The Interaction Of Sedlin Related To Spondyloepiphyseal Dysplasia Tarda And PAM14

Spondyloepiphyseal dysplasia tarda (SEDT, MIM 313400) is a genetically heterogeneous disorder of X-linked recessive (XR). The main clinical features are skeletal disorders including osteoarthritis, short stature dwarf due to platyspondyly and dysplasia of large joints.SEDT is caused by the mutation of SEDL gene (MIM 300202), the product of which is called as Sedlin composed of 140-amino acid residues. The SEDL gene is 20 kb in length containing the six exons, and is mapped in Xp22. The highly homology between its orthlogs is found from yeast to human. Sedlin's ortholog in yeast is Trs20p, a subunit of TRAPP (transport protein particle) complex. The TRAPP has two functional forms, TRAPPâ… and TRAPPâ…¡. TRAPPâ… localizes on the cis membrane of Golgi complex and plays a critical role in the vesical transport from endoplasmic reticulum to Golgi as the receptor of the COPâ…¡vesicle. TRAPPâ…¡mainly participate in the trafficking within the Golgi complex. Both of the TRAPPâ… and TRAPPâ…¡have the Trs20p subunit and its deletion has lethal effect on yeast cells that can be resecued by Sedlin with microinjection. So Sedlin may play important roles in the vesicle trafficking from the endoplasmic reticulum to the Golgi complex.To further examine the potential functions of Sedlin in mammalian cells, we performed the yeast two-hybrid screening and obtained some positive clones including CLIC(chloride intracellular channel), EBI3(Epstein-Barr virus-induced gene 3), and PAM14 (protein associated with MRG, 14 kD) , each of them manifest the wide variety of functions in cells.We have confirmed the Sedlin can be associated with CLIC1 in mammalian cells. Now we try to investigate whether Sedlin and PAM14 can interact each other in mammalian cells. Firstly, we constructed the recombinant plasmids pCDGFP-SEDL, pCDGFP-PAM14, and transfected them into mammalian cells, and the expression and localization of the Sedlin and PAM14 proteins were analyzed by western blot and fluorescence microscopy. Then we performed the GST-pull down assay with recombinant plasmid pGEX-3X-SEDL expressing the fusion protein GST-Sedlin. Finally, the interaction of Sedlin and PAM14 was confirmed with coimmuniprecipitation assay in HEK 293T cells, and immunofluorescence in COS7 cells was performed to investigate whether they are colocalized or not.The results showed the Sedlin can interact with PAM14 in vitro by GST-pull down assay and in vivo by coimmunoprecitation, and the colocalization of Sedlin and PAM14 was observed in COS7 cells. So we can conclude that Sedlin and PAM14 interact each other in mammalian cells.PAM14, a highly conserved protein composed of 127 amino acid residues, is associated with MRG15 and Rb which complexes with MRG15. Rb and MRG15 both function in transcriptional regulations and play important roles in cell growth, senescence and/or immortalization. Our results suggest Sedlin may have implications in transcription process.