Rab8a and Vps35 influence intracellular transport of vitamin E via α-Tocopherol transport protein in hepatocytes

Of all the forms of vitamin E, α-tocopherol is distinguished as the primary ligand for the α-tocopherol transport protein (α-TTP), a pivotal factor in its secretion into the bloodstream and subsequent systemic distribution. Nevertheless, the intricate molecular mechanisms governing the transport of α-tocopherol via α-TTP have yet to be fully elucidated. In this research, Co-Immunoprecipitation (Co-IP)/LC-MS and His-pull-down assays were utilized to identify proteins interacting with α-TTP. Immunofluorescence staining and Co-IP/Western blotting further confirmed these interactions. Meanwhile, RNA-seq was utilized to discover α-tocopherol-related genes. Genes knockdown was conducted to examine the influence of related genes on vitamin E transport. The concentrations of intracellular and extracellular vitamin E were quantified using LC-MS and specific assay kits. Immunofluorescence staining showed colocalization of Rab8a, SNX3, SNX5, SNX17, and SNX27 with α-TTP, whereas Co-IP/Western blot analysis indicated a specific interaction among Vps35, Rab8a and α-TTP. Notably, the knockdown of Rab8a, SNX5, SNX17, and SNX27 individually influenced the vitamin E content both intracellularly and extracellularly, whereas knockdown of SNX3 did not show such effects in hepatocytes. This research highlights the crucial roles of Rab8a and Vps35 in α-tocopherol's intracellular transport probably by direct interaction with α-TTP, and their association with Retromer-SNX27, Commander-SNX17, and ESCPE1 complexes.