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recombinant yeast or human gst-uba1/ube1  (R&D Systems)


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    R&D Systems recombinant yeast or human gst-uba1/ube1
    A126 mutations disrupt enzymatic activity and stability of Rad6. A , immunoblot of products of an in vitro ubiquitination assay with <t>recombinant</t> WT Rad6 or indicated mutants. Enzyme was incubated at 30 °C for the indicated time along with ubiquitin (Ub), Uba1, ATP/Mg 2+ , and yeast histone H2B (substrate). The reaction mix was then resolved by SDS-PAGE prior to immunoblotting. The asterisk indicates a cross-reacting protein. The catalytic-dead mutant rad6-C88A served as a control. B , left , immunoblot of Flag-tagged WT or mutant Rad6. A two-fold serial gradient of the extracts prepared from the indicated strains were resolved by SDS-PAGE prior to immunoblotting. Extract from the strain expressing Rad6 served as a ‘no tag’ control. Right , plot of mean fold-change in the steady-state levels of A126 mutant relative to WT Rad6 (± SEM from two independent experiments) based on densitometry quantitation, for which the signals in the numbered lanes were used. The signals for WT or mutant Rad6 were initially normalized to the signals for Pgk1, which serves as a loading control. C , left , immunoblot for analysis of stability of Flag epitope–tagged Rad6 or mutants grown in a medium containing raffinose (+Raf; uninduced) or galactose (+Gal; induced) and at different time points after transcription from the GAL1 promoter was halted by adding glucose. Pgk1 served as loading control and for normalization. Right , plot shows densitometric values for WT or mutant Rad6 protein at different time points after glucose-mediated transcriptional inhibition relative to that in the induced state (+Gal, 0 min). Asterisk , p-value <0.05 (Student’s t test). D , growth assay was conducted by spotting ten-fold serial dilution of the indicated yeast strains on synthetic medium without tryptophan (-TRP) or in medium containing 6% ethanol or 2% formamide and incubated at 30 °C. Cells spotted on -TRP medium were also subjected to heat stress by incubation at 37 °C. Rad6, Radiation sensitive 6; Ub 1 , monoubiquitinated H2B; Ub n , polyubiquitinated H2B.
    Recombinant Yeast Or Human Gst Uba1/Ube1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+gst+uba1+ube1/human+ube1+e1/pmc09630792-382-17-23
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    1) Product Images from "Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability"

