Journal:

Article Title: The Sesquiterpene Synthase from the Botrydial Biosynthetic Gene Cluster of the Phytopathogen Botrytis cinerea

doi: 10.1021/cb800225v

Figure Lengend Snippet: Biosynthesis of botrydial by Botrytis cinerea. a. Cyclization of farnesyl diphosphate (FPP) to presilphiperfolan-8β-ol (10) catalyzed by the BcBOT2 protein and biosynthesis of botrydial from 10. B. cinerea mutant bcbot1 does not form botrydial and other late stage intermediates but does accumulate 9 and other probotryanes, while mutant bcbot2 is blocked in the entire pathway. b. Cyclization of [1,1-2H2]farnesyl diphosphate to [5,5-2H2]presilphiperfolan-8β-ol.

Article Snippet: MALDI-TOF analysis of recombinant His 6 -tag-BcBOT2 protein was carried out on an Applied Biosystems Voyager-DE Pro spectrometer with bovine serum albumin (M D 66431) added as an internal standard: M D of 47221 ± 50 (calcd for His 6 -tag-BcBOT2 minus N -terminal Met-Gly M D 47246.)

Techniques: Mutagenesis

Journal:

Article Title: The Sesquiterpene Synthase from the Botrydial Biosynthetic Gene Cluster of the Phytopathogen Botrytis cinerea

doi: 10.1021/cb800225v

Figure Lengend Snippet: Metabolites identified in Botrytis cinerea wild-type strains, recipient strains ( ku mutants) and bcbot2 mutants (numbers correspond to compounds in ).

Article Snippet: MALDI-TOF analysis of recombinant His 6 -tag-BcBOT2 protein was carried out on an Applied Biosystems Voyager-DE Pro spectrometer with bovine serum albumin (M D 66431) added as an internal standard: M D of 47221 ± 50 (calcd for His 6 -tag-BcBOT2 minus N -terminal Met-Gly M D 47246.)

Techniques: Mutagenesis

Journal:

Article Title: The Sesquiterpene Synthase from the Botrydial Biosynthetic Gene Cluster of the Phytopathogen Botrytis cinerea

doi: 10.1021/cb800225v

Figure Lengend Snippet: Virulence of B. cinerea strains on bean and tomato leaves illustrated in . Values are means and standard deviation of at least 15 lesions resulting from 3 independent assays with 5 to 7 independent leaves per strain or mutant.

Article Snippet: MALDI-TOF analysis of recombinant His 6 -tag-BcBOT2 protein was carried out on an Applied Biosystems Voyager-DE Pro spectrometer with bovine serum albumin (M D 66431) added as an internal standard: M D of 47221 ± 50 (calcd for His 6 -tag-BcBOT2 minus N -terminal Met-Gly M D 47246.)

Techniques: Standard Deviation, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ^♦

doi: 10.1074/jbc.M113.542159

Figure Lengend Snippet: PDCD5 interacts with β-tubulin U2OS cells were transfected with FLAG-TEV-PDCD5 or empty vector. PDCD5 immunoprecipitates were analyzed for binding partners by tandem mass spectrometry. This table displays significant hits found in the proteomics screen. The values in the first 2 columns indicate the normalized spectral counts for each sample. Values in the 3rd column indicate the significance of the peptide hits (Decibans were calculated from Bayes factors).

Article Snippet: For recombinant protein purification, His 6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR.

Techniques: Transfection, Plasmid Preparation, Binding Assay, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ^♦

doi: 10.1074/jbc.M113.542159

Figure Lengend Snippet: PDCD5 interacts with PhLP1 HEK-293T cells were transfected with PhLP-TEV-myc or Pdc-TEV-myc for comparison. An empty vector transfection served as a negative control. Myc immunoprecipitates were analyzed for binding partners by tandem mass spectrometry. This table displays significant hits found in the proteomics screen. The numbers in the first 3 columns indicate the normalized spectral counts for each sample. Values in the 4th column indicate the significance of the peptide hits.

Article Snippet: For recombinant protein purification, His 6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR.

Techniques: Transfection, Plasmid Preparation, Negative Control, Binding Assay, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ^♦

doi: 10.1074/jbc.M113.542159

Figure Lengend Snippet: Three-dimensional reconstruction of the PDCD5·CCT complex. A, representative area of a micrograph of a vitrified sample of PDCD5·CCT complexes. Green circles show end-on views of the complex, and red rectangles mark side views. B, gallery of selected images of PDCD5·CCT complexes. C, average images corresponding to different classes used for the generation of the first volume. D, selected projection views (top panel) and the corresponding class averages (bottom panel) of the final model. E, Fourier shell correlation (FSC) plot of the PDCD5-CCT reconstruction showing 25 Å resolution at a 0.5 Fourier shell correlation value.

