mycoplasma lipophilum  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: The C7orf43/TRAPPC14 component links the TRAPPII complex to Rabin8 for preciliary vesicle tethering at the mother centriole during ciliogenesis

doi: 10.1074/jbc.RA119.008615

Figure Lengend Snippet: C7orf43 binds to the N-terminal region of Rabin8. A, left, silver stain of SDS-polyacrylamide gel (4–12% gradient) of LAP-tagged Rabin8 purified by TAP (anti-GFP antibodies, followed by S-tag beads) from RPE-1 Flp-In stably expressing cells. 14 equally spaced gel slices were cut and analyzed by LC-MS/MS from a single experiment. Right, full-length Rabin8– and Rabin81–142–associated proteins and TRAPPCII components and C7orf43 had high-percentage peptide coverage in gel slices analyzed by LC-MS/MS corresponding to their predicted molecular weight from RPE-1 and 293Trex cells. Shown is MS analysis of full-length Rabin8 from mIMCD3 and HEK293Trex cells and Rabin81–142 from HEK293Trex from Ref. 9. UP, unique peptides; %C, percentage of amino acid coverage from peptides identified. B, domain mapping of Rabin8 for C7orf43 binding. Left, GFP antibody immunoprecipitation (IP) of GFP-Rabin8 WT and truncated fragments with HA-C7orf43 co-expressed in HEK293 cells for 48 h. Blots were probed with HA and GFP antibodies. Representative results from four independent experiments are shown. Right, schematic representation of GFP-Rabin8 full-length and deletion constructs used in immunoprecipitation. C, in vitro binding assay pull-down of recombinant His-tagged C7orf43 by GST and GST-Rabin8. Representative results from two independent experiments are shown. D, domain mapping of C7orf43 binding to Rabin8. Left, HA-beads were used to immunoprecipitate GFP-Rabin8 using HA-tagged C7orf43 full-length and deletion fragments. Representative results from two independent experiments are shown. Right, schematic representation of HA-C7orf43 full-length and deletion constructs used.

Article Snippet: For endogenous immunoprecipitations with and without overexpressed proteins, low-salt Triton lysis buffer (30 m m Tris-HCl, pH 8.0, 75 m m NaCl, 10% glycerol, 1% Triton X-100 with 5 m m MgCl 2 , and protease and phosphatase inhibitors) was used to lyse cells, and a Rab3IP/Rabin8 (rabbit) (catalogue no. 12321-1-AP, Proteintech), C7orf43 (catalogue no. {"type":"entrez-protein","attrs":{"text":"PAB21203","term_id":"1236634272","term_text":"PAB21203"}} PAB21203 , Abnova), or GFP custom antibody was incubated with lysate overnight followed by the addition of Protein A/G magnetic beads and incubation for 3 h. Rab3IP/Rabin8 (mouse/clone OTI4A7) (catalogue no. TA808962, Origene) was used to blot for endogenous Rabin8 after immunoprecipitation using Protein A/G magnetic beads.

Techniques: Silver Staining, Purification, Stable Transfection, Expressing, Liquid Chromatography with Mass Spectroscopy, Molecular Weight, Binding Assay, Immunoprecipitation, Construct, In Vitro, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: The C7orf43/TRAPPC14 component links the TRAPPII complex to Rabin8 for preciliary vesicle tethering at the mother centriole during ciliogenesis

