Molecular Mechanism For ACBD And FAPP2 Cooperating To Transfer Glucosylceramide

Newly-made lipids in the endoplasmic reticulum are transported to the next destination via vesicular transport and lipid transporters to synthesize the more complex lipids or to perform biological functions there.Lipid transfer protein has been shown to play an important role in lipid transport.PI4 P adaptor protein FAPP2 is a glycolipid transporter,which transfers glucosylceramide from cis-Golgi to the trans-Golgi network(TGN).Due to many enzymes responsible for complex glycosphingolipid synthesis located in the lumen of the Golgi apparatus.Glucosylceramide can be transformed into lactosylceramide and more complex glycosphingolipids there.Therefore,FAPP2 plays a key role in the glycosphingolipid metabolic pathway,even affects complex glycosphingolipids biosynthesis.In the present studies,we utilized yeast two-hybrid assay to identify ACBD3,a Golgi resident protein,as a novel interaction partner of FAPP2.Our co-immunoprecipitation further verified the interaction between ACBD3 and FAPP2.Then we observed ACBD3 and FAPP2 co-localized in the region of the Golgi complex with a fluorescence microscope.In the mean time we mapped ACBD3(1-180)is a region responsible for the interaction of ACBD3 with FAPP2.Under electron microscopy the Golgi apparatus in wild type cells was flat membrane stacks continuously distributed around the nucleus,while the Golgi apparatus in ACBD3 knockdown cells had fragmented structural features.We further analyzed the results of immunofluorescence and found that the relative Golgi area and the relative FAPP2 distribution in ACBD3 knockdown cells were significantly increased compared with that of the control cells.Our lipidomics data indicated that,compared with control cells,the levels of Glc Cer in the ACBD3 knockdown cells were increased significantly.However,the contents of Lac Cer,GM3 and Gb3 were decreased.We then tried to rescue the defects caused by ACBD3 knockdown through expressing GFP-ACBD3-4M,GFP-ACBD3(1-180)and GFP-ACBD3(171-528)in ACBD3 depleted cells,respectively.We found that GFP-ACBD3-4M and GFP-ACBD3(171-528)could effectively rescue the Golgi morphological defects and changes of glycosphingolipids metabolism by the ACBD3 knockdown.However,expression of GFP-ACBD3(1-180)in ACBD3 knockdown cells failed to remedy the defects of Golgi morphology and the complex glycosphingolipids metabolism caused by ACBD3 knockdown.Taken together,the Golgi integrity maintained by the ACBD3 is required for FAPP2 transferring glucosylceramide for the complex glycosphingolipids biosynthesis.