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ABA and the ubiquitin E3 ligase KEEP ON GOING affect proteolysis of the Arabidopsis thaliana transcription factors ABF1 and ABF3.

Chen YT, Liu H, Stone S, Callis J - Plant J. (2013)

Bottom Line: Loss of ABF1 or ABF3 in the keg background has a phenotypic effect similar to the loss of ABI5, and there is no additional rescue of the keg phenotype in abf1 abf3 abi5 keg seedlings.This result suggests that the abundance of other substrates is altered in keg seedlings, affecting growth.In conclusion, ABF1 and ABF3 abundance is affected by ABA and KEG, and the conserved C4 region serves as a stabilizing element.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, UC-Davis, 1 Shields Ave, Davis, CA 95616, USA.

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In vitro degradation of ABF1 and ABF3 is slowed in keg.Bacterially expressed recombinant GST-FUS3 (a), His-Flag-ABF1 (b), His-Flag-ABF3 (c), His-Flag-ABF1ΔC4 (d) or His-Flag-ABF3ΔC4 (e) was incubated with a 7–day-old Col or a 14–day-old keg seedling extract over the indicated time courses [note the shorter times in (d) and (e) compared with (b) and (c)]. His-Flag-tagged protein levels were visualized by anti-Flag immunoblotting. Ponceau S staining was used as the loading control. (f) Data from independent experiments comparing the in vitro degradation of full-length proteins and their respective ΔC4 forms were plotted normalized to time 0 (ABF1, n = 3; ABF3, n = 2).(g) Protein at 90 or 10 min for full-length and ΔC4 forms, respectively, were normalized to time 0. Student's t–tests indicate that the loss of the same protein was significantly slower in keg extracts compared with the wild type (WT; n = 3; P < 0.03, P < 0.02 and P < 0.03 for ABF1, ABF1ΔC4 and ABF3 ΔC4, respectively). (Test not performed for ABF3, this specific time course was repeated twice, and a different time course, with the same results was performed once.) Note: cannot compare full-length and ΔC4 forms here because time points are different (see Figures 5f and 6 for these comparisons).
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fig05: In vitro degradation of ABF1 and ABF3 is slowed in keg.Bacterially expressed recombinant GST-FUS3 (a), His-Flag-ABF1 (b), His-Flag-ABF3 (c), His-Flag-ABF1ΔC4 (d) or His-Flag-ABF3ΔC4 (e) was incubated with a 7–day-old Col or a 14–day-old keg seedling extract over the indicated time courses [note the shorter times in (d) and (e) compared with (b) and (c)]. His-Flag-tagged protein levels were visualized by anti-Flag immunoblotting. Ponceau S staining was used as the loading control. (f) Data from independent experiments comparing the in vitro degradation of full-length proteins and their respective ΔC4 forms were plotted normalized to time 0 (ABF1, n = 3; ABF3, n = 2).(g) Protein at 90 or 10 min for full-length and ΔC4 forms, respectively, were normalized to time 0. Student's t–tests indicate that the loss of the same protein was significantly slower in keg extracts compared with the wild type (WT; n = 3; P < 0.03, P < 0.02 and P < 0.03 for ABF1, ABF1ΔC4 and ABF3 ΔC4, respectively). (Test not performed for ABF3, this specific time course was repeated twice, and a different time course, with the same results was performed once.) Note: cannot compare full-length and ΔC4 forms here because time points are different (see Figures 5f and 6 for these comparisons).

Mentions: We next used the cell-free degradation assay to determine whether ABF1 or ABF3 degradation is slowed in keg extracts in vitro, as previously observed in vivo. The equivalent in vitro proteolysis of GST-FUSCA3, a B3 domain-containing transcription factor involved in embryogenesis previously demonstrated to be unstable (Lu et al., 2010), in wild-type and keg extracts demonstrates active proteolysis in keg extracts (Figure 5a). In wild-type protein extracts, 12% of His-Flag-ABF1 (Figure 5b, lane 4) or 2% of His-Flag-ABF3 (Figure 5c, lane 4) remained after 90 or 20 min, respectively, whereas 87 and 87%, of His-Flag-ABF1 and His-Flag-ABF3, respectively, remained in keg extracts (Figure 5b,c, lanes 8). The slower in vivo (Figure 4) and in vitro (Figure 5) degradation of tagged ABF1 and ABF3 in keg indicates ABF1 and ABF3 degradation is modulated by KEG E3 ligase.


