L. Data are processed by ImageJ (NIH) and presented as the mean ± SEM (n=3), technical replicates. Statistical significance and p values were determined by one-way analysis of variance (ANOVA). ***p<0.001; ****p<0.0001; ns, not significant.
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Figure 1—source data 1
Uncropped SDS-gel shown in Figure 1B.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig1-data1-v1.zip
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Figure 1—source data 2
Excel file with data used to make Figure 1C, E, H and J.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig1-data2-v1.xlsx
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Figure 1—source data 3
Uncropped SDS-gel shown in Figure 1D.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig1-data3-v1.zip
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Figure 1—source data 4
Uncropped SDS-gel and Western blot shown in Figure 1G.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig1-data4-v1.zip
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Figure 1—source data 5
Uncropped SDS-gel shown in Figure 1I.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig1-data5-v1.zip
Figure 1—figure supplement 1
Size-exclusion chromatograph (SEC) and analytical ultracentrifugation (AUC) analysis of Rph3A FL protein.
(A) SEC elution profiles of Rph3A FL protein with Superdex 200 10/300 GL, red arrow represents the elution peak of Rph3A FL protein. (B) SV AUC analysis of Rph3A FL protein (left), the molecular weights were calculated based on sedimentation coefficient, obtained with SEDFIT software with a continuous c(s) model. The right panel represents Rph3A FL sample before the SV assay, resolved with SDS-PAGE.
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Figure 1—figure supplement 1—source data 1
Uncropped SDS-gel shown in Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig1-figsupp1-data1-v1.zip
Figure 1—figure supplement 2
Binding Kd between Rph3A FL and GST-SN25.
(A) Increasing concentrations of Rph3A FL were added as indicated (0, 0.5, 1, 2, 3, 4, 6, 8, and 10 µM) with 6 µM GST-SN25 or GST. The 15% quantity of the maximum Rph3A was added as input in the experiment. (B) Quantification of the bound Rph3A FL shown in (A). Data are fitted to the Hill equation, where Bmax (the intensity of the bound Rph3A FL when Rph3A was added at the concentration of 10 µM) was set to 1. Data are presented as mean ± SEM; n=3 technical replicates.
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Figure 1—figure supplement 2—source data 1
Uncropped SDS-gel shown in Figure 1—figure supplement 2A.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Excel file with data used to make Figure 1—figure supplement 2B.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig1-figsupp2-data2-v1.xlsx
Figure 1—figure supplement 3
The effect of G102A/L104A mutant on Rph3A–SN25 interaction.
(A) Binding of Rph3A Δ185–371 WT or G102A/L104A mutant to GST-SN25 measured by GST pull-down assay. Asterisk shows a band of bound protein. (B) Quantification of bound Rph3A Δ185–371 proteins in (A). Data are processed by ImageJ (NIH) and presented as the mean ± SEM (n=3), technical replicates. Statistical significance and p values were determined by one-way analysis of variance (ANOVA). ****p<0.0001. (C). The structure of Rph3A-RBD–Rab3A complex (PDB entry: 1ZBD). The G102/L104 site was mapped to Rph3A-RBD domain.
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Figure 1—figure supplement 3—source data 1
Uncropped SDS-gel shown in Figure 1—figure supplement 2A.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig1-figsupp3-data1-v1.zip
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Figure 1—figure supplement 3—source data 2
Excel file with data used to make Figure 1—figure supplement 2B.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig1-figsupp3-data2-v1.xlsx
Figure 2 with 4 supplements
Characterization of the Rph3A-binding sites in SN25.