doi: 10.1074/jbc.RA119.008615

Figure Lengend Snippet: C7orf43 is required for ciliogenesis in human cells and zebrafish embryos. A, Western blot analysis of lysates from RPE-1 cells treated with control and C7orf43 siRNAs for 72 h. C7orf43 and β-actin antibodies were used for immunoblotting. Protein levels of C7orf43 compared with siControl were determined by densitometry analysis and are shown below blots normalized for differences in actin expression between RNAi treatments. Representative results from three independent experiments are shown. B and C, quantification of ciliation in NeoHDF (B) and RPE-1 (C) cells treated with C7orf43 siRNAs as in A and serum-starved for the final 24 h. Cells were stained with acetylated tubulin (Actub) and pericentrin antibodies and 4′,6-diamidino-2-phenylindole and imaged by epifluorescence microscopy. Representative images (right) are shown in B. Scale bar, 10 μm. Cells were counted for NeoHDF, siControl (n = 607) and siC7orf43 (n = 521) (B). Cells were counted for RPE-1, siControl (n = 1287), and siC7orf43 (n = 1070) (C). Means ± S.E. (error bars) are shown from three independent experiments. Two-tailed, unpaired Student's t test was used. *, p < 0.05; **, p < 0.01; ***, p < 0.001. D, Western blot analysis of lysates from 2-day postfertilization (dpf) zebrafish embryos injected with c7orf43 MO and probed with C7orf43 and β-actin antibodies. Representative results from two independent experiments are shown. E, panels show representative brightfield images of zebrafish embryos treated as in D from three or four independent experiments. Rescued embryos (right) were co-injected with both c7orf43 MO and human C7ORF43 RNA (hC7orf43). F–H, top, immunostaining of ciliated organs from 2-dpf embryos treated as in D showing ciliation. White arrowheads, neuromasts cilia. Bottom, quantification of organs with abnormal cilia in 2-dpf embryos. n > 14 olfactory vesicles; n > 27 neuromasts; n > 10 olfactory placodes. Note that ciliation was rescued upon injection of human C7orf43 mRNA, validating MO specificity. Two-tailed, unpaired Student's t test was used. Means ± S.E. are shown from three or four independent experiments. **, p < 0.01.

Article Snippet: For endogenous immunoprecipitations with and without overexpressed proteins, low-salt Triton lysis buffer (30 m m Tris-HCl, pH 8.0, 75 m m NaCl, 10% glycerol, 1% Triton X-100 with 5 m m MgCl 2 , and protease and phosphatase inhibitors) was used to lyse cells, and a Rab3IP/Rabin8 (rabbit) (catalogue no. 12321-1-AP, Proteintech), C7orf43 (catalogue no. {"type":"entrez-protein","attrs":{"text":"PAB21203","term_id":"1236634272","term_text":"PAB21203"}} PAB21203 , Abnova), or GFP custom antibody was incubated with lysate overnight followed by the addition of Protein A/G magnetic beads and incubation for 3 h. Rab3IP/Rabin8 (mouse/clone OTI4A7) (catalogue no. TA808962, Origene) was used to blot for endogenous Rabin8 after immunoprecipitation using Protein A/G magnetic beads.

Techniques: Western Blot, Control, Expressing, Staining, Epifluorescence Microscopy, Two Tailed Test, Injection, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: The C7orf43/TRAPPC14 component links the TRAPPII complex to Rabin8 for preciliary vesicle tethering at the mother centriole during ciliogenesis

