Final Activity Report Summary - PLANTAPC (Plant Anaphase Promoting Complexes: unrevealing APC functions by APC substrates and activators)
The ubiquitin-dependent proteolysis by the 26S proteasome is a key mechanism in eukaryotes, which controls the degradation of short-lived proteins and regulates nearly all fundamental cellular processes including the cell cycle. The selection and specific timing of polyubiquitination of the target proteins are conferred by different E3 ubiquitin ligases. The APC is the most complex E3 ubiquitin ligase, has fundamental roles in the cell cycle; in the metaphase-anaphase transition, exit from mitosis, control of DNA replication and endoreduplication cycles by ordered destruction of various cell cycle proteins.
Substrate specificity and stage-specific activation of the APC are defined by binding of either of the two adaptor proteins, CDC20 or Cdh1 (CCS52 in plants). While CDC20 is restricted to mitotic cells, Cdh1 appears to be active both mitotic and postmitotic cells. In animals and human, there is a single CDC20 gene, which is thought to be exclusively active in proliferating cells. The function of the CDC20s in plants has not yet been studied.
Plants code for several isoforms of APC activators. In A. thaliana five genes code for highly homologous CDC20 proteins (CDC20-1 - At4g33270, CDC20-2 - At4g33260, CDC20-3 - At5g27080, CDC20-4 - At5g26900, CDC20-5 - At5g27570).
They can be grouped into two classes based on their gene structure, promoter region and binding domain patterns of their proteins. The first group contains CDC20-1 and CDC20-2 that likely result of gene duplication. The expression of these genes and the structure of their proteins are similar to other eukaryotic CDC20s. In the second group (CDC20-3, CDC20-4, CDC20-5) there is no intron in the genes, nevertheless, their protein contain most of the conserved motifs.
Expression pattern of CDC20-1 and CDC20-2 are overlapping during plant development and their cell cycle regulation is identical. In line with that, the single CDC20-1 and CDC20-2 mutants have no visible phenotypes, which suggest redundant function of these proteins. CDC20-1 and CDC20-2 RNAi lines were created for gradient reduce of gene expression, the analysis is under progress. Indeed, these two isoforms are real members of the spindle attachment checkpoint (SAC) protein complex that responsible for the appropriate chromatide separation in late mitosis. In spite of common function in the spindle checkpoint, the two proteins, which regulate also the level of mitotic cyclins, interacted differently with mitotic cyclins in Y2H, indicating that they share this function in the cell cycle in different cell types.
Microarray data indicated low expression for the other three CDC20s. CDC20-4 is expressed in G2 phase of the cell cycle, earlier then CDC20-1 and CDC20-2. In Y2H tests none of these three CDC20s interacted with the SAC proteins or mitotic cyclins, indicating that they might not play major role in the cell cycle or at least not in the way of the conserved CDC20 function. Whether CDC20-3 and CDC20-5 are pseudogenes or genes involved in specific processes is remain to be elucidated.
Substrate specificity and stage-specific activation of the APC are defined by binding of either of the two adaptor proteins, CDC20 or Cdh1 (CCS52 in plants). While CDC20 is restricted to mitotic cells, Cdh1 appears to be active both mitotic and postmitotic cells. In animals and human, there is a single CDC20 gene, which is thought to be exclusively active in proliferating cells. The function of the CDC20s in plants has not yet been studied.
Plants code for several isoforms of APC activators. In A. thaliana five genes code for highly homologous CDC20 proteins (CDC20-1 - At4g33270, CDC20-2 - At4g33260, CDC20-3 - At5g27080, CDC20-4 - At5g26900, CDC20-5 - At5g27570).
They can be grouped into two classes based on their gene structure, promoter region and binding domain patterns of their proteins. The first group contains CDC20-1 and CDC20-2 that likely result of gene duplication. The expression of these genes and the structure of their proteins are similar to other eukaryotic CDC20s. In the second group (CDC20-3, CDC20-4, CDC20-5) there is no intron in the genes, nevertheless, their protein contain most of the conserved motifs.
Expression pattern of CDC20-1 and CDC20-2 are overlapping during plant development and their cell cycle regulation is identical. In line with that, the single CDC20-1 and CDC20-2 mutants have no visible phenotypes, which suggest redundant function of these proteins. CDC20-1 and CDC20-2 RNAi lines were created for gradient reduce of gene expression, the analysis is under progress. Indeed, these two isoforms are real members of the spindle attachment checkpoint (SAC) protein complex that responsible for the appropriate chromatide separation in late mitosis. In spite of common function in the spindle checkpoint, the two proteins, which regulate also the level of mitotic cyclins, interacted differently with mitotic cyclins in Y2H, indicating that they share this function in the cell cycle in different cell types.
Microarray data indicated low expression for the other three CDC20s. CDC20-4 is expressed in G2 phase of the cell cycle, earlier then CDC20-1 and CDC20-2. In Y2H tests none of these three CDC20s interacted with the SAC proteins or mitotic cyclins, indicating that they might not play major role in the cell cycle or at least not in the way of the conserved CDC20 function. Whether CDC20-3 and CDC20-5 are pseudogenes or genes involved in specific processes is remain to be elucidated.