(A) Schematic diagram showing domain organization and variant fragments of SN25. NP, N-peptide of SN25 (residues, 1–10); SN1, SNARE motif 1; LR, linker region; SN2, SNARE motif 2. The relative Rph3A FL binding affinity (–, +++) derived from binding experiments is indicated on the right. (B, C) Binding of Rph3A FL to GST-SN25 FL or fragments measured by GST pull-down assay (B) and quantification of the bound Rph3A FL (C). Asterisk in (B) shows the band of degraded GST-SN25 (1–140) proteins. (D) Schematic diagram showing N-peptide deletion mutants of SN25 (1–82). The relative Rph3A FL binding affinity (–, ±, +++) derived from binding experiments is indicated on the right. (E, F) Binding of Rph3A FL to GST-SN25 (1–82) or N-peptide deletions (7–82 and 11–82) measured by GST pull-down assay (E) and quantification of the bound Rph3A FL (F). Data are processed by ImageJ (NIH) and presented as the mean± SEM (n=3), technical replicates. Statistical significance and p values were determined by one-way analysis of variance (ANOVA). ****p<0.0001; ns, not significant.
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Figure 2—source data 1
Uncropped SDS-gel shown in Figure 2B.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig2-data1-v1.zip
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Figure 2—source data 2
Excel file with data used to make Figure 2C and F.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig2-data2-v1.xlsx
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Figure 2—source data 3
Uncropped SDS-gel shown in Figure 2E.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig2-data3-v1.zip
Figure 2—figure supplement 1
Effect of middle region mutants in SN1 on Rph3A interaction.
(A) GST pull-down assays were performed with GST-SN25 (1–82) WT or EDR (E38A/D41A/R45A), DER (D51A/E55A/R59A) mutants and Rph3A FL protein. (B) Quantification of the relatively bound Rph3A FL shown in (A). All data are presented as mean ± SEM (n=3), technical replicates. Statistical significance and p values were determined by one-way analysis of variance (ANOVA). **p<0.01; ns, not significant.
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Figure 2—figure supplement 1—source data 1
Uncropped SDS-gel shown in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Excel file with data used to make Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig2-figsupp1-data2-v1.xlsx
Figure 2—figure supplement 2
Sequences alignment of SN25 using Clustal Omega Program and Escript 3.0.
(A) Alignment of the N-terminus sequences of SN25 across species. (B) Alignment of the N-terminus sequences of SN25 with SNAP-23, SNAP-29, and SNAP-47. (C) Alignment of the N-terminus sequences of SN25 among Qb SNAREs (Vti1A, Vti1B, GOSR1, GOSR2, and Sec20) in endosomal or ER-Golgi fusion. Uniprot entries are shown on the left of the chart. Residues with 100% consensus are shaded by red, residues which consensus level >80% are colored red. The green box represents the N-peptide region of SN25. Hs, Homo sapiens; Rn, Rattus norvegicus; Mm, Mus musculus; Dr, Danio rerio; Ce, Caenorhabditis elegans.
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Figure 2—figure supplement 2—source data 1
Uncropped SDS-gel shown in Figure 2—figure supplement 2A.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
Excel file with data used to make Figure 2—figure supplement 2B.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig2-figsupp2-data2-v1.xlsx
Figure 2—figure supplement 3
The role of conserved acidic residues in N-peptide of SN25 in Rph3A binding.
GST pull-down assays were performed with GST-SN25 (1–82) WT or E3A, D4A, D6A or Δ1–10 (11–82) mutants and Rph3A FL protein.
Figure 2—figure supplement 4
Lipid sedimentation assay to measure the binding of Rph3A–SN25 in the presence of negative membranes.
(A) Representative lipid sedimentation assay. Increasing concentrations of SN25 (1–206) or (11–206) (5–20 µM) were incubated with 5 µM Rph3A FL in the presence of lipid mixture (PC:PE:PS:PI(4,5)P2=58%:20%:20%:2%). The free (S) and bound (P) Rph3A FL and SN25 were analyzed by SDS-PAGE. (B) Quantification of the relative binding of Rph3A FL (dark gray bars) and SN25 (light gray bars) to liposomes as a function of the total Rph3A FL at 5 µM SN25. Values are normalized for the total protein added to each complete assay. Data are processed by Image J (NIH) and presented as the mean ± SEM (n = 3), technical replicates. Statistical significance and P values were determined by two-way analysis of variance (ANOVA). ****P < 0.0001.
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Figure 2—figure supplement 4—source data 1
Uncropped SDS-gel and statistic data shown in Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig2-figsupp4-data1-v1.zip
Figure 3
Importance of the N-peptide SN25 for DCV exocytosis in PC12 cells.