ABA and the ubiquitin E3 ligase KEEP ON GOING affect proteolysis of the Arabidopsis thaliana transcription factors ABF1 and ABF3.

Chen YT, Liu H, Stone S, Callis J - Plant J. (2013)

In vitro degradation of ABF1 and ABF3 is slowed in keg.Bacterially expressed recombinant GST-FUS3 (a), His-Flag-ABF1 (b), His-Flag-ABF3 (c), His-Flag-ABF1ΔC4 (d) or His-Flag-ABF3ΔC4 (e) was incubated with a 7–day-old Col or a 14–day-old keg seedling extract over the indicated time courses [note the shorter times in (d) and (e) compared with (b) and (c)]. His-Flag-tagged protein levels were visualized by anti-Flag immunoblotting. Ponceau S staining was used as the loading control. (f) Data from independent experiments comparing the in vitro degradation of full-length proteins and their respective ΔC4 forms were plotted normalized to time 0 (ABF1, n = 3; ABF3, n = 2).(g) Protein at 90 or 10 min for full-length and ΔC4 forms, respectively, were normalized to time 0. Student's t–tests indicate that the loss of the same protein was significantly slower in keg extracts compared with the wild type (WT; n = 3; P < 0.03, P < 0.02 and P < 0.03 for ABF1, ABF1ΔC4 and ABF3 ΔC4, respectively). (Test not performed for ABF3, this specific time course was repeated twice, and a different time course, with the same results was performed once.) Note: cannot compare full-length and ΔC4 forms here because time points are different (see Figures 5f and 6 for these comparisons).
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fig05: In vitro degradation of ABF1 and ABF3 is slowed in keg.Bacterially expressed recombinant GST-FUS3 (a), His-Flag-ABF1 (b), His-Flag-ABF3 (c), His-Flag-ABF1ΔC4 (d) or His-Flag-ABF3ΔC4 (e) was incubated with a 7–day-old Col or a 14–day-old keg seedling extract over the indicated time courses [note the shorter times in (d) and (e) compared with (b) and (c)]. His-Flag-tagged protein levels were visualized by anti-Flag immunoblotting. Ponceau S staining was used as the loading control. (f) Data from independent experiments comparing the in vitro degradation of full-length proteins and their respective ΔC4 forms were plotted normalized to time 0 (ABF1, n = 3; ABF3, n = 2).(g) Protein at 90 or 10 min for full-length and ΔC4 forms, respectively, were normalized to time 0. Student's t–tests indicate that the loss of the same protein was significantly slower in keg extracts compared with the wild type (WT; n = 3; P < 0.03, P < 0.02 and P < 0.03 for ABF1, ABF1ΔC4 and ABF3 ΔC4, respectively). (Test not performed for ABF3, this specific time course was repeated twice, and a different time course, with the same results was performed once.) Note: cannot compare full-length and ΔC4 forms here because time points are different (see Figures 5f and 6 for these comparisons).
Mentions: We next used the cell-free degradation assay to determine whether ABF1 or ABF3 degradation is slowed in keg extracts in vitro, as previously observed in vivo. The equivalent in vitro proteolysis of GST-FUSCA3, a B3 domain-containing transcription factor involved in embryogenesis previously demonstrated to be unstable (Lu et al., 2010), in wild-type and keg extracts demonstrates active proteolysis in keg extracts (Figure 5a). In wild-type protein extracts, 12% of His-Flag-ABF1 (Figure 5b, lane 4) or 2% of His-Flag-ABF3 (Figure 5c, lane 4) remained after 90 or 20 min, respectively, whereas 87 and 87%, of His-Flag-ABF1 and His-Flag-ABF3, respectively, remained in keg extracts (Figure 5b,c, lanes 8). The slower in vivo (Figure 4) and in vitro (Figure 5) degradation of tagged ABF1 and ABF3 in keg indicates ABF1 and ABF3 degradation is modulated by KEG E3 ligase.

Bottom Line: Loss of ABF1 or ABF3 in the keg background has a phenotypic effect similar to the loss of ABI5, and there is no additional rescue of the keg phenotype in abf1 abf3 abi5 keg seedlings.This result suggests that the abundance of other substrates is altered in keg seedlings, affecting growth.In conclusion, ABF1 and ABF3 abundance is affected by ABA and KEG, and the conserved C4 region serves as a stabilizing element.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, UC-Davis, 1 Shields Ave, Davis, CA 95616, USA.

Show MeSH