    Article Title: Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.102524

    A126 mutations disrupt enzymatic activity and stability of Rad6. A , immunoblot of products of an in vitro ubiquitination assay with recombinant WT Rad6 or indicated mutants. Enzyme was incubated at 30 °C for the indicated time along with ubiquitin (Ub), Uba1, ATP/Mg 2+ , and yeast histone H2B (substrate). The reaction mix was then resolved by SDS-PAGE prior to immunoblotting. The asterisk indicates a cross-reacting protein. The catalytic-dead mutant rad6-C88A served as a control. B , left , immunoblot of Flag-tagged WT or mutant Rad6. A two-fold serial gradient of the extracts prepared from the indicated strains were resolved by SDS-PAGE prior to immunoblotting. Extract from the strain expressing Rad6 served as a ‘no tag’ control. Right , plot of mean fold-change in the steady-state levels of A126 mutant relative to WT Rad6 (± SEM from two independent experiments) based on densitometry quantitation, for which the signals in the numbered lanes were used. The signals for WT or mutant Rad6 were initially normalized to the signals for Pgk1, which serves as a loading control. C , left , immunoblot for analysis of stability of Flag epitope–tagged Rad6 or mutants grown in a medium containing raffinose (+Raf; uninduced) or galactose (+Gal; induced) and at different time points after transcription from the GAL1 promoter was halted by adding glucose. Pgk1 served as loading control and for normalization. Right , plot shows densitometric values for WT or mutant Rad6 protein at different time points after glucose-mediated transcriptional inhibition relative to that in the induced state (+Gal, 0 min). Asterisk , p-value <0.05 (Student’s t test). D , growth assay was conducted by spotting ten-fold serial dilution of the indicated yeast strains on synthetic medium without tryptophan (-TRP) or in medium containing 6% ethanol or 2% formamide and incubated at 30 °C. Cells spotted on -TRP medium were also subjected to heat stress by incubation at 37 °C. Rad6, Radiation sensitive 6; Ub 1 , monoubiquitinated H2B; Ub n , polyubiquitinated H2B.
    Figure Legend Snippet: A126 mutations disrupt enzymatic activity and stability of Rad6. A , immunoblot of products of an in vitro ubiquitination assay with recombinant WT Rad6 or indicated mutants. Enzyme was incubated at 30 °C for the indicated time along with ubiquitin (Ub), Uba1, ATP/Mg 2+ , and yeast histone H2B (substrate). The reaction mix was then resolved by SDS-PAGE prior to immunoblotting. The asterisk indicates a cross-reacting protein. The catalytic-dead mutant rad6-C88A served as a control. B , left , immunoblot of Flag-tagged WT or mutant Rad6. A two-fold serial gradient of the extracts prepared from the indicated strains were resolved by SDS-PAGE prior to immunoblotting. Extract from the strain expressing Rad6 served as a ‘no tag’ control. Right , plot of mean fold-change in the steady-state levels of A126 mutant relative to WT Rad6 (± SEM from two independent experiments) based on densitometry quantitation, for which the signals in the numbered lanes were used. The signals for WT or mutant Rad6 were initially normalized to the signals for Pgk1, which serves as a loading control. C , left , immunoblot for analysis of stability of Flag epitope–tagged Rad6 or mutants grown in a medium containing raffinose (+Raf; uninduced) or galactose (+Gal; induced) and at different time points after transcription from the GAL1 promoter was halted by adding glucose. Pgk1 served as loading control and for normalization. Right , plot shows densitometric values for WT or mutant Rad6 protein at different time points after glucose-mediated transcriptional inhibition relative to that in the induced state (+Gal, 0 min). Asterisk , p-value <0.05 (Student’s t test). D , growth assay was conducted by spotting ten-fold serial dilution of the indicated yeast strains on synthetic medium without tryptophan (-TRP) or in medium containing 6% ethanol or 2% formamide and incubated at 30 °C. Cells spotted on -TRP medium were also subjected to heat stress by incubation at 37 °C. Rad6, Radiation sensitive 6; Ub 1 , monoubiquitinated H2B; Ub n , polyubiquitinated H2B.

    Techniques Used: Activity Assay, Western Blot, In Vitro, Ubiquitin Assay, Recombinant, Incubation, SDS Page, Mutagenesis, Expressing, Quantitation Assay, FLAG-tag, Inhibition, Growth Assay, Serial Dilution