(A) Immunoblotting assay to determine the SN25 expression level in control, SN25 KD, SN25 FL, or SN25 (11–206) rescued PC12 cells. Expression of SN25 and β-actin in PC12 cells was analyzed by 15% SDS-PAGE followed by immunoblotting with anti-SN25 antibody, and anti-β-actin antibody, respectively. (B) Representative TIRF images of control, SN25 KD, SN25 FL, or SN25 (11–206) rescued PC12 cells expressing indicator EGFP, and DCV content (NPY-td-mOrange2) are shown. Scale bars, 10 μm. (C) The density of docked vesicles was determined by counting the NPY-td-mOrange2 labeled vesicles in each image (n≥9 cells in each). (D) NPY-td-mOrange2 release events detected by TIRF microscopy during sustaining high K+ stimulation. The curves indicate the cumulative number of fusion events per µm2 in each cell. (E) Quantification of the results at 180 s in the experiments of (D). Data are presented as mean ± SEM; (n≥6 cells in each). Statistical significance and p values were determined by one-way analysis of variance (ANOVA). **p<0.01; ns, not significant.
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Figure 3—source data 1
Uncropped Western blot shown in Figure 3A.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig3-data1-v1.zip
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Figure 3—source data 2
Excel file with data used to make Figure 3C–E.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig3-data2-v1.xlsx
Figure 4 with 1 supplement
Importance of the C2B bottom α-helix of Rph3A FL for SN25 binding and DCV exocytosis in PC12 cells.
(A) Structural diagrams (left) and electrostatic surface potential (right) of Rph3A C2B (PDB entry: 5LOW). Interface I residues K651/K656/K663 on the bottom α-helix and interface II residues K590/K591/K593/K595 on the side are shown as magenta and cyan spheres, respectively. Black boxes display the basic patches that include the residues shown on the left. (B, C) Binding of Rph3A FL or mutations to the SN25 (1–82) measured by GST pull-down assay (B) and quantification of the Rph3A binding (C). K3, Rph3A FL protein bearing the K651A/K656A/K663A mutation in interface I; K4, Rph3A FL protein bearing the K590Q/K591Q/K593Q/K595Q mutation in interface II. Data are processed by ImageJ (NIH) and presented as the mean ± SEM (n=3), technical replicates. (D) Representative TIRF images of control, Rph3A KD, Rph3A WT, K3 mutant, or GLAA mutant rescued PC12 cells expressing indicator EGFP, and DCV content (NPY-td-mOrange2) are shown. Scale bars, 10 μm. (E) Immunoblotting assay to determine the Rph3A WT, Rph3A-K3, or Rph3A GLAA mutant expression level in PC12 cells. Expression of Rph3A WT or mutants and β-actin in PC12 cells was analyzed by 12% SDS-PAGE followed by immunoblotting with anti Rph3A antibody, and anti-β-actin antibody, respectively. The positions of the molecular mass markers are shown on the left. (F) The density of docked vesicles was determined by counting the NPY-td-mOrange2 labeled vesicles in each image (n≥13 cells in each). (G) NPY-td-mOrange2 release events detected by TIRF microscopy during sustaining high K+ stimulation. The curves indicate the cumulative number of fusion events per µm2 in each cell. (H) Quantification of the results at 180 s in the experiments of (G). Data are presented as mean ± SEM; (n≥9 cells in each). Statistical significance and p values were determined by one-way (in (F) and (H)) or two-way (in (C)) analysis of variance (ANOVA). **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.
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Figure 4—source data 1
Uncropped SDS-gel shown in Figure 4B.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig4-data1-v1.zip
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Figure 4—source data 2
Excel file with data used to make Figure 4C and F–H.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig4-data2-v1.xlsx
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Figure 4—source data 3
Uncropped Western blot shown in Figure 4E.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig4-data3-v1.zip
Figure 4—figure supplement 1
Effect of Rph3A interface I mutant on SN25 (1–140) binding.
