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AXR1-ECR1 and AXL1-ECR1 heterodimeric RUB-activating enzymes diverge in function in Arabidopsis thaliana.

Hotton SK, Eigenheer RA, Castro MF, Bostick M, Callis J - Plant Mol. Biol. (2011)

Bottom Line: Gene duplications are widespread in angiosperms, and in line with this observation, components of the RUB conjugation pathway are found in multiples in Arabidopsis.Using mass spectrometry, endogenous AXR1 and AXL1 proteins were found in complex with 3HA-RUB1, suggesting that AXR1 and AXL1 exist in parallel RUB E1 complexes in Arabidopsis.These results suggest that while both proteins function in the RUB pathway and are biochemically similar in RUB-ECR1 thioester formation, they are not functionally equivalent.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, 1 Shields Avenue, Davis, CA 95616, USA.

ABSTRACT
RELATED TO UBIQUITIN (RUB) modification of CULLIN (CUL) subunits of the CUL-RING ubiquitin E3 ligase (CRL) superfamily regulates CRL ubiquitylation activity. RUB modification requires E1 and E2 enzymes that are analogous to, but distinct from, those activities required for UBIQUITIN (UBQ) attachment. Gene duplications are widespread in angiosperms, and in line with this observation, components of the RUB conjugation pathway are found in multiples in Arabidopsis. To further examine the extent of redundancy within the RUB pathway, we undertook biochemical and genetic characterizations of one such duplication event- the duplication of the genes encoding a subunit of the RUB E1 into AUXIN RESISTANT1 (AXR1) and AXR1-LIKE1 (AXL1). In vitro, the two proteins have similar abilities to function with E1 C-TERMINAL-RELATED1 (ECR1) in catalyzing RUB1 activation and RUB1-ECR1 thioester formation. Using mass spectrometry, endogenous AXR1 and AXL1 proteins were found in complex with 3HA-RUB1, suggesting that AXR1 and AXL1 exist in parallel RUB E1 complexes in Arabidopsis. In contrast, AXR1 and AXL1 differ in ability to correct phenotypic defects in axr1-30, a severe loss-of-function AXR1 mutant, when the respective coding sequences are expressed from the same promoter, suggesting differential in vivo functions. These results suggest that while both proteins function in the RUB pathway and are biochemically similar in RUB-ECR1 thioester formation, they are not functionally equivalent.

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At day 70 post-plating, AXR1 corrects the inflorescence height defect of axr1-30 more than AXL1. AXR1 and AXL lines were grown for 70 days and height was measured. Representative pictures of AXL lines (a–d), AXR1 lines (e–f), axr1-30 (g), and Columbia (h) are shown. Scale bar represents 5 cm
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Fig5: At day 70 post-plating, AXR1 corrects the inflorescence height defect of axr1-30 more than AXL1. AXR1 and AXL lines were grown for 70 days and height was measured. Representative pictures of AXL lines (a–d), AXR1 lines (e–f), axr1-30 (g), and Columbia (h) are shown. Scale bar represents 5 cm

Mentions: Finally, inflorescence height at ten weeks was measured for all lines. Here the difference in ability to restore the phenotype to Columbia between AXR1 and AXL1 expression is most apparent. Both AXR1 lines show good restoration of height, averaging 45.8 cm and 37.3 cm, compared to axr1-30 and Columbia, which average 25.2 cm and 52.1 cm, respectively (Figs. 4b, 5). Of the AXL lines, only line 1 that averages 29.3 cm in height shows a moderate correction of axr1-30 height. Statistical analyses confirm that AXL line 1, AXR1 line 1, and AXR1 line 2 are significantly taller than axr1-30, though none are the same height as Columbia (Figs. 4b, 5 and Online Resource 11). Both AXR1 lines (P < 0.0001) are significantly taller than AXL line 1, and AXR line 1 is taller than AXR1 line 2 (P < 0.0001), using a Student’s t-test with Bonferroni correction and α = 0.00313.Fig. 5


AXR1-ECR1 and AXL1-ECR1 heterodimeric RUB-activating enzymes diverge in function in Arabidopsis thaliana.