    A126 mutation adversely affects solubility and activity of UBE2B. A , assessment of solubility of His6-tagged UBE2B or UBE2B-A126F ( arrowhead ) by SDS-PAGE of uninduced (Un) and induced (In) bacterial cells, whole-cell lysate (WCL), and soluble and pellet (or insoluble) fractions. B , in vitro ubiquitination assay using recombinant WT UBE2B or the UBE2B-A126F mutant was performed essentially as described for <xref ref-type=Figure 3 A ; except, reactions were incubated at 37 °C for the indicated time. Reaction without the E2 enzyme (-) served as a control. C , in vitro ubiquitin chain formation assay was performed using recombinant WT UBE2B or the UBE2B-A126F mutant for the indicated time points. Control reactions without human Uba1 (- E1 ) or UBE2B (- E2 ) or omitting ATP were also performed. Blots were probed with antibodies recognizing ubiquitin, mono-, or poly-ubiquitinated proteins (clone FK2) or UBE2B. Plot shows quantitation of immunoblots by densitometry. Fold-change in the immunoblot signals obtained for the anti-ubiquitin antibody for either UBE2B or UBE2B-A126F at the various incubation times are shown relative to that obtained for UBE2B after 30 min incubation (set as 1) and were obtained from three independent experiments. The asterisk indicates p-value <0.05 computed using Student’s t test. M, protein ladder; In panel b, Ub n , ubiquitin chains or polyubiquitinated UBE2B; In panel C, Ub , ubiquitin; Ub 1 , monoubiquitinated H2B; Ub 2 , diubiquitin; Ub n , polyubiquitinated H2B. " title="... fractions. B , in vitro ubiquitination assay using recombinant WT UBE2B or the UBE2B-A126F mutant was performed ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: A126 mutation adversely affects solubility and activity of UBE2B. A , assessment of solubility of His6-tagged UBE2B or UBE2B-A126F ( arrowhead ) by SDS-PAGE of uninduced (Un) and induced (In) bacterial cells, whole-cell lysate (WCL), and soluble and pellet (or insoluble) fractions. B , in vitro ubiquitination assay using recombinant WT UBE2B or the UBE2B-A126F mutant was performed essentially as described for Figure 3 A ; except, reactions were incubated at 37 °C for the indicated time. Reaction without the E2 enzyme (-) served as a control. C , in vitro ubiquitin chain formation assay was performed using recombinant WT UBE2B or the UBE2B-A126F mutant for the indicated time points. Control reactions without human Uba1 (- E1 ) or UBE2B (- E2 ) or omitting ATP were also performed. Blots were probed with antibodies recognizing ubiquitin, mono-, or poly-ubiquitinated proteins (clone FK2) or UBE2B. Plot shows quantitation of immunoblots by densitometry. Fold-change in the immunoblot signals obtained for the anti-ubiquitin antibody for either UBE2B or UBE2B-A126F at the various incubation times are shown relative to that obtained for UBE2B after 30 min incubation (set as 1) and were obtained from three independent experiments. The asterisk indicates p-value <0.05 computed using Student’s t test. M, protein ladder; In panel b, Ub n , ubiquitin chains or polyubiquitinated UBE2B; In panel C, Ub , ubiquitin; Ub 1 , monoubiquitinated H2B; Ub 2 , diubiquitin; Ub n , polyubiquitinated H2B.

    Techniques Used: Mutagenesis, Solubility, Activity Assay, SDS Page, In Vitro, Ubiquitin Assay, Recombinant, Incubation, Tube Formation Assay, Quantitation Assay, Western Blot

    Related Articles

    Ubiquitin Proteomics:

    Article Title: Conserved α-helix-3 is crucial for structure and functions of Rad6 E2 ubiquitin-conjugating enzymes
    Article Snippet: The samples were then resolved by 15% SDS-PAGE and proteins were visualized by staining using SimplyBlueTM Safe Stain (Invitrogen). .. The ubiquitination reaction contained 1X Reaction buffer and 5mM Mg-ATP (Ubiquitylation Assay Kit; Abcam), 0.1 μM recombinant yeast or human GST-Uba1/UBE1 (E1, R&D Systems), 2.5 μM recombinant yeast or human ubiquitin (R&D Systems), 0.1 μM wild-type or mutant Rad6 or UBE2B (E2), and 2 μM substrate recombinant yeast histone H2B. ..

    Article Title: Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability
    Article Snippet: The samples were then resolved by 15% SDS-PAGE and proteins were visualized by staining using SimplyBlue Safe Stain (Invitrogen). .. The ubiquitination reaction contained 1× reaction buffer and 5 mM Mg-ATP (Ubiquitylation Assay Kit; Abcam), 0.1 μM recombinant yeast or human GST-Uba1/UBE1 (E1, R&D Systems), 2.5 μM recombinant yeast or human ubiquitin (R&D Systems), 0.1 μM WT or mutant Rad6 or UBE2B (E2), and 2 μM substrate recombinant yeast histone H2B. .. For yeast Rad6 or its mutant derivatives, the reaction was performed at 30 °C for 15 min, 2 h, or 10 h. For UBE2B or UBE2B-A126F, the reaction was performed at 37 °C for 2 h, 4 h, or 16 h. The reactions were stopped by adding 2× Laemmli sample buffer (Bio-Rad) and resolved in a 12% SDS-PAGE prior to immunoblotting with anti-monoubiquitinated and polyubiquitinated protein antibody (clone FK2), anti-yeast H2B antibody, or anti-Rad6 or UBE2B antibody (see details below).

