The Role Of TfR-Ubi And Tom1L1 In Endosomal Sorting

Endocytosis refers to the trafficking of extracellular material into the cell though the cell membrane depression and completes various biological processes.The endocytosis compartment is a complex heterogeneous organelle composed of a large number of tube bubble cells,extending from the surface of the cell member to the nucleus.The intake and sorting of nutrients,metabolites,hormone and growth factors are started from endocytosis,in addition,endocytosis also maintains the distribution of function domain of polar plasma membrane and the systemic circulation of receptor and ligand.After the receptor binds to the ligand,some of them go back to the membrane surface from the early endosome,and the other are transferred to late endosome/ the lysosome for degradation.The ubiquitin molecule is a signal for the receptor sorted into different pathways,and the identification of the ubiquitin signal depends on the endosomal sorting complex required for transport(ESCRT).The ubiquitination receptors selected by ESCRT entry into multivesicular body(MVB),and were subsequently transported into late endosome/ the lysosome to lower the cell signal.Tom1L1 is implicated as the substrate of Src family protein kinase to mediate endocytosis of EGF receptor,intracellular sorting and signal transduction.Studies have shown that EGF-stimulated Src family promoted the phosphorylation of Tom1L1 combining with Grb2.EGF recruited Tom1L1 from the cytoplasm to the plasma membrane and into the early intracellular body with EGFR.But Tom1L1 mutants were able to inhibit the endocytosis of EGFR.Tom1L1 was essential for the process of endocytosis and intracellular sorting.But there is no explanation that how Tom1L1 is involved in the ESCRT,so in this study we firstly build transferrin receptor-ubiquitin as intracellular markers,to investigate the role of Tom1L1?ESCRT subunits and the ubiquitin protein in intracellular sorting.We observe that the role of Tom1L1 in intracellular sorting,degradation pathways and the MVB forming process.It provides molecular pathologic basis for many diseases and new ideas for clinical treatment strategies.Method 1.Construction of transferrin receptor – ubiquitin vector and Tom1L1 vector.According to specific primers design,transferrin receptor?ubiquitin and Tom1L1 was amplified by PCR.Purified gene was cloned into p CI-neo-HA vector.And the recombinant expression vector was sequenced exactly.2.The expression of transferrin receptor – ubiquitin and Tom1L1.After sequenced correctly,the recombinant expression vector was transfected into A431 cells.After purification of cell supernatant,they were detected by Western Blot.3.Transferrin receptor – ubiquitin were detected by immunofluorescence.In the A431 cells with stable expression of transferrin receptor – ubiquitin,transferrin stimulated and immunofluorescence microscopy was used to observe the localization of transferrin receptor – ubiquitin in different time periods.4.The relationship between transferrin receptor – ubiquitin and Tom1L1.When Tom1L1 was overexpressed in the stable cell strain of A431,transferrin stimulated,immunofluorescence microscopy was used to observe the localization of ubiquitin-transferrin receptor and Tom1L1.In the A431 cells with Tom1L1 knock-out,the transferrin stimulated and immunofluorescence microscopy was used to observe the localization of the ubiquitin-transferrin receptor.5.Effects of Hrs between transferrin receptor – ubiquitin and Tom1L1.The expression of Tom1L1 was detected by immunofluorescence in A431 cells with Hrs over-expressed.In the MEF cells with Hrs knocked-out,theimmunofluorescence detected transferrin receptor – ubiquitin and Tom1L1.Result: 1.Construction,expression and identification of transferrin receptor – ubiquitin,Tom1L1 and its mutant gene vectors.Ubiquitin,Transferrin receptor,Tom1L1 and its mutant gene fragments were amplified by PCR according to specific designed primers as expected.Purified products were cloned into p CI-neo-HA vector.The recombinant expression vector were sequenced exactly and transfected into A431 cells.After purification of cell supernatant,transferrin receptor-ubiquitin was confirmed as one band of 100 k Da by Western Blot.Tom1L1 was confirmed as the band of 55 k Da.2.Localization of the transferrin receptor – ubiquitin in cells.Immunofluorescence results showed that the transferrin receptor modified by ubiquitin did not return to the cell surface,but was sorted to the intracellular body.3.The relationship between transferrin receptor-ubiquitin and Tom1L1.The results of immunofluorescence showed that Tom1L1 and transferrin re-ceptor-ubiquitin colocated intracellular body,and the localization of transferri n receptor-ubiquitin to intracellular body was influenced with Tom1L1 knock ed down.4.Effect of Hrs between transferrin receptor – ubiquitin and Tom1L1.In A431 cells with Hrs over-expressed,Tom1L1 is attracted to the intracellular body.In the Hrs-KO MEF cells,the number of intracellular body structures with transferrin receptor-ubiquitin was decreased,and the volume of endosome was larger,then Tom1L1 was no longer attracted to the intracellular body.Conclusion: 1.Tf R-Ubi provides experimental tools for further study of ESCRT and subsequent mechanism research.2.Tf R-Ubi localization to the body structure depends on the interaction between Tom1L1 and Hrs.3.Tom1L1 can participate in the separation of the protein cells in the presence of Hrs.