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.
Highlights
Proliferating Cell Nuclear Antigen (PCNA) is the fundamental eukaryotic protein which is present mainly in the nuclei of dividing cells
Molecular studies on plant organisms have demonstrated that the genomes of some species, e.g., carrot (Hata et al, 1992), maize (Lopez et al, 1997) and Arabidopsis (Arabidopsis Genome Initiative, 2000) have two genes coding for PCNA1 and PCNA2 proteins
In this study we present the results from the analysis of Arabidopsis CycD/PCNA complexes
Summary
Proliferating Cell Nuclear Antigen (PCNA) is the fundamental eukaryotic protein which is present mainly in the nuclei of dividing cells. Molecular studies on plant organisms have demonstrated that the genomes of some species, e.g., carrot (Hata et al, 1992), maize (Lopez et al, 1997) and Arabidopsis (Arabidopsis Genome Initiative, 2000) have two genes coding for PCNA1 and PCNA2 proteins. An analysis of PCNA amino acid sequences from different plant species, including rice (Suzuka et al, 1991), maize (Lopez et al, 1997), common bean (Strzalka and Ziemienowicz, 2007), and runner bean (Strzalka et al, 2010) showed that the identity between these proteins is over 85%. The amino acid sequence identity between Arabidopsis/yeast and Arabidopsis/human PCNA is 40 and 65% respectively, crystallographic data demonstrated that these proteins have a very similar and conserved three dimensional architecture (Gulbis et al, 1996; Strzalka et al, 2009). A PCNA monomer, a 29 kDa polypeptide composed of two structural domains
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