Article Snippet: For recombinant protein purification, His 6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ^♦

doi: 10.1074/jbc.M113.542159

Figure Lengend Snippet: PDCD5 forms a complex with PhLP1 and CCT. A, binding of PDCD5 to phosducin family members was measured by co-immunoprecipitation from HEK-293T cells transfected with PDCD5-FLAG along with Myc-tagged phosducin family members as indicated. After 48 h, cells were lysed, immunoprecipitated with an anti-Myc antibody, and immunoblotted for PDCD5-FLAG. B, binding of purified PDCD5 to PhLP1 or CK2-phosphorylated PhLP1 was assessed by co-immunoprecipitation in vitro. Phosphorylated PhLP1, unphosphorylated PhLP1, or no PhLP1 was incubated with PDCD5, immunoprecipitated with an Myc antibody, and blotted as indicated. C, simultaneous binding of PDCD5 and PhLP1 was measured by co-immunoprecipitation. HEK-293T cells were transfected with PDCD5-FLAG or empty vector, immunoprecipitated with FLAG, and blotted for endogenous PhLP1 and CCTϵ (left panel). Endogenous CCTϵ was also immunoprecipitated and blotted for endogenous PhLP1 and PDCD5-FLAG (right panel). A nontargeting Myc antibody served as a negative control. D, effect of CCT knockdown on PDCD5 binding to PhLP1 was measured by co-immunoprecipitation of PhLP1-Myc from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5 and PhLP1-Myc. The ratio of the PDCD5 band to the PhLP1 band was calculated and normalized to the control. Bars represent the average ± S.E. of the mean from at least three experiments. Representative blots are shown below the graphs. E, formation of a PhLP1·PDCD5·CCT complex was demonstrated in double immunoprecipitation experiments from HEK-293T cells transfected with PDCD5-FLAG along with PhLP1-TEV-Myc or empty vector.

Article Snippet: For recombinant protein purification, His 6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR.

Techniques: Binding Assay, Immunoprecipitation, Transfection, Purification, In Vitro, Incubation, Plasmid Preparation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ^♦

doi: 10.1074/jbc.M113.542159

Figure Lengend Snippet: PhLP1 and PDCD5 bind CCT independently of each other. PDCD5 was either overexpressed (A) or knocked down (B), along with PhLP1-Myc overexpression in HEK-293T cells. Cell lysates were immunoprecipitated with anti-CCTϵ (A) or anti-Myc (B) and blotted as indicated. PhLP1 was either overexpressed (C) or knocked down (D), along with PDCD5-FLAG overexpression in HEK-293T cells. Cell lysates were immunoprecipitated with anti-CCTϵ (C) or anti-FLAG (D) and blotted as indicated. Bars represent the average ± S.E. from at least three experiments. Cell lysates were blotted for PDCD5-FLAG, endogenous PDCD5, PhLP1-Myc, or endogenous PhLP1 as indicated to verify the overexpression and knockdowns. Representative blots are shown below the graphs.

Article Snippet: For recombinant protein purification, His 6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR.

Techniques: Over Expression, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ^♦

doi: 10.1074/jbc.M113.542159

Figure Lengend Snippet: PDCD5 inhibits β-tubulin folding. A, rate of association or dissociation from CCT complexes was measured by pulse-chase immunoprecipitations of CCTϵ from HEK-293T cells transfected with PDCD5-FLAG. The rate of association of CCTα and -γ subunits (black, t½ = 112 ± 18 min) and PDCD5 (red, t½= 44 ± 2 min) was calculated along with the rate of dissociation for tubulin (blue, t½ = 39 ± 1 min). B, binding of β-tubulin to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells transfected with FLAG-PDCD5 or empty vector. C, effect of CCT knockdown on β-tubulin binding to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5. The ratio of the β-tubulin band to the PDCD5 band was calculated and normalized to the control. D, folding of the indicated proteins by CCT was measured by pulse-chase co-immunoprecipitations from HEK-293T cells treated with PDCD5 siRNA or negative control as indicated (see “Experimental Procedures”). E and F, effect of PDCD5 knockdown (E) or overexpression (F) on β-tubulin binding to CCT was measured by co-immunoprecipitation with CCTϵ and immunoblotting as indicated. The ratio of the β-tubulin band to the CCTϵ band was calculated and normalized to the control. In all experiments, bars represent the average ± S.E. from at least three experiments. Representative gels or blots are shown below each graph. PDCD5 knockdown averaged between 65 and 80% as measured by immunoblotting.