doi: 10.1074/jbc.RA119.008615

Figure Lengend Snippet: C7orf43 is required for Rabin8 preciliary trafficking to the centrosome. A, live-cell imaging using spinning-disk confocal microscopy of 24-h transiently co-expressed LAP-C7orf43 and tRFP-Rabin8 in RPE-1 cells grown in the presence of serum or serum-starved for 1 h. Representative images are shown from three independent experiments. Scale bars, 5 μm. B, RPE-1 cells transiently expressing C7orf43-LAP and tRFP-Rabin8 for 24 h and serum-starved the last hour, fixed and stained with antibodies marking the centrosome (pericentrin) and Actub (cilia). Merged refers to green, red, and blue channels. The white dashed boxes in the left panels show the full cell corresponding to magnified panels to the right. The top left image displays a white outline of two cells, one with (top cell for magnified images in the top right panels) and the other without (bottom cell for magnified images in the middle right panels) tRFP-Rabin8 expression. The bottom left image displays a cell with a cilia (magnified images in the bottom right panels) expressing C7orf43-LAP and tRFP-Rabin8. Representative images are shown from three independent experiments. Scale bars, 5 μm (left image) and 0.5 μm (magnified right images). C, representative images from time lapses of RPE-1 GFP-Rabin8 cells treated with either siControl (left panels) or siC7orf43 (right panels), serum-starved for 1–2 h and imaged by live epifluorescence microscopy from three independent experiments. Scale bar, 10 μm. Top insets, zoom of GFP-Rabin8 centrosomal accumulation in cells 1, 2, and 3 at time point 0 s. Bottom insets, to better visualize vesicle movement, zoom images of insets from time points 0, 2, and 4 s were pseudocolored red (0 s), green (2 s), and blue (4 s), respectively, and merged. Scale bars, 5 μm. D, corresponding quantification of GFP-Rabin8 centrosomal and vesicular accumulation from C. Two-tailed, unpaired Student's t test was used. Means ± S.E. (error bars) are shown from three independent experiments with n > 150 cells quantified in total. **, p < 0.01; ***, p < 0.001. E, representative images of live RPE-1 cells stably expressing GFP-Rabin8 and tRFP-CENTRIN2, treated with either siControl or siC7orf43, serum-starved at 72 h post-transfection for 2 h to promote Rabin8 trafficking, and imaged by epifluorescence microscopy from three independent experiments. GFP-Rabin8 signal was inverted to better visualize membrane vesicles. White arrow, tRFP-CENTRIN2 localizing to mother and daughter centrioles. Scale bar, 5 μm.

Article Snippet: For endogenous immunoprecipitations with and without overexpressed proteins, low-salt Triton lysis buffer (30 m m Tris-HCl, pH 8.0, 75 m m NaCl, 10% glycerol, 1% Triton X-100 with 5 m m MgCl 2 , and protease and phosphatase inhibitors) was used to lyse cells, and a Rab3IP/Rabin8 (rabbit) (catalogue no. 12321-1-AP, Proteintech), C7orf43 (catalogue no. {"type":"entrez-protein","attrs":{"text":"PAB21203","term_id":"1236634272","term_text":"PAB21203"}} PAB21203 , Abnova), or GFP custom antibody was incubated with lysate overnight followed by the addition of Protein A/G magnetic beads and incubation for 3 h. Rab3IP/Rabin8 (mouse/clone OTI4A7) (catalogue no. TA808962, Origene) was used to blot for endogenous Rabin8 after immunoprecipitation using Protein A/G magnetic beads.

Techniques: Live Cell Imaging, Confocal Microscopy, Expressing, Staining, Epifluorescence Microscopy, Two Tailed Test, Stable Transfection, Transfection, Membrane

Journal: The Journal of Biological Chemistry

Article Title: The C7orf43/TRAPPC14 component links the TRAPPII complex to Rabin8 for preciliary vesicle tethering at the mother centriole during ciliogenesis

doi: 10.1074/jbc.RA119.008615

Figure Lengend Snippet: C7orf43 specifically associates with the TRAPPII complex. A, PSM and percent coverage (%C) values from MS analysis of C7orf43-binding proteins immunoprecipitated with a GFP antibody from HEK293 cells transiently expressing either LAP alone or C7orf43-LAP from a single experiment. B, immunoblot (IB) showing immunoprecipitation of endogenous C7orf43 from HEK293 cells with either IgG control or C7orf43 antibody. The blot was probed with antibodies against TRAPPC proteins as indicated on the right. Representative results from two independent experiments are shown. C, immunoprecipitation of LAP-tagged proteins from HEK293 cells transfected with LAP, TRAPPC10-LAP, or TRAPPC11-LAP is shown. GFP, C7orf43, TRAPPC9, and TRAPPC4 antibodies were used for immunoblotting. Representative results from two independent experiments are shown. D, domain mapping of HA-C7orf43 for TRAPPC10-LAP binding as performed in Fig. 1D. A representative blot from three independent experiments is shown. E, immunoblotting of size-exclusion chromatography on HEK293 cell lysate using Rabin8, C7orf43, and TRAPPC antibodies. Representative results from three independent experiments are shown. F, yeast two-hybrid analysis of the GAL4 DNA-binding domain (BD) fused to C7orf43 co-transformed with GAL4 activating domain (AD) control or TRAPPC fusions. Control double selection (leucine and tryptophan) and quadruple selection (leucine, tryptophan, histidine, and adenine) are shown. Five independent colonies are shown, and the results are representative from two or three independent experiments.

