Journal: Infection and Immunity

Article Title: Surface Immunolabeling and Consensus Computational Framework To Identify Candidate Rare Outer Membrane Proteins of Treponema pallidum ▿ †

doi: 10.1128/IAI.00834-10

Figure Lengend Snippet: Predicted candidate T. pallidum rare outer membrane proteins

Article Snippet: Depicted is a cartoon representation of the model in which β-strands, loops, and α-helices are shown in yellow, green, and red, respectively. (B) Amino acid sequence alignment of TprC and TprK generated by MacVector using a PAM matrix.

Techniques: Membrane, Sequencing

Journal: Infection and Immunity

Article Title: Surface Immunolabeling and Consensus Computational Framework To Identify Candidate Rare Outer Membrane Proteins of Treponema pallidum ▿ †

doi: 10.1128/IAI.00834-10

Figure Lengend Snippet: (A) β-Barrel structure of TprC (TP0117) predicted by TMBpro (108). Depicted is a cartoon representation of the model in which β-strands, loops, and α-helices are shown in yellow, green, and red, respectively. (B) Amino acid sequence alignment of TprC and TprK generated by MacVector using a PAM matrix. Identical residues are shown in boxes; above the aligned sequences are the β-sheets predicted for TprC by TMBPro.

Article Snippet: Depicted is a cartoon representation of the model in which β-strands, loops, and α-helices are shown in yellow, green, and red, respectively. (B) Amino acid sequence alignment of TprC and TprK generated by MacVector using a PAM matrix.

Techniques: Sequencing, Generated

Journal: The Journal of Biological Chemistry

Article Title: VCIP135 associates with both the N- and C-terminal regions of p97 ATPase

doi: 10.1016/j.jbc.2023.105540

Figure Lengend Snippet: VCIP135 has two p97-binding sites. A , VCIP135 binds to p97 in a nucleotide-dependent manner. Endogenous p97 was biotinylated and immobilized onto streptavidin beads. The p97-immobilized beads (p97-beads; p97, 0.40 μg) were incubated with His-tagged VCIP135 (1.0 μg) in the presence of the indicated nucleotide (1 mM), and the bound proteins were fractionated by SDS-PAGE, followed by Western blotting with antibodies to p97 and His-tag. B , both the N- and the C-terminal halves of VCIP135 bind to p97. p97-immobilized beads (p97-beads; p97, 0.75 μg) were incubated with either His-tagged full-length VCIP135 (1.2 μg), its N-terminal half [(1-743), 1.1 μg] or C-terminal half [(744-1221), 0.60 μg] in the presence of 1 mM AMP-PNP. The blots were probed with antibodies to p97 and His-tag. C , the p97-binding site in the N-terminal half of VCIP135. p97-immobilized beads (p97-beads; p97, 0.50 μg) were incubated with either the N-terminal half of VCIP135 or its fragments [(1-743), 1.1 μg; (1-390), 0.67 μg; (361-743), 0.77 μg] in the presence of 1 mM AMP-PNP, and analyzed, as in ( B ). D , the p97-binding site in the C-terminal half of VCIP135. p97-immobilized beads (p97-beads; p97, 0.75 μg) were incubated with either the C-terminal half of VCIP135 or its fragments [(744-1221), 0.60 μg; (744-999), 0.33 μg; (903-1053), 0.50 μg; (1054-1221), 0.40 μg] in the presence of 1 mM AMP-PNP, and analyzed, as in ( B ). E , VCIP135 has two distinct p97-binding sites, (1-390) and (903-1053).

Article Snippet: B , amino acid sequence alignment of VCIP135 orthologues (rn, Rattus norvegicus ; hs, Homo sapiens ; gg, Gallus gallus ; xt, Xenopus tropicalis ; dr, Danio rerio ; sp, Strongylocentrotus purpuratus ; ed, Exaiptasia diaphana ; aq, Amphimedon queenslandica ).

