Figure 1. Characterization of the COP9/Signalosome in C. elegant.(A) Two-hybrid interaction matrix of the C. elegans COP9/signalosome subunits. Yeast cells (MAV103) expressing CSN-1-CSN-6 (fused to the Gal-4 DNA binding domain, DB) and CSN-1-CSN-5 (CSN subunit fused to the activation domain, AD) were grown on filters for the β-galactosidase assay.(B) A schematic representation of the network of interaction defined by two-hybrid experiments. The gray circles represent subunits with MPN domains; the white circles represent subunits with a PCI domain. The bold lines indicate that the interaction was found to be positive by using two independent reporter genes (LACZ and HIS3); the dashed lines indicate that the interactions were found to be positive by using the HIS3 reporter gene. It is important to note that several interactions were found to be positive in one direction but not in the other.(C) CSN-5 is able to partially restore deneddylation of Cdc53 in the
rri1delta strain. Protein extracts from wild-type (K699, lane1),
rri1delta (YLP89, lane 2), or
rri1delta expressing CSN-5 under the control of the constitutive ADH promoter (YLP108, lane 3) were loaded on SDS/PAGE and were transferred to a membrane. The membrane was probed with anti-Cdc53 antibodies. The arrows point to the position of rubylated and nonrubylated Cdc53.(D) CSN-5 localized to the nucleus and the cytoplasm. Confocal micrographs of CSN-5 visualized by indirect immunofluorescence with an affinity-purified anti-CSN-5 antibody and chromosomal DNA in (a and b) wild-type embryos during pronuclear migration, in (c and d) two-cell embryos, and in (e and f) four-cell embryos. For control, a
csn-5(RNAi) embryo is shown in (g). Note that CSN-5 is mainly cytoplasmic during mitosis (example: AB cell of the two-cell embryo, [c]).(E) The affinity-purified anti-CSN-5 antibody recognized a specific band at the expected size of 41 kDa. Embryo extracts from wild-type were loaded on SDS-PAGE and were transferred to a membrane. The membrane was probed with preimmune serum (lane 1) or affinity-purified anti-CSN-5 antibody (lane 2).