Article Snippet: For endogenous immunoprecipitations with and without overexpressed proteins, low-salt Triton lysis buffer (30 m m Tris-HCl, pH 8.0, 75 m m NaCl, 10% glycerol, 1% Triton X-100 with 5 m m MgCl 2 , and protease and phosphatase inhibitors) was used to lyse cells, and a Rab3IP/Rabin8 (rabbit) (catalogue no. 12321-1-AP, Proteintech), C7orf43 (catalogue no. {"type":"entrez-protein","attrs":{"text":"PAB21203","term_id":"1236634272","term_text":"PAB21203"}} PAB21203 , Abnova), or GFP custom antibody was incubated with lysate overnight followed by the addition of Protein A/G magnetic beads and incubation for 3 h. Rab3IP/Rabin8 (mouse/clone OTI4A7) (catalogue no. TA808962, Origene) was used to blot for endogenous Rabin8 after immunoprecipitation using Protein A/G magnetic beads.

Techniques: Binding Assay, Immunoprecipitation, Expressing, Western Blot, Control, Transfection, Size-exclusion Chromatography, Transformation Assay, Selection

Journal: The Journal of Biological Chemistry

Article Title: The C7orf43/TRAPPC14 component links the TRAPPII complex to Rabin8 for preciliary vesicle tethering at the mother centriole during ciliogenesis

doi: 10.1074/jbc.RA119.008615

Figure Lengend Snippet: C7orf43 is a TRAPPII complex component but is not required for the core complex integrity. A, immunoblotting of size-exclusion chromatography fractions performed using lysate from either siControl or siC7orf43(#1+#2)–treated HEK293 cells. Blots were probed with antibodies against C7orf43/TRAPPC14, TRAPPC proteins, and actin. Comparative protein ratios of siC7orf43/siControl were determined by densitometry and are shown below blots for expressing fractions. Representative results from two independent experiments are shown. B, immunoprecipitation analysis of transiently expressed TRAPPC10-LAP after siControl and siC7orf43/TRAPPC14(#1+#2) treatments in HEK293 cells. Blots were probed with antibodies against GFP and TRAPPC proteins. Protein levels compared with siControl in WCL and immunoprecipitations (IP) were determined by densitometry analysis and are shown below blots normalized for differences in GFP expression between RNAi treatments. Representative results from two independent experiments are shown. C, PSM and %C from MS analysis of immunoprecipitated TRAPPC10-LAP from transient expression in HEK293 cells after treatment with siControl or siC7orf43(#1+#2) for 72 h from a single experiment.

Article Snippet: For endogenous immunoprecipitations with and without overexpressed proteins, low-salt Triton lysis buffer (30 m m Tris-HCl, pH 8.0, 75 m m NaCl, 10% glycerol, 1% Triton X-100 with 5 m m MgCl 2 , and protease and phosphatase inhibitors) was used to lyse cells, and a Rab3IP/Rabin8 (rabbit) (catalogue no. 12321-1-AP, Proteintech), C7orf43 (catalogue no. {"type":"entrez-protein","attrs":{"text":"PAB21203","term_id":"1236634272","term_text":"PAB21203"}} PAB21203 , Abnova), or GFP custom antibody was incubated with lysate overnight followed by the addition of Protein A/G magnetic beads and incubation for 3 h. Rab3IP/Rabin8 (mouse/clone OTI4A7) (catalogue no. TA808962, Origene) was used to blot for endogenous Rabin8 after immunoprecipitation using Protein A/G magnetic beads.

Techniques: Western Blot, Size-exclusion Chromatography, Expressing, Immunoprecipitation