Techniques: Binding Assay, Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: VCIP135 associates with both the N- and C-terminal regions of p97 ATPase

doi: 10.1016/j.jbc.2023.105540

Figure Lengend Snippet: The N- and C-terminal halves of VCIP135 bind to the C- and N-terminal regions of p97, respectively. A , full-length VCIP135 binds to both the N- and C-terminal regions of p97. GST-tagged p97 fragments (4.0 μg) were immobilized on glutathione beads and incubated with full-length VCIP135 (2.0 μg). The bound proteins were analyzed by Western blotting with antibodies to His-tag and GST-tag. B , the N-terminal half of VCIP135 binds to the C-terminal region of p97. GST-tagged p97 fragments (4.0 μg) immobilized on glutathione beads were incubated with the N-terminal half of VCIP135 [(1-743), 1.0 μg], and the bound proteins were analyzed, as in ( A ). C , the C-terminal half of VCIP135 binds to the N-terminal region of p97. GST-tagged p97 fragments (4.0 μg) immobilized on glutathione beads were incubated with the C-terminal half of VCIP135 [(744-1221), 1.0 μg], and the bound proteins were analyzed, as in ( A ).

Article Snippet: B , amino acid sequence alignment of VCIP135 orthologues (rn, Rattus norvegicus ; hs, Homo sapiens ; gg, Gallus gallus ; xt, Xenopus tropicalis ; dr, Danio rerio ; sp, Strongylocentrotus purpuratus ; ed, Exaiptasia diaphana ; aq, Amphimedon queenslandica ).

Techniques: Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: VCIP135 associates with both the N- and C-terminal regions of p97 ATPase

doi: 10.1016/j.jbc.2023.105540

Figure Lengend Snippet: The L133S mutation in the N-terminal p97-binding site of VCIP135 abolishes its binding to p97. A , VCIP135(1-743)(L133S) does not interact with p97 in the yeast two-hybrid experiment. Yeast cells expressing either AD-tagged VCIP135(1-743)wt or its mutant (L133S) and BD-tagged p97 were grown on a histidine-depleted selection plate containing 0.5 mM 3AT ( right panel ). B , amino acid sequence alignment of VCIP135 orthologues (rn, Rattus norvegicus ; hs, Homo sapiens ; gg, Gallus gallus ; xt, Xenopus tropicalis ; dr, Danio rerio ; sp, Strongylocentrotus purpuratus ; ed, Exaiptasia diaphana ; aq, Amphimedon queenslandica ). Identical residues between the homologues are marked with asterisks. The arrowhead indicates Leu-133 in rat VCIP135. C , the N-terminal half of VCIP135 with the L133S mutation has no binding affinity for p97. Either His-tagged VCIP135(1-743)wt or its L133S mutant (2.0 μg) was incubated with p97-immobilized beads (p97-beads; p97, 0.50 μg) in the presence of 1 mM AMP-PNP. The blots were probed with antibodies to p97 and the His-tag.

Article Snippet: B , amino acid sequence alignment of VCIP135 orthologues (rn, Rattus norvegicus ; hs, Homo sapiens ; gg, Gallus gallus ; xt, Xenopus tropicalis ; dr, Danio rerio ; sp, Strongylocentrotus purpuratus ; ed, Exaiptasia diaphana ; aq, Amphimedon queenslandica ).

Techniques: Mutagenesis, Binding Assay, Expressing, Selection, Sequencing, Incubation

Journal: The Journal of Biological Chemistry

Article Title: VCIP135 associates with both the N- and C-terminal regions of p97 ATPase