Hotton SK, Eigenheer RA, Castro MF, Bostick M, Callis J - Plant Mol. Biol. (2011)

At day 70 post-plating, AXR1 corrects the inflorescence height defect of axr1-30 more than AXL1. AXR1 and AXL lines were grown for 70 days and height was measured. Representative pictures of AXL lines (a–d), AXR1 lines (e–f), axr1-30 (g), and Columbia (h) are shown. Scale bar represents 5 cm
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044220&req=5

Fig5: At day 70 post-plating, AXR1 corrects the inflorescence height defect of axr1-30 more than AXL1. AXR1 and AXL lines were grown for 70 days and height was measured. Representative pictures of AXL lines (a–d), AXR1 lines (e–f), axr1-30 (g), and Columbia (h) are shown. Scale bar represents 5 cm
Mentions: Finally, inflorescence height at ten weeks was measured for all lines. Here the difference in ability to restore the phenotype to Columbia between AXR1 and AXL1 expression is most apparent. Both AXR1 lines show good restoration of height, averaging 45.8 cm and 37.3 cm, compared to axr1-30 and Columbia, which average 25.2 cm and 52.1 cm, respectively (Figs. 4b, 5). Of the AXL lines, only line 1 that averages 29.3 cm in height shows a moderate correction of axr1-30 height. Statistical analyses confirm that AXL line 1, AXR1 line 1, and AXR1 line 2 are significantly taller than axr1-30, though none are the same height as Columbia (Figs. 4b, 5 and Online Resource 11). Both AXR1 lines (P < 0.0001) are significantly taller than AXL line 1, and AXR line 1 is taller than AXR1 line 2 (P < 0.0001), using a Student’s t-test with Bonferroni correction and α = 0.00313.Fig. 5

Bottom Line: Gene duplications are widespread in angiosperms, and in line with this observation, components of the RUB conjugation pathway are found in multiples in Arabidopsis.Using mass spectrometry, endogenous AXR1 and AXL1 proteins were found in complex with 3HA-RUB1, suggesting that AXR1 and AXL1 exist in parallel RUB E1 complexes in Arabidopsis.These results suggest that while both proteins function in the RUB pathway and are biochemically similar in RUB-ECR1 thioester formation, they are not functionally equivalent.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, 1 Shields Avenue, Davis, CA 95616, USA.

ABSTRACT
RELATED TO UBIQUITIN (RUB) modification of CULLIN (CUL) subunits of the CUL-RING ubiquitin E3 ligase (CRL) superfamily regulates CRL ubiquitylation activity. RUB modification requires E1 and E2 enzymes that are analogous to, but distinct from, those activities required for UBIQUITIN (UBQ) attachment. Gene duplications are widespread in angiosperms, and in line with this observation, components of the RUB conjugation pathway are found in multiples in Arabidopsis. To further examine the extent of redundancy within the RUB pathway, we undertook biochemical and genetic characterizations of one such duplication event- the duplication of the genes encoding a subunit of the RUB E1 into AUXIN RESISTANT1 (AXR1) and AXR1-LIKE1 (AXL1). In vitro, the two proteins have similar abilities to function with E1 C-TERMINAL-RELATED1 (ECR1) in catalyzing RUB1 activation and RUB1-ECR1 thioester formation. Using mass spectrometry, endogenous AXR1 and AXL1 proteins were found in complex with 3HA-RUB1, suggesting that AXR1 and AXL1 exist in parallel RUB E1 complexes in Arabidopsis. In contrast, AXR1 and AXL1 differ in ability to correct phenotypic defects in axr1-30, a severe loss-of-function AXR1 mutant, when the respective coding sequences are expressed from the same promoter, suggesting differential in vivo functions. These results suggest that while both proteins function in the RUB pathway and are biochemically similar in RUB-ECR1 thioester formation, they are not functionally equivalent.

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