    Ubiquitin Assay:

    Article Title: Conserved α-helix-3 is crucial for structure and functions of Rad6 E2 ubiquitin-conjugating enzymes
    Article Snippet: The samples were then resolved by 15% SDS-PAGE and proteins were visualized by staining using SimplyBlueTM Safe Stain (Invitrogen). .. The ubiquitination reaction contained 1X Reaction buffer and 5mM Mg-ATP (Ubiquitylation Assay Kit; Abcam), 0.1 μM recombinant yeast or human GST-Uba1/UBE1 (E1, R&D Systems), 2.5 μM recombinant yeast or human ubiquitin (R&D Systems), 0.1 μM wild-type or mutant Rad6 or UBE2B (E2), and 2 μM substrate recombinant yeast histone H2B. ..

    Article Title: Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability
    Article Snippet: The samples were then resolved by 15% SDS-PAGE and proteins were visualized by staining using SimplyBlue Safe Stain (Invitrogen). .. The ubiquitination reaction contained 1× reaction buffer and 5 mM Mg-ATP (Ubiquitylation Assay Kit; Abcam), 0.1 μM recombinant yeast or human GST-Uba1/UBE1 (E1, R&D Systems), 2.5 μM recombinant yeast or human ubiquitin (R&D Systems), 0.1 μM WT or mutant Rad6 or UBE2B (E2), and 2 μM substrate recombinant yeast histone H2B. .. For yeast Rad6 or its mutant derivatives, the reaction was performed at 30 °C for 15 min, 2 h, or 10 h. For UBE2B or UBE2B-A126F, the reaction was performed at 37 °C for 2 h, 4 h, or 16 h. The reactions were stopped by adding 2× Laemmli sample buffer (Bio-Rad) and resolved in a 12% SDS-PAGE prior to immunoblotting with anti-monoubiquitinated and polyubiquitinated protein antibody (clone FK2), anti-yeast H2B antibody, or anti-Rad6 or UBE2B antibody (see details below).

    Recombinant:

    Article Title: Conserved α-helix-3 is crucial for structure and functions of Rad6 E2 ubiquitin-conjugating enzymes
    Article Snippet: The samples were then resolved by 15% SDS-PAGE and proteins were visualized by staining using SimplyBlueTM Safe Stain (Invitrogen). .. The ubiquitination reaction contained 1X Reaction buffer and 5mM Mg-ATP (Ubiquitylation Assay Kit; Abcam), 0.1 μM recombinant yeast or human GST-Uba1/UBE1 (E1, R&D Systems), 2.5 μM recombinant yeast or human ubiquitin (R&D Systems), 0.1 μM wild-type or mutant Rad6 or UBE2B (E2), and 2 μM substrate recombinant yeast histone H2B. ..

    Article Title: Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability
    Article Snippet: The samples were then resolved by 15% SDS-PAGE and proteins were visualized by staining using SimplyBlue Safe Stain (Invitrogen). .. The ubiquitination reaction contained 1× reaction buffer and 5 mM Mg-ATP (Ubiquitylation Assay Kit; Abcam), 0.1 μM recombinant yeast or human GST-Uba1/UBE1 (E1, R&D Systems), 2.5 μM recombinant yeast or human ubiquitin (R&D Systems), 0.1 μM WT or mutant Rad6 or UBE2B (E2), and 2 μM substrate recombinant yeast histone H2B. .. For yeast Rad6 or its mutant derivatives, the reaction was performed at 30 °C for 15 min, 2 h, or 10 h. For UBE2B or UBE2B-A126F, the reaction was performed at 37 °C for 2 h, 4 h, or 16 h. The reactions were stopped by adding 2× Laemmli sample buffer (Bio-Rad) and resolved in a 12% SDS-PAGE prior to immunoblotting with anti-monoubiquitinated and polyubiquitinated protein antibody (clone FK2), anti-yeast H2B antibody, or anti-Rad6 or UBE2B antibody (see details below).

