Subcellular localization of wild type and mutated forms of the recombinant CSAFlag-HA protein
The subcellular localization of the recombinant CSAFlag-HA protein, either in its wild type (wtCSAFlag-HA) or mutated forms (E52V-, Q106P-, or K174A-CSAFlag-HA), was investigated by immunofluorescence with antibodies raised against the HA epitope tag (anti-HA). The analysis was performed on the isogenic cell lines previously generated: the CS3BE-wtCSAFlag-HA, CS3BE-E52V-CSAFlag-HA, CS3BE-Q106P-CSAFlag-HA, CS3BE-K174A-CSAFlag-HA, and CS3BE-cassette1 cells, the last ones serving as negative control (Fig. 1). We found that the wtCSAFlag-HA protein is mainly localized in the nucleus with a less intense, but not omitted, staining inside the nucleolus, in agreement with previous observations [22, 23]. Differently, CSAFlag-HA proteins with the single amino acid change E52V or Q106P localized mainly in the cytoplasmic and perinuclear region, while the CSAFlag-HA protein carrying the K174A substitution equally distributed in the cytoplasmic and nuclear compartments with a clear accumulation in nucleoli. Overall, these results revealed that single amino acid substitutions affect the subcellular localization of the wtCSAFlag-HA and increase its accumulation in the nucleolus.
Subcellular localization of the TRiC complex in the isogenic cell lines expressing the wild type or mutated forms of CSAFlag-HA
Previous studies in the laboratory have shown that CSA interacts with the TRiC chaperonin complex. In particular, it was shown a direct interaction of CSA with the TCP1, CCT3, and CCT8 subunits of the TRiC complex (Uggè et al., in preparation). Therefore, we investigated whether amino acids changes in CSA, which affect the cellular localization of the protein itself, may also impact the cellular distribution of the TRiC complex. To address this issue, we looked at the subcellular localization of the complex by immunofluorescence analysis using antibodies raised against the TCP1, CCT3, and CCT8 subunits in the parental cell line CS3BE-cassette1 as well as in all the isogenic cell lines expressing either the wild type or mutated forms of CSAFlag-HA. Since all the primary antibodies (anti-TCP1, anti-CCT3, and anti-CCT8) were originated in rabbit, we first performed a control experiment using only the secondary anti-rabbit antibody. As shown in Fig. S2, no fluorescence background signal was observed in CS3BE-wtCSAFlag-HA cells.
Next, we used anti-CCT3 antibody to investigate the cellular localization of this subunit of the TRiC complex. In all the isogenic cell lines, we found that the protein is localized mainly in the cytoplasm and perinuclear region, where protein synthesis and folding occurs. Also, we observed a very faint nuclear staining with occasional fluorescent dots of accumulations. Relevant of note, by comparing the immunofluorescence pattern distribution of CCT3 in the different cell lines, we did not observe any significant alteration. The protein is similarly distributed in CS3BE-cassette1, which lacks a functional CSA protein, and in all the isogenic cell lines expressing either the wild type or the mutated forms of CSAFlag-HA (Fig. S3).
Antibodies raised against the CCT8 subunit of the TRiC complex revealed that CCT8 shows a similar cellular distribution as CCT3. Also, this subunit of the TRiC complex localizes mainly in the cytosol and perinuclear region, with no evident exclusion from the nuclear compartment (Fig. 2; S4). Moreover, similarly to CCT3, no alterations were observed by comparing the immunofluorescence pattern distribution of CCT8 in the different cell lines. Overall, these data allowed us to conclude that the functionality of CSA protein does not impact the subcellular localization of the CCT3 or CCT8 proteins.
Then, we investigated the cellular localization of the TCP1 subunit of the TRiC complex by using anti-TCP1 antibody. We found that this subunit reveals a different cellular distribution compared to CCT3 and CCT8. Indeed, TCP1 is more abundantly present in the nuclei where it tends to accumulate in specific nuclear structures that we will refer to as nuclear bodies (NBs). It is also possible that these structures may represent Cajal bodies (CBs), according to literature data showing that TRiC subunits are required for the proper assembly and trafficking of telomerase to CBs [21]. Once more, by comparing the immunofluorescence pattern distribution of TCP1 in the different cell lines, we did not find major differences either in CS3BE-cassette1 or in the other isogenic cell lines expressing the wild type or mutated forms of CSAFlag-HA (Fig. 3; S5).
Cellular co-localization of the recombinant wtCSAFlag-HA or the mutated forms of CSAFlag-HA protein with the TRiC complex
As a result of the similarity between CCT3 and CCT8, the cellular co-localization of wtCSAFlag-HA or the mutated forms of CSAFlag-HA with the TRiC complex was further investigated by double immunofluorescence staining with anti-HA and anti-CCT8 or anti-TCP1 antibodies in CS3BE-cassette1 and all the isogenic cell lines. We found that the nuclear wtCSAFlag-HA recombinant protein showed some restricted co-localization signal with the faint nuclear fluorescence staining of CCT8 (Fig. 2), which per se is more abundantly found in the cytoplasm (Fig. 2). Notably, all the mutated forms of wtCSAFlag-HA that tend to accumulate in the cytoplasm (Fig. 2), present a stronger co-localization with the cytoplasmic fluorescence of CCT8 subunit. A different pattern is observed for the TCP1 subunit that it is more abundantly found in the nucleus. The nuclear wtCSAFlag-HA co-localizes with nuclear TCP1 and the TCP1-positive nuclear bodies (NBs) whereas the mutated CSAFlag-HA proteins showed very mild co-localization signals (Fig. 3).