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Arabidopsis PCNAs form complexes with selected D-type cyclins.

Strzalka WK, Aggarwal C, Krzeszowiec W, Jakubowska A, Sztatelman O, Banas AK - Front Plant Sci (2015)

Bottom Line: Moreover, our results indicate that the formation of CycD3;2/PCNA and CycD4;1/PCNA complexes can be regulated by other as yet unidentified factor(s).Additionally, FLIM-FRET analyses suggested that in planta the distance between PCNA1/CycD4;1, PCNA1/CycD6;1, PCNA1/CycD7;1, and PCNA2/CycD4;2 proteins was shorter than that between PCNA2/CycD4;1, PCNA2/CycD6;1, PCNA2/CycD7;1, and PCNA1/CycD4;2 pairs.These data indicate that the nine amino acid differences between PCNA1 and PCNA2 have an impact on the architecture of Arabidopsis CycD/PCNA complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University Krakow, Poland ; The Bioremediation Department, Malopolska Centre of Biotechnology, Jagiellonian University Krakow, Poland.

ABSTRACT
Proliferating Cell Nuclear Antigen (PCNA) is a key nuclear protein of eukaryotic cells. It has been shown to form complexes with cyclin dependent kinases, cyclin dependent kinase inhibitors and the D-type cyclins which are involved in the cell cycle control. In Arabidopsis two genes coding for PCNA1 and PCNA2 proteins have been identified. In this study by analyzing Arabidopsis PCNA/CycD complexes we tested the possible functional differentiation of PCNA1/2 proteins in cell cycle control. Most out of the 10 cyclins investigated showed only nuclear localization except CycD2;1, CycD4;1, and CycD4;2 which were observed both in the nucleus and cytoplasm. Using the Y2H, BiFC and FLIM-FRET techniques we identified D-type cyclins which formed complexes with either PCNA1 or PCNA2. Among the candidates tested only CycD1;1, CycD3;1, and CycD3;3 were not detected in a complex with the PCNA proteins. Moreover, our results indicate that the formation of CycD3;2/PCNA and CycD4;1/PCNA complexes can be regulated by other as yet unidentified factor(s). Additionally, FLIM-FRET analyses suggested that in planta the distance between PCNA1/CycD4;1, PCNA1/CycD6;1, PCNA1/CycD7;1, and PCNA2/CycD4;2 proteins was shorter than that between PCNA2/CycD4;1, PCNA2/CycD6;1, PCNA2/CycD7;1, and PCNA1/CycD4;2 pairs. These data indicate that the nine amino acid differences between PCNA1 and PCNA2 have an impact on the architecture of Arabidopsis CycD/PCNA complexes.

No MeSH data available.


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Analysis of interactions between Arabidopsis PCNA1/2 and D-type cyclins using the split-uiquitin Y2H system. The interactions were tested in the following combinations. (A) PCNA1 (bait)/D-type cyclins (prey), (B) PCNA2 (bait)/D-type cyclins (prey), and (C) D-type cyclins (bait)/PCNA1/2 (prey). The transformed yeast cells were plated on either an SC-Leu-Trp control solid medium or an SC-Leu-Trp-His selection solid medium supplemented with 10 mM 3-aminotriazol (3-AT). For the beta-galactosidase assay, the yeast transformants were grown on a nitrocellulose filter placed on the surface of a YPAD solid medium followed by incubation with X-gal. The results are representative of three independently repeated experiments.
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Figure 3: Analysis of interactions between Arabidopsis PCNA1/2 and D-type cyclins using the split-uiquitin Y2H system. The interactions were tested in the following combinations. (A) PCNA1 (bait)/D-type cyclins (prey), (B) PCNA2 (bait)/D-type cyclins (prey), and (C) D-type cyclins (bait)/PCNA1/2 (prey). The transformed yeast cells were plated on either an SC-Leu-Trp control solid medium or an SC-Leu-Trp-His selection solid medium supplemented with 10 mM 3-aminotriazol (3-AT). For the beta-galactosidase assay, the yeast transformants were grown on a nitrocellulose filter placed on the surface of a YPAD solid medium followed by incubation with X-gal. The results are representative of three independently repeated experiments.

Mentions: At the first stage of this study the Y2H technique was employed to identify which Arabidopsis D-type cyclin candidates may form complexes with PCNA1 and/or PCNA2. The interactions between PCNA1/2 and D-type cyclins were tested in two different combinations. In the first combination PCNA1/2 proteins were used as bait. The result of this analysis demonstrated that PCNA1 formed a complex with CycD3;2, CycD4;1, and CycD4;2 (Figure 3A) while PCNA2 showed interaction only with CycD4;1 and CycD4;2 but not CycD3;2 (Figure 3B). Testing the opposite combination, where D-type cyclins were expressed as bait, interactions between CycD2;1/PCNA1, CycD3;2/PCNA1, CycD4;2/PCNA1, CycD2;1/PCNA2, CycD3;2/PCNA2, and CycD4;2/PCNA2 were observed (Figure 3C). Moreover, complex formation was also observed for CycD5;1/PCNA2 and CycD6;1/PCNA2 (Figure 3C).


