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Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

Ramadan A, Nemoto K, Seki M, Shinozaki K, Takeda H, Takahashi H, Sawasaki T - BMC Plant Biol. (2015)

Bottom Line: In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity.The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form.In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.

View Article: PubMed Central - PubMed

Affiliation: Proteo-Science Center, Ehime University, Matsuyama, 790-8577, Japan. z862004m@mails.cc.ehime-u.ac.jp.

ABSTRACT

Background: Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, cannot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised.

Results: Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time.

Conclusion: The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.

No MeSH data available.


Related in: MedlinePlus

Immunoblot analysis of the N-terminal FLAG-tagged RING protein library expressed by the wheat germ cell-free system. For analysis, 2 μL of crude recombinant RING proteins with N-terminus FLAG tag was loaded onto SDS-PAGE and detected by anti-FLAG-HRP antibody. A total of 204 out of 208 RING proteins analysed were detected. Arrows show the expected signal for each RING protein. Blue asterisks refer to proteins with high molecular smears, while red asterisks refer to RING proteins did not show high molecular smears and were subsequently used in the in vitro ubiquitination analysis (Fig. 5, Fig. 6)
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Fig3: Immunoblot analysis of the N-terminal FLAG-tagged RING protein library expressed by the wheat germ cell-free system. For analysis, 2 μL of crude recombinant RING proteins with N-terminus FLAG tag was loaded onto SDS-PAGE and detected by anti-FLAG-HRP antibody. A total of 204 out of 208 RING proteins analysed were detected. Arrows show the expected signal for each RING protein. Blue asterisks refer to proteins with high molecular smears, while red asterisks refer to RING proteins did not show high molecular smears and were subsequently used in the in vitro ubiquitination analysis (Fig. 5, Fig. 6)

Mentions: The Arabidopsis genome is predicted to encode more than 470 RING domain-containing proteins [14]. To construct a protein library of Arabidopsis RING proteins, we collected 274 cDNA clones from the RAFL library [21] according to the annotated RING proteins [14] and annotated genes in RAFL database [21]. We prepared transcription templates with an N-terminal FLAG-tag sequence using the ‘split-primer’ PCR method (Additional file 2). We were able to construct transcription templates for 208 RING clones (about 75 % of the clones collected) (Additional file 3). Following expression using the bilayer mode of the wheat germ cell-free system, expression was confirmed for 204 of the 208 RING protein-encoding mRNAs by immunoblot analysis (Fig. 3). Fifteen RNAs were expressed at relatively low levels. We compared the sizes of the expressed proteins against the expected molecular weights, as recorded in the RAFL database. We note that not all cDNA clones from RAFL matched the representative gene model in TAIR v10. Therefore, we mainly used the RAFL information to make comparisons since it was the source of the cDNAs used in the synthesis of our RING protein library. Accordingly, all but seven of the 204 expressed proteins had molecular weights that match those predicted (+/- 20 KDa). These seven proteins were > 20 KDa smaller than their expected RAFL sizes and were considered to be truncated (Additional file 3). Interestingly, upon detection of expressed RING proteins, we noticed a group of 31 proteins with anti-FLAG high molecular smears or with immunoreactivity at very high molecular masses near the top of the resolving gel (Fig. 3, with blue asterisks; Table 2). Because RING proteins are predicted to function as Ub E3 ligases and the wheat germ extract contains endogenous E1, E2, and Ub, we hypothesized that these smears and high molecular mass forms result from Ub ligase activity.Fig. 3


Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

Ramadan A, Nemoto K, Seki M, Shinozaki K, Takeda H, Takahashi H, Sawasaki T - BMC Plant Biol. (2015)

Immunoblot analysis of the N-terminal FLAG-tagged RING protein library expressed by the wheat germ cell-free system. For analysis, 2 μL of crude recombinant RING proteins with N-terminus FLAG tag was loaded onto SDS-PAGE and detected by anti-FLAG-HRP antibody. A total of 204 out of 208 RING proteins analysed were detected. Arrows show the expected signal for each RING protein. Blue asterisks refer to proteins with high molecular smears, while red asterisks refer to RING proteins did not show high molecular smears and were subsequently used in the in vitro ubiquitination analysis (Fig. 5, Fig. 6)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4641371&req=5

Fig3: Immunoblot analysis of the N-terminal FLAG-tagged RING protein library expressed by the wheat germ cell-free system. For analysis, 2 μL of crude recombinant RING proteins with N-terminus FLAG tag was loaded onto SDS-PAGE and detected by anti-FLAG-HRP antibody. A total of 204 out of 208 RING proteins analysed were detected. Arrows show the expected signal for each RING protein. Blue asterisks refer to proteins with high molecular smears, while red asterisks refer to RING proteins did not show high molecular smears and were subsequently used in the in vitro ubiquitination analysis (Fig. 5, Fig. 6)
Mentions: The Arabidopsis genome is predicted to encode more than 470 RING domain-containing proteins [14]. To construct a protein library of Arabidopsis RING proteins, we collected 274 cDNA clones from the RAFL library [21] according to the annotated RING proteins [14] and annotated genes in RAFL database [21]. We prepared transcription templates with an N-terminal FLAG-tag sequence using the ‘split-primer’ PCR method (Additional file 2). We were able to construct transcription templates for 208 RING clones (about 75 % of the clones collected) (Additional file 3). Following expression using the bilayer mode of the wheat germ cell-free system, expression was confirmed for 204 of the 208 RING protein-encoding mRNAs by immunoblot analysis (Fig. 3). Fifteen RNAs were expressed at relatively low levels. We compared the sizes of the expressed proteins against the expected molecular weights, as recorded in the RAFL database. We note that not all cDNA clones from RAFL matched the representative gene model in TAIR v10. Therefore, we mainly used the RAFL information to make comparisons since it was the source of the cDNAs used in the synthesis of our RING protein library. Accordingly, all but seven of the 204 expressed proteins had molecular weights that match those predicted (+/- 20 KDa). These seven proteins were > 20 KDa smaller than their expected RAFL sizes and were considered to be truncated (Additional file 3). Interestingly, upon detection of expressed RING proteins, we noticed a group of 31 proteins with anti-FLAG high molecular smears or with immunoreactivity at very high molecular masses near the top of the resolving gel (Fig. 3, with blue asterisks; Table 2). Because RING proteins are predicted to function as Ub E3 ligases and the wheat germ extract contains endogenous E1, E2, and Ub, we hypothesized that these smears and high molecular mass forms result from Ub ligase activity.Fig. 3

Bottom Line: In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity.The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form.In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.

View Article: PubMed Central - PubMed

Affiliation: Proteo-Science Center, Ehime University, Matsuyama, 790-8577, Japan. z862004m@mails.cc.ehime-u.ac.jp.

ABSTRACT

Background: Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, cannot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised.

Results: Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time.

Conclusion: The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.

No MeSH data available.


Related in: MedlinePlus