| The C0P9 signalosome (CSN) complex is a conserved protein complex, typically consisting of eight subunits designated CSN1-CSN8. CSN complex is originally identified based on mutants with a constitutive photomorphogenic (cop) phenotype in Arabidopsis thaliana seedlings.CSN, COP1 and C0P10-DDB1-DET1 (CDD) complex component are key regulators of plant light responses and development. COP1 can respond to light signals by differentially partitions between nuclear and cytoplasmic compartments. Previous genetic analysis in Arabidopsis indicates that nuclear localization of COP1 requires CSN. However the mechanism underlying the functional relationship between COP1 and CSN is unknown.In this report, we report that, in onion epidermal cells, expression of CSN1 can stimulate nuclear localization of GUS-COP1, and the N-terminal domain of CSN1 is necessary and sufficient for this function. We also provide genetic evidence that CSN1 N-terminal domain is specifically required for COP1 nuclear localization in Arabidopsis hypocotyls cells. Further, we demonstrate that CSN1, via its N-terminal domain (NTD), directly interacts with COP1 at the coiled-coil domain in yeast two hybrid system, and confirm that COP1 weakly associates with CSN in vivo. Moreover, CSN1-induced COP1 nuclear localization requires COP1's own nuclear-localization sequences as well as its coiled-coil domain, which contains both the cytoplasmic localization sequences and the CSN1 interacting domain. These findings provide a mechanistic insight into the interrelationship between COP1 and the CSN complex. Our findings set a foundation for further investigations into the mechanism of COP1 subcellular localization in plants.CSN complex is initially defined as a repressor of photomorphogenesis in Arabidopsis, and it has now been found to participate in diverse cellular and developmental processes in various eukaryotic organisms. CSN1 contains a large PCI domain located at the C-terminal half of the protein, which is necessary for subunit-subunit interaction and complex assembly. The NTD of CSN1, on the other hand, does not have a structural role, but is involved in a critical function of CSN that is not yet understood molecularly.To get more information about the functional contribution of CSNl-NTD in plant development, yeast two-hybrid screening is used to identify proteins able to interact with the NTD of CSNl. At last, five different genes are identified by sequencing the positive clones. The proteins of these genes are PAD2, TSA1, eIF-2 family protein,60S ribosomal protein L35 and C2 domain-containing protein. We focus on the study of the functions of PAD2, TSA1 with CSN1-NTD.PAD2 is one of the 20S proteasome alph subunits. By yeast two-hybrid screening, we got the NTD truncated version of PAD2. In Arabidopsis, PAD2 has a homology, named PAD1. We find that the C-terminal domain (CTD) of PAD1 can also interact with CSN1-NTD in yeast two-hybrid system. Moreover, the interaction between COP1 and the CTD of PAD1, but not the CTD of PAD2, is detected by yeast two-hybrid assay.TSA1 is a caldesmon-related protein, and has Ca2+-binding activity. By yeast two-hybrid screening, we got the NTD truncated version of TSA1. The interaction between full length TSA1 and CSN1-NTD is confirmed by yeast two-hybrid assay. To study the biological function of TSA1, T-DNA insertion mutant is obtained from the SALK T-DNA collection. In the line, T-DNA insertion is 103bp before the ATG of AtTSAl. The expression level of AtTSA1 in the mutant is examined by half-quantitative RT-PCR, which shows lower TSA1 transcript in the mutant than in the wild type. In normal light growth conditions, tsal mutants do not exhibit any detectable abnormality throughout the whole life span. While in the dark, the mutants exhibit slightly decreased hypocotyls, compared with the wild type.Further studies are required to del ineate the functional relationship between CSNl and these interaction proteins. |
PDF Full Text Request