    Mutagenesis:

    Article Title: Conserved α-helix-3 is crucial for structure and functions of Rad6 E2 ubiquitin-conjugating enzymes
    Article Snippet: The samples were then resolved by 15% SDS-PAGE and proteins were visualized by staining using SimplyBlueTM Safe Stain (Invitrogen). .. The ubiquitination reaction contained 1X Reaction buffer and 5mM Mg-ATP (Ubiquitylation Assay Kit; Abcam), 0.1 μM recombinant yeast or human GST-Uba1/UBE1 (E1, R&D Systems), 2.5 μM recombinant yeast or human ubiquitin (R&D Systems), 0.1 μM wild-type or mutant Rad6 or UBE2B (E2), and 2 μM substrate recombinant yeast histone H2B. ..

    Article Title: Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability
    Article Snippet: The samples were then resolved by 15% SDS-PAGE and proteins were visualized by staining using SimplyBlue Safe Stain (Invitrogen). .. The ubiquitination reaction contained 1× reaction buffer and 5 mM Mg-ATP (Ubiquitylation Assay Kit; Abcam), 0.1 μM recombinant yeast or human GST-Uba1/UBE1 (E1, R&D Systems), 2.5 μM recombinant yeast or human ubiquitin (R&D Systems), 0.1 μM WT or mutant Rad6 or UBE2B (E2), and 2 μM substrate recombinant yeast histone H2B. .. For yeast Rad6 or its mutant derivatives, the reaction was performed at 30 °C for 15 min, 2 h, or 10 h. For UBE2B or UBE2B-A126F, the reaction was performed at 37 °C for 2 h, 4 h, or 16 h. The reactions were stopped by adding 2× Laemmli sample buffer (Bio-Rad) and resolved in a 12% SDS-PAGE prior to immunoblotting with anti-monoubiquitinated and polyubiquitinated protein antibody (clone FK2), anti-yeast H2B antibody, or anti-Rad6 or UBE2B antibody (see details below).