Article Snippet: For recombinant protein purification, His 6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR.

Techniques: Pulse Chase, Transfection, Binding Assay, Immunoprecipitation, Plasmid Preparation, Negative Control, Over Expression, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ^♦

doi: 10.1074/jbc.M113.542159

Figure Lengend Snippet: PDCD5 binds CCT near the δ subunit. A, gel analysis of the PDCD5·CCT complex. PDCD5 was mixed with CCT at a 10:1 molar ratio in the absence of nucleotide, and the mixture was resolved on a native polyacrylamide gel. The CCT oligomer (∼960 kDa), which runs with a mobility clearly distinct from that of PDCD5 (∼14 kDa), was excised and run on a denaturing acrylamide gel revealing bands corresponding to the eight CCT subunits and a band corresponding to PDCD5. B–D, average electron microscopy images obtained from negatively stained particles. B, PDCD5-CCT; C, apo-CCT; D, anti-CCTδ-PDCD5. Images were averaged from 1356, 1128, and 1018 particles, respectively.

Article Snippet: For recombinant protein purification, His 6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR.

Techniques: Acrylamide Gel Assay, Electron Microscopy, Staining

Journal: The Journal of Biological Chemistry

Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ^♦

doi: 10.1074/jbc.M113.542159

Figure Lengend Snippet: PDCD5 binds the apical domain of one CCT subunit. A, two orthogonal views of the three-dimensional reconstruction of the PDCD5·CCT complex carried out with 13,000 particles at 25 Å resolution. B, same two views showing the docking of the crystal structure of the open form of CCT colored by subunit (Protein Data Bank code 2XSM) and the atomic structure of PDCD5 in green (Protein Data Bank code 2K6B). C, binding of various C-terminal truncations of PDCD5 to CCT was measured by co-immunoprecipitation and immunoblotting.

Article Snippet: For recombinant protein purification, His 6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR.

Techniques: Binding Assay, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ^♦

doi: 10.1074/jbc.M113.542159

Figure Lengend Snippet: Unnatural amino acid incorporation into PDCD5. A, scheme of unnatural amino acid UV cross-linking using BzF. B, BzF incorporation into Amber codon variants of PDCD5 and the G protein β-subunit. HEK-293T cells were transfected with the indicated FLAG-PDCD5 constructs, BzF tRNA and BzF amino acid synthetase, and treated with 1 mm BzF 3 h post-transfection. Lysates were immunoblotted to determine the level of expression of the variants. The percent incorporation was measured as the ratio of the truncated protein band, resulting from a lack of BzF incorporation into the Amber codon, to the full-length protein band, resulting from BzF incorporation into the Amber codon. PDCD5 averaged 6% incorporation compared with 75% for Gβ1.

Article Snippet: For recombinant protein purification, His 6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR.

Techniques: Transfection, Construct, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ^♦

doi: 10.1074/jbc.M113.542159

Figure Lengend Snippet: PDCD5 interacts directly with CCTβ. A, cross-linking of PDCD5 to CCT. HEK-293T cells were transfected with the indicated FLAG-PDCD5 constructs, BzF tRNA, and BzF amino acid synthetase and treated with 1 mm BzF 3 h post-transfection. Lysates were exposed ± UV light prior to immunoprecipitation and blotting for FLAG-PDCD5. The asterisk indicates a PDCD5 cross-linked band. HC, heavy chain. B, cross-linking of PDCD5 to the CCT subunits was measured by co-immunoprecipitation from HEK-293T cells treated as in A and exposed to UV light prior to immunoprecipitation with a CCTϵ antibody and blotting for each of the CCT subunits. The asterisk indicates a CCTβ cross-linked band. The arrows mark the position of the CCT subunits. NS, nonspecific band. C, PDCD5 cross-linking to CCTβ depends on an interaction with the PDCD5 C terminus. HEK-293T cells were treated as in A and lysates were exposed ± UV light prior to immunoprecipitation with a CCTϵ antibody and blotting for CCTβ. The asterisk indicates a CCTβ cross-linked band. D, BzF catalyzes PDCD5 cross-linking to CCT without incorporation into Amber codon sites. HEK-293T cells were transfected with FLAG-PDCD5 (WT) in the absence of BzF tRNA and BzF AA synthetase. Cells were treated ± 1 mm BzF 3 h post-transfection. Lysates were exposed ± UV light prior to immunoprecipitation with CCTϵ and immunoblotting for CCTβ. The asterisk indicates a CCTβ cross-linked band.

Article Snippet: For recombinant protein purification, His 6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR.

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot