Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 1. The amino acid residues of hCRM1 required for Rex ac- tivity. (A) The ability of CRM1s to support Rex activity. HeLa cells were transfected with the indicated plasmids. After cell lysis, the amount of CAT and the activity of -Gal were measured and CAT/ -Gal ratios were calculated. The ratio for the control sample without pSRTAgRexM64 and pSRCRM1s was arbitrarily set at 1. The amount of CAT and the -Gal activity in control samples were over 300 pg and 3.0 103 U, respectively. Error bars represent standard deviations. (B) Restoration of Rex activity by overexpression of CRM1s in HeLa cells. The experimental procedure was the same as that described for panel A. (C) Effect of overexpressing CRM1s on Rex activity in REF52 cells. REF52 cells were transfected with the indicated plasmids. At 24 h of posttransfection, CAT/-Gal ratios were calculated. The ratio for the sample without pSRCRM1s was arbi- trarily set at 1. (D) Western blot analysis of various CRM1s. A fraction of each sample used in the experiments described for panel C was subjected to Western blot analysis using the anti-hCRM1 antibody to examine CRM1 protein synthesis. This antibody was raised with the peptide, which represents the carboxy-terminal region of hCRM1 and has a sequence different from that of rCRM1, so it does not recognize endogenous rCRM1. (E) Effect of CRM1s on Rex-mediated Gag expression from HTLV-1 molecular clone. At 48 h posttransfection, Gag/-Gal ratios were calculated. The ratio for the sample without pSRCRM1s was arbitrarily set at 1. (F) A schematic representation of hCRM1 functional domains. The amino acid sequence of hCRM1 is shown. Experiments characterizing the RanGTP binding domain (bro- ken line) (33), LMB binding residue (arrowhead) (24), Rev-interacting amino acids (asterisks) (2), and the domain binding to NES (under- line) (33) have been previously reported. The residues 411, 414, 474, and 481 in hCRM1 are indicated in bold characters, and the corre- sponding residues of rCRM1 are indicated under the hCRM1 se- quence in single-letter amino acid code.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: Activity Assay, Transfection, Lysis, Control, Over Expression, Western Blot, Sequencing, Expressing, Functional Assay, Binding Assay, Residue

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 2. In vivo interaction of Rex with CRM1 mutants in which one amino acid is replaced. REF52 cells were transfected with the plasmid expressing GAL-CRM1s in combination with pRexVP, pG5BCAT, and pCDM-gal. The cells were harvested and subjected to CAT and -Gal assays, and CAT/-Gal ratios were calculated. The ratio for the control sample, which detected the interaction between GAL-hCRM1 and Rex-VP, was arbitrarily set at 1. The amount of CAT and -Gal activity in control samples were over 400 pg and 3.0 103 U, respec- tively. GAL4 nonfusion protein, expressing only a GAL4 region, was used as a negative control. A fraction of each sample was subjected to Western blot analysis using the anti-GAL4 monoclonal antibody to examine GAL-CRM1 expression.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Control, Activity Assay, Negative Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein

doi: 10.1128/mcb.23.23.8751-8761.2003

Figure Lengend Snippet: FIG. 6. Binding characterization of two amino acid-substituted CRM1s. (A) In vivo interaction of CRM1s with Rex. REF52 cells were treated as described for Fig. 2 except for the plasmids that expressed two amino acid-substituted CRM1s as a GAL4 fusion protein. A portion of each sample was subjected to Western blot analysis to confirm GAL-CRM1 expression. (B) In vitro binding of h411/414 to RanBP3. As shown in Fig. 5A, in vitro-translated CRM1s were incubated with GST or GST-RanBP3 immobilized on glutathione-Sepharose 4B.

Article Snippet: Mouse anti-GAL4 monoclonal antibody (Santa Cruz Biotechnology) and affinity-purified chicken antihCRM1 antibody (18) were used as primary antibodies to detect GAL-fused proteins and CRM1s, respectively.

