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.