3.6 CRCT forms a complex with 14-3-3 proteins
To clarify the structural component of the CRCT complex, an immunoprecipitation was carried out using the FLAG-CRCT overexpression line (driven by the Actin promoter) and CRCT knockout line. At the beginning of this experiment, both the CRCT antibody and the FLAG antibody were tested, so the knockout mutant was used as the reference instead of the wild type. There were no apparent differences in the band pattern of WT and CRCT knock-out mutant after SDS-PAGE followed by Coomassie blue staining (Figure 6a). The FLAG antibody was used going forward because it was more specific than the CRCT antibody. The FLAG-CRCT fusion protein was detected by immunoblot analysis both in the input and immunoprecipitation with the FLAG antibody (Figure 7). Some silver stain bands (Band 1, 2 and 3) were detected only in the FLAG-CRCT overexpression line and not in CRCT knockout line (Figure 7). MS analysis with MASCOT search indicated that Band 1 contained 60S acidic ribosomal protein and guanine nucleotide-binding protein β. Bands 2 and 3 contained three 14-3-3-like proteins (GF14B, GF14E, GF14F) and two 14-3-3-like proteins (GF14A, GF14C), respectively (Table 1). These five 14-3-3-like proteins showed higher MS scores than the proteins from band 1 and mutually share high amino acid sequence identity (Yao, Du, Jiang, & Liu, 2007). Thus, we selected GF14B from band 2 and GF14A from band 3 for subsequent analyses of interaction with CRCT.
A yeast two-hybrid analysis was performed to confirm the interactions between CRCT and the 14-3-3 proteins (Figure 8). Full length CRCT and 14-3-3 proteins (GF14A and GF14B) were fused to the GAL4 activation domain and GAL4 DNA binding domain, respectively. The growth of yeast with empty vectors was not observed on the SD3- plate, whereas the growth of yeast with CRCT and 14-3-3 proteins were observed under the condition, indicating that CRCT could interact with both GF14A and GF14B. The 14-3-3 protein binding motif was reported to be (R/K)X1-3(S/T)XP and the S/T in this motif should be phosphorylated to bind to 14-3-3 proteins (Chen et al., 2019). CRCT contains the 14-3-3 binding motif KEKPSVP upstream of the CCT domain (Figure S7 and S8; Table S3). The CCT domain of CRCT and the 14-3-3 proteins were fused to the GAL4 DNA binding domain and GAL4 activation domain, respectively. The growth of yeast with these vectors was not observed in the SD3- plate (Figure S8b), indicating that the CCT domain alone could not interact with the 14-3-3 proteins, and it is likely that the 14-3-3 binding motif found in CRCT is necessary for the interaction between CRCT and 14-3-3 proteins.
The interaction between CRCT and GF14A was further investigated by a bimolecular fluorescence complementation (BiFC) assay (Figure 9). Among GFP variants, the superfolder GFP (sfGFP) was used for this BiFC analysis because sfGFP-based BiFC is the brightest in plants compared with other GFP variants used in BiFC (Fujii, & Kodama, 2015). The N- and C-terminals of sfGFP were fused to the N-terminal ends of CRCT and GF14A and expressed in onion epidermal cells. Fluorescence from the reconstituted sfGFP protein was detected when CRCT and GF14A were coexpressed, and this fluorescence matched with the position of nuclei, as visualized by DAPI staining. The 14-3-3 proteins were previously reported to be present as dimers in the nuclear and non-nuclear compartments other than vacuoles (Bihn et al., 1997). Thus, we used GF14A as a positive control of BiFC. As expected, a significant fluorescent signal was observed in the nuclear and non-nuclear compartments when GF14A fused to both the N- and C-terminal parts of sfGFP were coexpressed. In contrast, sfGFP fluorescence was quite low when CRCT fused to both the N- and C-terminal parts of sfGFP were coexpressed, suggesting that the oligomerization of CRCT is not likely. In our BiFC analysis, self-assembly of the sfGFP was unlikely because sfGFP fluorescence was quite low when 7-methylxantine methyltransferase (MXMT), an arbitrary cytosolic protein was coexpressed with CRCT. These results indicate that CRCT is mostly in close contact with 14-3-3 proteins in the nucleus.