Arabidopsis PCNAs form complexes with selected D-type cyclins.

Strzalka WK, Aggarwal C, Krzeszowiec W, Jakubowska A, Sztatelman O, Banas AK - Front Plant Sci (2015)

Analysis of interactions between Arabidopsis PCNA1/2 and D-type cyclins using the split-uiquitin Y2H system. The interactions were tested in the following combinations. (A) PCNA1 (bait)/D-type cyclins (prey), (B) PCNA2 (bait)/D-type cyclins (prey), and (C) D-type cyclins (bait)/PCNA1/2 (prey). The transformed yeast cells were plated on either an SC-Leu-Trp control solid medium or an SC-Leu-Trp-His selection solid medium supplemented with 10 mM 3-aminotriazol (3-AT). For the beta-galactosidase assay, the yeast transformants were grown on a nitrocellulose filter placed on the surface of a YPAD solid medium followed by incubation with X-gal. The results are representative of three independently repeated experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4550699&req=5

Figure 3: Analysis of interactions between Arabidopsis PCNA1/2 and D-type cyclins using the split-uiquitin Y2H system. The interactions were tested in the following combinations. (A) PCNA1 (bait)/D-type cyclins (prey), (B) PCNA2 (bait)/D-type cyclins (prey), and (C) D-type cyclins (bait)/PCNA1/2 (prey). The transformed yeast cells were plated on either an SC-Leu-Trp control solid medium or an SC-Leu-Trp-His selection solid medium supplemented with 10 mM 3-aminotriazol (3-AT). For the beta-galactosidase assay, the yeast transformants were grown on a nitrocellulose filter placed on the surface of a YPAD solid medium followed by incubation with X-gal. The results are representative of three independently repeated experiments.
Mentions: At the first stage of this study the Y2H technique was employed to identify which Arabidopsis D-type cyclin candidates may form complexes with PCNA1 and/or PCNA2. The interactions between PCNA1/2 and D-type cyclins were tested in two different combinations. In the first combination PCNA1/2 proteins were used as bait. The result of this analysis demonstrated that PCNA1 formed a complex with CycD3;2, CycD4;1, and CycD4;2 (Figure 3A) while PCNA2 showed interaction only with CycD4;1 and CycD4;2 but not CycD3;2 (Figure 3B). Testing the opposite combination, where D-type cyclins were expressed as bait, interactions between CycD2;1/PCNA1, CycD3;2/PCNA1, CycD4;2/PCNA1, CycD2;1/PCNA2, CycD3;2/PCNA2, and CycD4;2/PCNA2 were observed (Figure 3C). Moreover, complex formation was also observed for CycD5;1/PCNA2 and CycD6;1/PCNA2 (Figure 3C).

Bottom Line: Moreover, our results indicate that the formation of CycD3;2/PCNA and CycD4;1/PCNA complexes can be regulated by other as yet unidentified factor(s).Additionally, FLIM-FRET analyses suggested that in planta the distance between PCNA1/CycD4;1, PCNA1/CycD6;1, PCNA1/CycD7;1, and PCNA2/CycD4;2 proteins was shorter than that between PCNA2/CycD4;1, PCNA2/CycD6;1, PCNA2/CycD7;1, and PCNA1/CycD4;2 pairs.These data indicate that the nine amino acid differences between PCNA1 and PCNA2 have an impact on the architecture of Arabidopsis CycD/PCNA complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University Krakow, Poland ; The Bioremediation Department, Malopolska Centre of Biotechnology, Jagiellonian University Krakow, Poland.

ABSTRACT
Proliferating Cell Nuclear Antigen (PCNA) is a key nuclear protein of eukaryotic cells. It has been shown to form complexes with cyclin dependent kinases, cyclin dependent kinase inhibitors and the D-type cyclins which are involved in the cell cycle control. In Arabidopsis two genes coding for PCNA1 and PCNA2 proteins have been identified. In this study by analyzing Arabidopsis PCNA/CycD complexes we tested the possible functional differentiation of PCNA1/2 proteins in cell cycle control. Most out of the 10 cyclins investigated showed only nuclear localization except CycD2;1, CycD4;1, and CycD4;2 which were observed both in the nucleus and cytoplasm. Using the Y2H, BiFC and FLIM-FRET techniques we identified D-type cyclins which formed complexes with either PCNA1 or PCNA2. Among the candidates tested only CycD1;1, CycD3;1, and CycD3;3 were not detected in a complex with the PCNA proteins. Moreover, our results indicate that the formation of CycD3;2/PCNA and CycD4;1/PCNA complexes can be regulated by other as yet unidentified factor(s). Additionally, FLIM-FRET analyses suggested that in planta the distance between PCNA1/CycD4;1, PCNA1/CycD6;1, PCNA1/CycD7;1, and PCNA2/CycD4;2 proteins was shorter than that between PCNA2/CycD4;1, PCNA2/CycD6;1, PCNA2/CycD7;1, and PCNA1/CycD4;2 pairs. These data indicate that the nine amino acid differences between PCNA1 and PCNA2 have an impact on the architecture of Arabidopsis CycD/PCNA complexes.

No MeSH data available.


Related in: MedlinePlus