doi: 10.1016/j.jbc.2023.105540

Figure Lengend Snippet: The F1024L/L1031P mutation in the C-terminal p97-binding site of VCIP135 abolishes its binding to p97. A , VCIP135(744-1221) (F1024L, L1031P) does not interact with p97 in the yeast two-hybrid experiment. Yeast cells expressing either AD-tagged VCIP135(744-1221)wt or its mutant (F1024L/L1031P) and BD-tagged p97 were grown on a histidine-depleted selection plate containing 0.5 mM 3AT ( right panel ). B , amino acid sequence alignment of VCIP135 orthologues (rn, Rattus norvegicus : hs, Homo sapiens ; gg, Gallus gallus ; xt, Xenopus tropicali s; dr, Danio rerio ; sp, Strongylocentrotus purpuratus ; ed, Exaiptasia diaphana ; aq, Amphimedon queenslandica ). Identical residues between the homologues are marked with asterisks . The arrowheads indicate Phe-1024 and Leu-1031 in rat VCIP135. C , the C-terminal half of VCIP135 with the F1024L/L1031P mutation shows no binding affinity for p97. Either His-tagged VCIP135(744-1221)wt or its F1024L/L1031P mutant (1.0 μg) was incubated with p97-immobilized beads (p97-beads; p97, 0.50 μg) in the presence of 1 mM AMP-PNP. The blots were probed with antibodies to p97 and the His-tag. D , amino acid sequence alignment of VCIP135, p47, p37 and Ufd1. Identical residues are marked with asterisks . The arrowheads indicate Phe-1024 and Leu-1031 in VCIP135.

Article Snippet: B , amino acid sequence alignment of VCIP135 orthologues (rn, Rattus norvegicus ; hs, Homo sapiens ; gg, Gallus gallus ; xt, Xenopus tropicalis ; dr, Danio rerio ; sp, Strongylocentrotus purpuratus ; ed, Exaiptasia diaphana ; aq, Amphimedon queenslandica ).

Techniques: Mutagenesis, Binding Assay, Expressing, Selection, Sequencing, Incubation

Journal: The Journal of Biological Chemistry

Article Title: VCIP135 associates with both the N- and C-terminal regions of p97 ATPase

doi: 10.1016/j.jbc.2023.105540

Figure Lengend Snippet: Full-length VCIP135 associates with p97 through their two distinct interactions. A , full-length VCIP135 binds to p97 using either its N- or C-terminal binding site. Either His-tagged full-length VCIP135wt or its mutant (1.0 μg) was incubated with p97-immobilized beads (p97-beads; p97, 0.40 μg) in the presence of 1 mM AMP-PNP. The blots were probed with antibodies to p97 and His-tag. B , two full-length VCIP135 molecules can bind to one p97 molecule at its N- and C-terminal regions. GST-tagged VCIP135(F1024L/L1031P) (0.90 μg) was incubated with either His-tagged p97wt (1.2 μg) or p97 (199-806) (p97ΔN, 2.0 μg), and isolated on glutathione beads. The resulting beads were then incubated with His-tagged VCIP135wt (1.0 μg). All reactions were carried out in the presence of 1 mM AMP-PNP. The blots were probed with antibodies to GST-tag and His-tag. C , nucleotide dependency of the binding of the N-terminal binding site in VCIP135 to p97. p97-mmobilized beads (p97-beads; p97, 0.40 μg) were incubated with His-tagged VCIP135 (F1024L/L1031P) (1.0 μg) in the presence of the indicated nucleotide (1 mM). The blots were probed with antibodies to p97 and His-tag. D , nucleotide dependency of the binding of the C-terminal binding site in VCIP135 to p97. p97-immobilized beads (p97-beads; p97, 0.50 μg) were incubated with His-tagged VCIP135 (L133S) (1.0 μg) in the presence of the indicated nucleotide (1 mM), and analyzed as in ( C ).

Article Snippet: B , amino acid sequence alignment of VCIP135 orthologues (rn, Rattus norvegicus ; hs, Homo sapiens ; gg, Gallus gallus ; xt, Xenopus tropicalis ; dr, Danio rerio ; sp, Strongylocentrotus purpuratus ; ed, Exaiptasia diaphana ; aq, Amphimedon queenslandica ).