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    recombinant yeast or human gst-uba1/ube1  (R&D Systems)
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    A126 mutations disrupt enzymatic activity and stability of Rad6. A , immunoblot of products of an in vitro ubiquitination assay with <t>recombinant</t> WT Rad6 or indicated mutants. Enzyme was incubated at 30 °C for the indicated time along with ubiquitin (Ub), Uba1, ATP/Mg 2+ , and yeast histone H2B (substrate). The reaction mix was then resolved by SDS-PAGE prior to immunoblotting. The asterisk indicates a cross-reacting protein. The catalytic-dead mutant rad6-C88A served as a control. B , left , immunoblot of Flag-tagged WT or mutant Rad6. A two-fold serial gradient of the extracts prepared from the indicated strains were resolved by SDS-PAGE prior to immunoblotting. Extract from the strain expressing Rad6 served as a ‘no tag’ control. Right , plot of mean fold-change in the steady-state levels of A126 mutant relative to WT Rad6 (± SEM from two independent experiments) based on densitometry quantitation, for which the signals in the numbered lanes were used. The signals for WT or mutant Rad6 were initially normalized to the signals for Pgk1, which serves as a loading control. C , left , immunoblot for analysis of stability of Flag epitope–tagged Rad6 or mutants grown in a medium containing raffinose (+Raf; uninduced) or galactose (+Gal; induced) and at different time points after transcription from the GAL1 promoter was halted by adding glucose. Pgk1 served as loading control and for normalization. Right , plot shows densitometric values for WT or mutant Rad6 protein at different time points after glucose-mediated transcriptional inhibition relative to that in the induced state (+Gal, 0 min). Asterisk , p-value <0.05 (Student’s t test). D , growth assay was conducted by spotting ten-fold serial dilution of the indicated yeast strains on synthetic medium without tryptophan (-TRP) or in medium containing 6% ethanol or 2% formamide and incubated at 30 °C. Cells spotted on -TRP medium were also subjected to heat stress by incubation at 37 °C. Rad6, Radiation sensitive 6; Ub 1 , monoubiquitinated H2B; Ub n , polyubiquitinated H2B.
    Recombinant Yeast Or Human Gst Uba1/Ube1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+gst+uba1+ube1/human+ube1+e1/pmc09630792-382-17-23
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    human gst uba1 ube1  (R&D Systems)
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    R&D Systems human gst uba1 ube1
    A126 mutations disrupt enzymatic activity and stability of Rad6. A , immunoblot of products of an in vitro ubiquitination assay with <t>recombinant</t> WT Rad6 or indicated mutants. Enzyme was incubated at 30 °C for the indicated time along with ubiquitin (Ub), Uba1, ATP/Mg 2+ , and yeast histone H2B (substrate). The reaction mix was then resolved by SDS-PAGE prior to immunoblotting. The asterisk indicates a cross-reacting protein. The catalytic-dead mutant rad6-C88A served as a control. B , left , immunoblot of Flag-tagged WT or mutant Rad6. A two-fold serial gradient of the extracts prepared from the indicated strains were resolved by SDS-PAGE prior to immunoblotting. Extract from the strain expressing Rad6 served as a ‘no tag’ control. Right , plot of mean fold-change in the steady-state levels of A126 mutant relative to WT Rad6 (± SEM from two independent experiments) based on densitometry quantitation, for which the signals in the numbered lanes were used. The signals for WT or mutant Rad6 were initially normalized to the signals for Pgk1, which serves as a loading control. C , left , immunoblot for analysis of stability of Flag epitope–tagged Rad6 or mutants grown in a medium containing raffinose (+Raf; uninduced) or galactose (+Gal; induced) and at different time points after transcription from the GAL1 promoter was halted by adding glucose. Pgk1 served as loading control and for normalization. Right , plot shows densitometric values for WT or mutant Rad6 protein at different time points after glucose-mediated transcriptional inhibition relative to that in the induced state (+Gal, 0 min). Asterisk , p-value <0.05 (Student’s t test). D , growth assay was conducted by spotting ten-fold serial dilution of the indicated yeast strains on synthetic medium without tryptophan (-TRP) or in medium containing 6% ethanol or 2% formamide and incubated at 30 °C. Cells spotted on -TRP medium were also subjected to heat stress by incubation at 37 °C. Rad6, Radiation sensitive 6; Ub 1 , monoubiquitinated H2B; Ub n , polyubiquitinated H2B.
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    A126 mutations disrupt enzymatic activity and stability of Rad6. A , immunoblot of products of an in vitro ubiquitination assay with recombinant WT Rad6 or indicated mutants. Enzyme was incubated at 30 °C for the indicated time along with ubiquitin (Ub), Uba1, ATP/Mg 2+ , and yeast histone H2B (substrate). The reaction mix was then resolved by SDS-PAGE prior to immunoblotting. The asterisk indicates a cross-reacting protein. The catalytic-dead mutant rad6-C88A served as a control. B , left , immunoblot of Flag-tagged WT or mutant Rad6. A two-fold serial gradient of the extracts prepared from the indicated strains were resolved by SDS-PAGE prior to immunoblotting. Extract from the strain expressing Rad6 served as a ‘no tag’ control. Right , plot of mean fold-change in the steady-state levels of A126 mutant relative to WT Rad6 (± SEM from two independent experiments) based on densitometry quantitation, for which the signals in the numbered lanes were used. The signals for WT or mutant Rad6 were initially normalized to the signals for Pgk1, which serves as a loading control. C , left , immunoblot for analysis of stability of Flag epitope–tagged Rad6 or mutants grown in a medium containing raffinose (+Raf; uninduced) or galactose (+Gal; induced) and at different time points after transcription from the GAL1 promoter was halted by adding glucose. Pgk1 served as loading control and for normalization. Right , plot shows densitometric values for WT or mutant Rad6 protein at different time points after glucose-mediated transcriptional inhibition relative to that in the induced state (+Gal, 0 min). Asterisk , p-value <0.05 (Student’s t test). D , growth assay was conducted by spotting ten-fold serial dilution of the indicated yeast strains on synthetic medium without tryptophan (-TRP) or in medium containing 6% ethanol or 2% formamide and incubated at 30 °C. Cells spotted on -TRP medium were also subjected to heat stress by incubation at 37 °C. Rad6, Radiation sensitive 6; Ub 1 , monoubiquitinated H2B; Ub n , polyubiquitinated H2B.

