The Further Study On The Interaction Of Sedlin And PAM14

Objective To investigate the critical interaction region between PAM14 and Sedlin by yeast two-hybrid assay; Construction the eukaryocyte expression vectors of PAM14 protein and protein N -C terminal deletion mutants and Sedlin protein ,co-transfected into COS7 cells to observe in mammals Co-localization of cells; Co-transfect PAM14-N protein tagged with HA and GFP-tagged Sedlin protein into HEK293T cells together for Immunoprecipitation to detect PAM14-N protein can interact with Sedlin.protein in mammalian cells. Construction of the four point mutants S30A, V130D of Sedlin protein to pcDGFP eukaryocyte expression vector, transfected into COS7 cells to observe them single localization difference with the wild type, then,co-transfected with deletion mutants of PAM14 protein with HA tag into COS7 cells together to observe in mammals co-localization of cells,respectively.Methods The full length cDNA fragment of PAM14 was amplified by PCR from pACT2-PAM14. PCR product and pGBKT7 vector were digested by appropriate restriction enzymes respectively, then pGBKT7 -PAM14 was constructed. As also, pGBKT7 -PAM14-N, pGBKT7 -PAM14-C, pCDNA3.1-PAM14-N-HA and pCDNA3.1-HA-PAM14-C were constructed by PCR(N-terminal encoding 84 AA, labeled PAM14-N).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, pGADT7-Sedlin /pGBKT7,pGADT7-Sedlin /pGBKT7-PAM14 pGADT7- Sedlin / pGBKT7-PAM14-N ,pGADT7- Sedlin / pGBKT7-PAM14-C pGADT7- Sedlin / pGBKT7-PAM14-N-N,pGADT7- Sedlin / pGBKT7-PAM14-N-C pGADT7-PAM14/pGBKT7-Sedlin(S30A),pGADT7-PAM14/pGBKT7-Sedlin(V130D),pGADT7-PAM14/pGBKT7-Sedlin(F83L),pGADT7-PAM14/pGBKT7-Sedlin(S73L) pGADT7-PAM14/pGBKT7-Sedlin(D47Y).Then to detect the activity ofα-galactosidase by Clony-lift Filter Assay. The blue clones were positive, which shows protein-protein interactation.pCDNA3.1-PAM14-N-HA/pCDGFP-Sedlin and pCDNA3.1-HA-PAM14/ pCDGFP-Sedlin(S30A) were detected in COS7 cells by fluorescence microscopy. HEK293T cells were cotransfected with pCDNA3.1-PAM14-N-HA and pCDGFP-Sedlin and extractec cells lysis, we try to investigate whether PAM14-N and Sedlin can interact each other in mammalian cells with coimmuniprecipitation assay .Results Plasmids were constructed correctly in each group by sequence analysis.α-galactosidase activity tests and nutrition screening show that, pGBKT7-PAM14/ pGADT7-Sedlin,pGBKT7-PAM14-N/pGADT7-Sedlin,pGBKT7-PAM14/pGADT7-SedlinS(S30A) were positive clones. pCDNA3.1-PAM14-N-HA and pCDGFP-Sedlin conlocalization in the nucleus of COS7 cells. pCDNA3.1-HA-PAM14 and pCDGFP-Sedlin(S30A) conlocalization in the nucleus of COS7 cells . Coimmunoprecipitation remains to be shown the interaction of PAM14-N protein and Sedlin protein in HEK293T cellsConclusion Successfully used yeast-two-hybrid system 3 to identify the interactation between PAM14 and Sedlin. N-terminal 84 amino acid residues of PAM14 is necessary for interaction with Sedlin, Sedlin S30 for their interaction is not necessary.