The Preliminary Study On The Interaction Of Sedlin And PAM14

Objective To investigate the interaction between Sedlin and PAM14, and define the interaction region of Sedlin with PAM14 by yeast two-hybrid system. To construct PAM14 deletion mutants and Sedlin site-directed mutants, and to find the binding sites in yeast. Then, to investigate the colocalization of PAM14 and Sedlin in appropriate mammalian cell lines.Methods The full length cDNA fragment of PAM14 was amplified by PCR from pCDNA3.1- PAM14. PCR product and pACT2 vector were digested by appropriate restriction enzymes respectively, then pACT2-PAM14 was constructed. As also, pCDNA3.1-HA-PAM14 and pCDGFP-Sedlin were constructed by PCR. The deletion mutant pACT2-Pam14-N was constructed by SmaⅠdigestion. In addition, the other deletion mutant pACT2-Pam14-C was obtained by PCR. Coding sequences of the Sedlin mutant were construced by modified method of Stratagene Site-Directed Mutagenesis Kit with pAS-Sedlin as a template. Positive clones have been selected with restriction enzyme fragment analysis and confirmed by nucleotide sequencing.Those yeast expression vectors were classified into there groups, and their interactions were investigated in the yeast. Yeast Y190 cells were cotransformed with the following plasmids, the first group pACT2 / pAS-Sedlin, pACT2 / pAS-Sedlin-N, pACT2-PAM14 / pAS-Sedlin, pACT2-PAM14 / pAS-Sedlin-N. Next pACT2/pAS- Sedlin, pACT2-PA M14/pAS-Sedlin, pACT2-PAM14-N/pAS-Sedlin, pACT2-PAM14 -C / pAS-Sedlin. The third group the ACT2 / pAS-Sedlin, pACT2-PAM14 / pAS-Sedlin, pACT2-PAM14 /pAS-SedlinS. Then to detect the activity ofβ-galactosidase by Clony-lift Filter Assay. The blue clones were positive, which shows protein-protein interactation.pCDNA3.1-HA-PAM14, pCDGFP-Sedlin were detected in COS7 cells by fluorescence microscopy. Nucleoli were extracted from the cotransfected HEK 293T cells by sucrose density gradient centrifugation. The nucleoli were checked by western-blot and fluorescence microscopy.Results The Sedlin S124A has been confirmed by sequence analysis.β-galactosidase activity tests and nutrition screening show that, only pACT2-PAM14/pAS-Sedlin, pACT2-PAM14-N/pAS-Sedlin, pACT2-PAM14/pAS-SedlinS were positive clones. pCDNA3.1-HA-PAM14 and pCDGFP-Sedlin conlocal- ization in the nucleus of COS7 cells . Nucleoli of the cotransfected cells were extracted by sucrose gradient centrifuga- tion. Western-blot and fluorescence microscopy all show that PAM14 and Sedlin are both expressed in the nucleoli.Conclusion Successfully used yeast-two-hybrid system 2 to identify the interactation between PAM14 and Sedlin. N-terminal 84 amino acid residues of PAM14 is necessary for interaction with Sedlin, Sedlin S124 for their interaction is not necessary. Sedlin interaction with PAM14 in HEK 293T cells, and they also conlocalization in nucleoli in the COS7 cells.