(A, B) Binding of Rph3A WT or K3, K4 mutants to the GST-SN25 (1–140) measured by GST pull-down assay (A) and quantification of the Rph3A binding (B). All data are presented as mean ± SEM (n=3), technical replicates. Statistical significance and p values were determined by one-way analysis of variance (ANOVA). **p<0.01; ns, not significant.
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Figure 4—figure supplement 1—source data 1
Uncropped SDS-gel shown in Figure 4—figure supplement 1A.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Excel file with data used to make Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig4-figsupp1-data2-v1.xlsx
Figure 5 with 1 supplement
The function of Rph3A in SNARE complex assembly (A) Schematic diagram of SNARE complex assembly in the presence of Syx1 (2–253), Syb2 (29–93, S61C), and SN25 FL (1–206, R59C) or SN25 fragment (11–206, R59C).
The FRET signal between Syb2 S61C-BDPY (donor) and SN25 R59C-TMR (acceptor) was monitored. (B) Effects of the K3 and/or K4 mutations on Rph3A-promoted SNARE complex assembly detected by FRET assay. (C) Quantification of the results at 600 s in the experiments of (B). (D) Effects on SN25 (11–206) on Rph3A-promoted SNARE complex assembly detected by FRET assay. Curve Rph3A (SN25 FL) (green color) represents the SN25 FL mediated SNARE complex assembly in the presence of Rph3A FL. (E) Quantification of the results at 600 s in the experiments of (D). (F) Schematic diagram of the trans-SNARE complex formation between Syx1 liposomes and Syb2 liposomes in the presence of SN25 and Rph3A FL or K3 and GLAA mutants. BDPY and TMR, which act as fluorescent donor and acceptor as above, were separately labeled to Syb2 and SN25 Δ9 or SN25 (11–206 & Δ9) truncation, respectively. (G) FRET assay for monitoring trans-SNARE complex assembly on membranes in the presence of the SN25 Δ9 and Rph3A FL or K3 and GLAA mutants. (H) Quantification of the results at 2300s in the experiments of (G). (I) FRET assay for monitoring trans-SNARE complex assembly on membranes in the presence of the SN25 (11–206 & Δ9) and Rph3A FL. (J) Quantification of the results at 2500s in the experiments of (I). Data are presented as mean ± SEM (n=3), technical replicates. Statistical significance and p values were determined by one-way analysis of variance (ANOVA). ***p<0.001; ns, not significant. FRET, fluorescence resonance energy transfer.
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Figure 5—source data 1
Excel file with data used to make Figure 5C, E and G–J.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
The function of Rph3A in SNARE-mediated membrane fusion.
(A) Illustration of lipid mixing assay in the presence of acceptor liposome (57% POPC, 20% POPE, 20% DOPS, 1% PI(4,5)P2, and 2% DiD) contained membrane-embedded Syx1 (1–288), donor liposome (58% POPC, 20% POPE, 20% DOPS, and 2% DiI) contained Syb2 and SN25. FRET signal between membrane associate DiD and DiI was monitored. (B) Effects of the Rph3A FL or K3 and GLAA mutants on SNARE-mediated liposome fusion in the presence of SN25 (1–206). (C) Quantification of the results at 2300s in the experiments of (B). (D) Effects of the Rph3A FL on SNARE-mediated liposome fusion in the presence of SN25 (11–206). (E) Quantification of the results at 2300s in the experiments of (D). (F) Effects of Rph3A FL on SNARE-mediated liposome fusion in the presence of SN23. (G) Quantification of the results at 2500s in the experiments of (F). Data are presented as mean ± SEM (n=3), technical replicates. Statistical significance and p values were determined by one-way analysis of variance (ANOVA). **p<0.01; ***p<0.001; ns, not significant.
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Figure 5—figure supplement 1—source data 1
Excel file with data used to make Figure 5—figure supplement 1C,E,G.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 2 supplements
Conformation change of SN25 induced by Rph3A.