Techniques: Binding Assay, In Vivo, Western Blot, Expressing, In Vitro, Incubation

Journal: PLoS Genetics

Article Title: Mechanistic insights into global suppressors of protein folding defects

doi: 10.1371/journal.pgen.1010334

Figure Lengend Snippet: (A) CcdB proteins exhibit biphasic refolding kinetics with a fast and slow phase whereas (B) unfolding of CcdB proteins follows single exponential kinetics. The experimental kinetic traces obtained at different GdnCl concentrations are shown in black, while the fits are shown in red. (C) Interaction between native (black), native protein in GdnCl (grey) and refolded (white) CcdB mutant proteins and labeled GyrA14 analyzed by MST. (D) The difference in apparent thermal melting temperatures ( Δ T m = T m M u t a n t − T m W T ) for native proteins in GdnCl (cyan), and refolded proteins (grey). The error bars wherever shown represent the standard deviation from two independent experiments, each performed in duplicates. (E) Binding of 500 nM WT and mutant CcdB to immobilised GyrA14 measured by passing the same concentrations of the analyte (CcdB proteins), after heat stress at two different temperature (40 and 80°C), followed by cooling back to 25°C. A room temperature control (25°C) was also used. The residual active fraction was calculated as described in the materials section. (F) The SEC profiles of a few of the CcdB mutants are shown. The PIM L36A shows aggregation as well as degradation as compared to the WT, and E11R and S12G suppressors. The L36A-S12G has a similar profile like the WT and S12G. Kinetic parameters from the fits are listed in S3 Table and values extrapolated to zero denaturant are listed in S4 Table .

Article Snippet: Briefly, the amount of CcdB protein expressed on the yeast cell surface was estimated by chicken anti-HA antibodies from Bethyl labs (1˸600 dilution) and the GyrA14 binding activity on the yeast cell surface was estimated by incubating the induced CcdB mutants with 100 nM FLAG tagged GyrA14, followed by washing with FACS buffer and subsequent incubation with mouse anti-FLAG antibodies, at a dilution ratio of 1˸300 as described previously [ ].

Techniques: Mutagenesis, Labeling, Standard Deviation, Binding Assay, Control

Journal: PLoS Genetics

Article Title: Mechanistic insights into global suppressors of protein folding defects

doi: 10.1371/journal.pgen.1010334

Figure Lengend Snippet: (A) Analysis of yeast cell surface expression and GyrA14 binding of different CcdB mutants and WT. CcdB WT and mutant plots (blue), are overlaid with plot of uninduced cells (black). In the last two panels, V20F-M32T, L36A-M32T plots (red) are overlaid with plots of V20F-L42E, L36A-L42E (purple) and V20F-S43T, L36A-S43T (green), only M32T is able to suppress the deleterious effects of the V20F and L36A mutations. (B-I) Kinetic and thermodynamic characterisation of CcdB mutants. (B) Thermal unfolding profiles of 5 μM of CcdB-WT, M32T, L42E and S43T mutants carried out by nanoDSF. (C) Equilibrium GdnCl denaturation profiles of 5 μM of CcdB-WT, M32T, L42E and S43T mutants carried out by nanoDSF. (D) Difference in thermal ΔT m ( Δ T m = T m M u t a n t − T m W T ) (in cyan), and thermodynamic stability assayed by chemical denaturation, ΔΔG° ( Δ Δ G ° = Δ G M u t a n t ° − Δ G W T ° ) (in grey) of the CcdB mutants. (E) Thermal unfolding profiles of 5 μM of native proteins in 1.5 M GdnCl (dotted lines) and refolded CcdB proteins in the same concentration of GdnCl (dashed lines). (F-G) The observed rate constants and amplitudes of the fast phase (black) and slow phase (grey) of refolding (1.5 M GdnCl) of WT and CcdB mutants (see also S9 Table ). (H-I) The observed rate constants and amplitudes of unfolding (3.5 M GdnCl) of WT and CcdB mutants. The error bars wherever shown represent the standard deviation from two independent experiments, each performed in duplicates.

Article Snippet: Briefly, the amount of CcdB protein expressed on the yeast cell surface was estimated by chicken anti-HA antibodies from Bethyl labs (1˸600 dilution) and the GyrA14 binding activity on the yeast cell surface was estimated by incubating the induced CcdB mutants with 100 nM FLAG tagged GyrA14, followed by washing with FACS buffer and subsequent incubation with mouse anti-FLAG antibodies, at a dilution ratio of 1˸300 as described previously [ ].

Techniques: Expressing, Binding Assay, Mutagenesis, Nano Differential Scanning Fluorimetry, Concentration Assay, Standard Deviation