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.