(A, D) Illustration of the conformation change from random coils to α-helix in the SN1 of SN25 induced by Syx1 (2–253) and Syb2 (29–93), or by Rph3A, monitored by bimane-tryptophan quenching assay. Tryptophan was introduced at residue E55 and bimane fluorescence was labeled on residue R59C in SN25 FL (A) or SN25 11–206 (D). (B, C) Quenching of bimane fluorescence on the SN25 E55W/R59C with the addition of Syx1 and Syb2, and the addition of Rph3A FL or Rph3A-K3 mutant (B) and quantification of the results observed at 474 nm (C). (E, F) Quenching of bimane fluorescence on the SN25 (11–206) E55W/R59C with the addition of Syx1 and Syb2, or the addition of Rph3A FL (E) and quantification of the results observed at 474 nm (F). Data are presented as mean ± SEM (n=3), technical replicates. The significance were examined by one-way analysis of variance (ANOVA). *p<0.05; **p<0.01; ***p<0.001; ns, not significant.
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Figure 6—source data 1
Excel file with data used to make Figure 6C and F.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Assembly of the SNARE complex measured by fluorescence anisotropy.
The SNARE complex assembly was performed with Syb2 (29–93, S61C-BDPY) and Syx1 in the presence of SN25 FL, SN25 W55W/R59C, or SN25 (11–206) E55W/R59C.
Figure 6—figure supplement 2
The intrinsic bimane fluorescence quenching by E55W mutant on SN25.
(A) Comparison of bimane fluorescence between SN25 R59C labeling and SN25 E55W/R59C labeling. (B) Comparison of bimane fluorescence between SN25 11–206 R59C labeling and SN25 11–206 E55W/R59C labeling. Quantification of the bimane fluorescence observed at 474 nm in (A, B) is shown on the bottom. All data are presented as mean ± SEM (n=3), technical replicates. Differences among groups by unpaired Student’s t-test. ***p<0.001.
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Figure 6—figure supplement 2—source data 1
Excel file with data used to make Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/79926/elife-79926-fig6-figsupp2-data1-v1.xlsx
Figure 7
Model of Rph3A function in prefusion steps of exocytosis.
Stage i, Rph3A is associated with trafficking vesicles via binding to GTP-Rab3A. Stage ii, Rph3A promotes vesicle docking via binding to plasma membrane-bound SN25 and vesicle-bound Rab3A together. Stage iii, upon binding to the N-peptide of SN25, Rph3A induces a conformational change of SN1 from random coils to α-helix. Stage iv, Rph3A accelerates SNARE complex assembly via SN25 interaction, which promote vesicle priming. As indicated, multiple SNARE regulatory proteins, for example, Munc18-1, Munc13-1, Doc2, and Syt1 are also involved in stages i–iv.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21 (DE3) | Agilent | Cat#: 230245 | Strain for expressing recombinant proteins |
| Cell line (Rattus Norvegicus) | PC-12 | The cell bank of the typical culture deposit committee of Chinese academy of sciences | Cat#: TCR 8 | |
| Cell line (human) | Human embryo kidney (HEK) 293T | ATCC | Cat#: CRL-3216; RRID: CVCL_0063 | |
| Transfected construct (Rattus Norvegicus) | L309-shRph3A | This paper | Lentiviral construct to transfect and express the shRNA for Rph3A knockdown | |
| Transfected construct (human) | L309-shSN25 | This paper | Lentiviral construct to transfect and express for SN25 knockdown | |
| Transfected construct (Rattus Norvegicus) | L309-Rph3A | This paper | Lentiviral construct to transfect and express the Rph3A | |
| Transfected construct (Rattus Norvegicus) | L309-Rph3A-K3 | This paper | Lentiviral construct to transfect and express the Rph3A-K3 | |
