We have previously identified and characterized a maize Rpd3-type histone deacetylase (ZmRpd3I; Rossi, V, et al., Mol Gen Genet 258:288-296, 1998). In addition, the identification of components of the pRb/E2F pathway in plants suggests that the basic molecular control of cell cycle has been conserved from animals to plants (reviewed in Huntley, RP and Murray, JAH, Curr Opin Plant Biol 2:440-446, 1999). Analysis of the protein interactions between ZmRpd3I, maize retinoblastoma-related (ZmRBR1) and retinoblastoma associated (ZmRbAp1) proteins was performed by means of in vitro GST-pull down assays. The results indicate that ZmRBR1 interacts with ZmRpd3I and that ZmRbAp1 can bind both ZmRBR1 and ZmRpd3I. Deletions and site-specific mutants were used to analyze the regions of ZmRBR1 and ZmRpd3I involved in the protein interaction. We observed that the integrity of both A/B pocket and C-terminal domains of ZmRBR1 are required for the binding to ZmRpd3I. The same domains are also responsible for the association with ZmRbAp1, although mutations affecting the structure of the A/B pocket did not reduce the binding. It is noteworthy that the A/B pocket of pRb is highly conserved in higher eukaryotes. Particularly, it is believed that the LXCXE binding site, located within the pocket, is required in mediating the interaction with several pRb-associated proteins containing the LXCXE domain, including two of the three mammalian Rpd3-type HDACs identified so far (Harbour, JW and Dean, DC, Genes Dev 14:2393-2409, 2000). Because ZmRpd3I does not contain the LXCXE domain, we carried out GST-pull downs in which a synthetic LXCXE peptide was added to the reaction mix to detect its effect on binding. Our results suggest that the ZmRBR1 LXCXE binding site is not involved in the interaction with ZmRpd3I and ZmRbAp1. Hence, different domains in the A/B pocket and C-terminal region of ZmRBR1 are likely to be required to mediate these protein interactions.
Deletions in the ZmRpd3I sequence showed that the simultaneous removal of both C- and N- termini abolished the interaction with ZmRBR1 and ZmRbAp1, indicating that there are multiple contacts between these proteins. The two ZmRpd3I regions required for association with ZmRBR1 are partially overlapping with those involved in the interaction with ZmRbAp1. This finding suggests that ZmRbAp1 may mediate, at least in part, the contacts between ZmRpd3I and ZmRBR1. Accordingly with this scenario, we observed that addition of recombinant ZmRbAp1 protein in the reaction mix in GST-pull downs increased the amount of in vitro translated ZmRBR1 specifically retained by the GST-ZmRpd3I fusion protein. Similar results were obtained in reciprocal experiments. Conversely, addition of ZmRpd3I recombinant protein did not alter the association between ZmRbAp1 and ZmRBR1.
These results provide the first, although
still preliminary, evidence of plant multiprotein complexes containing
Rpd3-type HDACs and regulators that play a pivotal role in controlling
cell cycle progression.
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