3.3 Subcellular localization of CRCT
CRCT contains a CCT domain which usually has an NLS. Prediction of the subcellular localization of CRCT by NLS mapper (http://nls-mapper.iab.keio.ac.jp) yielded a moderate score (5.4), suggesting localization to both the nucleus and the cytoplasm and identified putative bipartite NLS within the CCT domain (Figure S7). To clarify the exact subcellular localization of CRCT, the fusion proteins of the full-length CRCT with GFP (FL-CRCT::GFP) and the CCT domain with GFP (CCT::GFP) were expressed in epidermal cells of onion (Allium cepa ). The GFP fluorescence of FL-CRCT::GFP was observed at the nucleus and outside the nucleus, possibly the cytosol (Figure 4). The GFP fluorescence of nucleus in FL-CRCT::GFP was more predominant than that in GFP alone. In addition, the GFP fluorescence at the nucleus was more specifically detected in CCT::GFP. These findings indicate that the CRCT protein can localize to the nucleus, and the CCT domain of CRCT is involved in the nuclear localization.