| Transfected construct (Rattus Norvegicus) | L309-Rph3A-GLAA | This paper | Lentiviral construct to transfect and express the Rph3A-GLAA | |
| Transfected construct (human) | L309-SN25 | This paper | Lentiviral construct to transfect and express the SN25 | |
| Transfected construct (human) | L309-SN25 11–206 | This paper | Lentiviral construct to transfect and express the SN25 11–206 | |
| Antibody | Anti-β-actin (Mouse monoclonal) | Proteintech | Cat#: 66009-1-Ig; RRID: AB_11232599 | WB (1:10,000) |
| Antibody | Anti-His6 (Mouse monoclonal) | Proteintech | Cat#: 66005-1-lg; RRID: AB_11232599 | WB (1:10,000) |
| Antibody | Anti-SN25 (Rabbit polyclonal) | Proteintech | Cat#: 14903-1-AP; RRID: AB_2192051 | WB (1:5000) |
| Antibody | Anti-Rph3A (Rabbit polyclonal) | Proteintech | Cat#: 11396-1-AP; RRID: AB_2181145 | WB (1:1000) |
| Antibody | Anti-Mouse IgG(H+L) (Goat polyclonal) | Proteintech | Cat#: SA00001-1; RRID: AB_2722565 | WB (1:10,000) |
| Antibody | Anti-Rabbit IgG(H+L) (Goat polyclonal) | Proteintech | Cat#: SA00001-2; RRID: AB_2722564 | WB (1:10,000) |
| Recombinant DNA reagent | pEXP5-NT/TOPO-Rph3A FL | This paper | 6xHis-tagged Rph3A FL (aa 1–681) for protein expression | |
| Recombinant DNA reagent | pEXP5-NT/TOPO-Rph3A (Δ185–371) | This paper | 6xHis-tagged Rph3A (Δ185–371) for protein expression | |
| Recombinant DNA reagent | pEXP5-NT/TOPO-Rph3A (Δ185–371) | This paper | 6xHis-tagged Rph3A (Δ185–371) for protein expression | |
| Recombinant DNA reagent | pEXP5-NT/TOPO-Rph3A (Δ185–371) G102A/L104A | This paper | 6xHis-tagged Rph3A (Δ185–371) G102A/L104A for protein expression | |
| Recombinant DNA reagent | pEXP5-NT/TOPO-Rph3A-K3 | This paper | 6xHis-tagged Rph3A K3 mutant for protein expression | |
| Recombinant DNA reagent | pEXP5-NT/TOPO-Rph3A-K4 | This paper | 6xHis-tagged Rph3A K4 mutant for protein expression | |
| Recombinant DNA reagent | pEXP5-NT/TOPO-Rph3A-GLAA | This paper | 6xHis-tagged Rph3A GLAA mutant for protein expression | |
| Recombinant DNA reagent | pET28a-Rph3A 1–281 | This paper | 6xHis-tagged Rph3A 1–281 for protein expression | |
| Recombinant DNA reagent | pET28a-Rph3A 182–681 | This paper | 6xHis-tagged Rph3A 182–681 for protein expression | |
| Recombinant DNA reagent | pET28a-Rph3A 282–681 | This paper | 6xHis-tagged Rph3A 282–681 for protein expression | |
| Recombinant DNA reagent | pET28a-Rph3A 372–681 | This paper | 6xHis-tagged Rph3A 372–681 for protein expression | |
| Recombinant DNA reagent | pET28a-SN25 | This paper | 6xHis-tagged SN25 for protein expression | |
| Recombinant DNA reagent | pET28a-SN25 11–206 | This paper | 6xHis-tagged SN25 11–206 for protein expression | |
| Recombinant DNA reagent | pGEX-6P-1-SN25 | This paper | GST-tagged SN25 for protein expression | |
| Recombinant DNA reagent | pGEX-6P-1-SN25 1–140 | This paper | GST-tagged SN25 1–140 for protein expression | |
| Recombinant DNA reagent | pGEX-6P-1-SN25 1–82 | This paper | GST-tagged SN25 1–82 for protein expression | |
| Recombinant DNA reagent | pGEX-6P-1-SN25 11–82 | This paper | GST-tagged SN25 11–82 for protein expression | |
| Recombinant DNA reagent | pGEX-6P-1-SN25 7–82 | This paper | GST-tagged SN25 7–82 for protein expression | |
| Software, algorithm | ImageJ | National Institutes of Health (NIH) | ||
| Software, algorithm | Prism 8.0.0 | GraphPad | ||
| Software, algorithm | Icy | BioImage Analysis unit Institut Pasteur |
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Rabphilin 3A binds the N-peptide of SNAP-25 to promote SNARE complex assembly in exocytosis
eLife 11:e79926.
https://doi.org/10.7554/eLife.79926