    Journal: The Journal of Biological Chemistry

    Article Title: Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability

    doi: 10.1016/j.jbc.2022.102524

    Figure Lengend Snippet: A126 mutations disrupt enzymatic activity and stability of Rad6. A , immunoblot of products of an in vitro ubiquitination assay with recombinant WT Rad6 or indicated mutants. Enzyme was incubated at 30 °C for the indicated time along with ubiquitin (Ub), Uba1, ATP/Mg 2+ , and yeast histone H2B (substrate). The reaction mix was then resolved by SDS-PAGE prior to immunoblotting. The asterisk indicates a cross-reacting protein. The catalytic-dead mutant rad6-C88A served as a control. B , left , immunoblot of Flag-tagged WT or mutant Rad6. A two-fold serial gradient of the extracts prepared from the indicated strains were resolved by SDS-PAGE prior to immunoblotting. Extract from the strain expressing Rad6 served as a ‘no tag’ control. Right , plot of mean fold-change in the steady-state levels of A126 mutant relative to WT Rad6 (± SEM from two independent experiments) based on densitometry quantitation, for which the signals in the numbered lanes were used. The signals for WT or mutant Rad6 were initially normalized to the signals for Pgk1, which serves as a loading control. C , left , immunoblot for analysis of stability of Flag epitope–tagged Rad6 or mutants grown in a medium containing raffinose (+Raf; uninduced) or galactose (+Gal; induced) and at different time points after transcription from the GAL1 promoter was halted by adding glucose. Pgk1 served as loading control and for normalization. Right , plot shows densitometric values for WT or mutant Rad6 protein at different time points after glucose-mediated transcriptional inhibition relative to that in the induced state (+Gal, 0 min). Asterisk , p-value <0.05 (Student’s t test). D , growth assay was conducted by spotting ten-fold serial dilution of the indicated yeast strains on synthetic medium without tryptophan (-TRP) or in medium containing 6% ethanol or 2% formamide and incubated at 30 °C. Cells spotted on -TRP medium were also subjected to heat stress by incubation at 37 °C. Rad6, Radiation sensitive 6; Ub 1 , monoubiquitinated H2B; Ub n , polyubiquitinated H2B.

    Article Snippet: The ubiquitination reaction contained 1× reaction buffer and 5 mM Mg-ATP (Ubiquitylation Assay Kit; Abcam), 0.1 μM recombinant yeast or human GST-Uba1/UBE1 (E1, R&D Systems), 2.5 μM recombinant yeast or human ubiquitin (R&D Systems), 0.1 μM WT or mutant Rad6 or UBE2B (E2), and 2 μM substrate recombinant yeast histone H2B.