Techniques: Binding Assay, Mutagenesis, Incubation, Isolation

Journal: The Journal of Biological Chemistry

Article Title: VCIP135 associates with both the N- and C-terminal regions of p97 ATPase

doi: 10.1016/j.jbc.2023.105540

Figure Lengend Snippet: The two binding interactions between VCIP135 and p97 are required for Golgi biogenesis in vivo . A , rescue experiments in VCIP135 siRNA-treated cells. HeLa cells were either mock transfected with water or transfected with siRNA duplexes specific to human VCIP135, and cultured for 46 h. In some groups, the indicated HA-tagged VCIP135wt/mutants, which were insensitive to VCIP135 siRNA, were expressed 18 h after the treatment of the cells with VCIP135 siRNA, and the cells were further cultured for 28 h. Levels of endogenous and exogenous VCIP135 in the cultured cells are presented in Fig. S2 A . The resulting cells were fixed and stained with a polyclonal antibody to GM130 ( panels a–f ) and a monoclonal antibody to HA ( panels g–j ). The treatment of cells with VCIP135 siRNA caused Golgi fragmentation ( panel b ). In VCIP135 siRNA-treated cells, the Golgi morphology was rescued by the expression of VCIP135wt ( panel c , white arrowheads ), but not by the expression of VCIP135 mutants in which either or both of the N- and C-terminal p97-binding sites were mutated ( panels d – f ). Scale bar = 5 μm. B , results of the quantification of ( A ). Results are expressed as the mean ± SD of six sets of independent experiments, with 100 cells counted in each group of each set. Asterisks indicate a significant difference at p < 0.01 compared with mock-treated cells (Bonferroni method).

Article Snippet: B , amino acid sequence alignment of VCIP135 orthologues (rn, Rattus norvegicus ; hs, Homo sapiens ; gg, Gallus gallus ; xt, Xenopus tropicalis ; dr, Danio rerio ; sp, Strongylocentrotus purpuratus ; ed, Exaiptasia diaphana ; aq, Amphimedon queenslandica ).

Techniques: Binding Assay, In Vivo, Transfection, Cell Culture, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: VCIP135 associates with both the N- and C-terminal regions of p97 ATPase

doi: 10.1016/j.jbc.2023.105540

Figure Lengend Snippet: The two binding interactions between VCIP135 and p97 are necessary for ER biogenesis in vivo . A , effects of the expression of VCIP135 mutants on ER structure in VCIP135 siRNA-treated cells. The HeLa cells stably expressing GFP-tagged HSP47 were used, and their ER structures were observed in living cells. The HeLa cells were either mock transfected with water or transfected with siRNA duplexes specific to human VCIP135 and cultured for 44 h. In some groups, the indicated mCherry2-fused VCIP135wt/mutants, which were insensitive to VCIP135 siRNA, were expressed 16 h after the treatment of cells with VCIP135 siRNA, and the cells were further cultured for 28 h. Levels of endogenous and exogenous VCIP135 in the cultured cells are presented in Fig. S2 B . Cells expressing mCherry2-fused VCIP135 were used to obtain images. Confocal images of cultured cells were obtained at the height half way between the base and equatorial plane of the nucleus. In VCIP135 siRNA-treated cells, the fine-meshed network of the ER was found to be disrupted in numerous areas, resulting in the appearance of large holes ( panel b ). The ER morphology was rescued by the expression of VCIP135wt ( panel c ), but not by the expression of VCIP135 mutants in which either or both of the N- and C-terminal p97-binding sites were mutated (panels d-f). Scale bar = 5 μm. B , results of the quantification of ( A ). Results are expressed as the mean ± SD of five sets of independent experiments, with 100 cells counted in each group of each set. Asterisks indicate a significant difference at p < 0.01 compared with mock-treated cells (Bonferroni method).

Article Snippet: B , amino acid sequence alignment of VCIP135 orthologues (rn, Rattus norvegicus ; hs, Homo sapiens ; gg, Gallus gallus ; xt, Xenopus tropicalis ; dr, Danio rerio ; sp, Strongylocentrotus purpuratus ; ed, Exaiptasia diaphana ; aq, Amphimedon queenslandica ).

Techniques: Binding Assay, In Vivo, Expressing, Stable Transfection, Transfection, Cell Culture