    Techniques: Activity Assay, Western Blot, In Vitro, Ubiquitin Assay, Recombinant, Incubation, SDS Page, Mutagenesis, Expressing, Quantitation Assay, FLAG-tag, Inhibition, Growth Assay, Serial Dilution

    A126 mutation adversely affects solubility and activity of UBE2B. A , assessment of solubility of His6-tagged UBE2B or UBE2B-A126F ( arrowhead ) by SDS-PAGE of uninduced (Un) and induced (In) bacterial cells, whole-cell lysate (WCL), and soluble and pellet (or insoluble) fractions. B , in vitro ubiquitination assay using recombinant WT UBE2B or the UBE2B-A126F mutant was performed essentially as described for <xref ref-type=Figure 3 A ; except, reactions were incubated at 37 °C for the indicated time. Reaction without the E2 enzyme (-) served as a control. C , in vitro ubiquitin chain formation assay was performed using recombinant WT UBE2B or the UBE2B-A126F mutant for the indicated time points. Control reactions without human Uba1 (- E1 ) or UBE2B (- E2 ) or omitting ATP were also performed. Blots were probed with antibodies recognizing ubiquitin, mono-, or poly-ubiquitinated proteins (clone FK2) or UBE2B. Plot shows quantitation of immunoblots by densitometry. Fold-change in the immunoblot signals obtained for the anti-ubiquitin antibody for either UBE2B or UBE2B-A126F at the various incubation times are shown relative to that obtained for UBE2B after 30 min incubation (set as 1) and were obtained from three independent experiments. The asterisk indicates p-value <0.05 computed using Student’s t test. M, protein ladder; In panel b, Ub n , ubiquitin chains or polyubiquitinated UBE2B; In panel C, Ub , ubiquitin; Ub 1 , monoubiquitinated H2B; Ub 2 , diubiquitin; Ub n , polyubiquitinated H2B. " width="100%" height="100%">

    Journal: The Journal of Biological Chemistry

    Article Title: Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability

    doi: 10.1016/j.jbc.2022.102524

    Figure Lengend Snippet: A126 mutation adversely affects solubility and activity of UBE2B. A , assessment of solubility of His6-tagged UBE2B or UBE2B-A126F ( arrowhead ) by SDS-PAGE of uninduced (Un) and induced (In) bacterial cells, whole-cell lysate (WCL), and soluble and pellet (or insoluble) fractions. B , in vitro ubiquitination assay using recombinant WT UBE2B or the UBE2B-A126F mutant was performed essentially as described for Figure 3 A ; except, reactions were incubated at 37 °C for the indicated time. Reaction without the E2 enzyme (-) served as a control. C , in vitro ubiquitin chain formation assay was performed using recombinant WT UBE2B or the UBE2B-A126F mutant for the indicated time points. Control reactions without human Uba1 (- E1 ) or UBE2B (- E2 ) or omitting ATP were also performed. Blots were probed with antibodies recognizing ubiquitin, mono-, or poly-ubiquitinated proteins (clone FK2) or UBE2B. Plot shows quantitation of immunoblots by densitometry. Fold-change in the immunoblot signals obtained for the anti-ubiquitin antibody for either UBE2B or UBE2B-A126F at the various incubation times are shown relative to that obtained for UBE2B after 30 min incubation (set as 1) and were obtained from three independent experiments. The asterisk indicates p-value <0.05 computed using Student’s t test. M, protein ladder; In panel b, Ub n , ubiquitin chains or polyubiquitinated UBE2B; In panel C, Ub , ubiquitin; Ub 1 , monoubiquitinated H2B; Ub 2 , diubiquitin; Ub n , polyubiquitinated H2B.

    Article Snippet: The ubiquitination reaction contained 1× reaction buffer and 5 mM Mg-ATP (Ubiquitylation Assay Kit; Abcam), 0.1 μM recombinant yeast or human GST-Uba1/UBE1 (E1, R&D Systems), 2.5 μM recombinant yeast or human ubiquitin (R&D Systems), 0.1 μM WT or mutant Rad6 or UBE2B (E2), and 2 μM substrate recombinant yeast histone H2B.

    Techniques: Mutagenesis, Solubility, Activity Assay, SDS Page, In Vitro, Ubiquitin Assay, Recombinant, Incubation, Tube Formation Assay, Quantitation Assay, Western Blot