Screening Of The Small GTPase That Targets CD36 To The Plasma Membrane

Due to the occurrence of post-translational modifications of proteins,proteins have more complete functions and more complex structures.As one of the post-translational modifications of protein,palmitoylation modification can regulate the interaction between protein and protein,regulate the stability and activity of protein,promote protein localization of membrane lipid raft,etc.Conduction,apoptosis,disease occurrence,and canceration all play important roles.CD36 plays an important role as a palmitoylated protein containing four palmitoylation sites,and also as a fatty acid translocase,which transports long-chain fatty acids and plays an important role in lipid metabolism.Preliminary research in the laboratory found that CD36 was palmitoylated by palmitoyl transferases DHHC4 and DHHC5 and then sorted into the plasma membrane for fatty acid uptake.However,it is not clear how the palmitoylated CD36 was transported to the plasma membrane.Small GTPases play an important role in the transport of intracellular vesicles.In this study,we first screened 3T3-L1 cells for small GTPases with relatively high abundance expression in adipose tissue by shRNA,and it was found that two small GTPases,RAB43 and ARF6,were necessary to maintain the plasma membrane localization of CD36.Subsequently,after knocking down ARF6 in HeLa cells,CD36 was localized in the Golgi apparatus,and after knocking down RAB43,CD36 was localized in the endoplasmic reticulum,which further proved the importance of these two proteins for CD36 transport.In addition to changes in localization,we also tested whether palmitoylation levels of CD36 were affected by knockdown of these two genes.The results also showed that the palmitoylation level of CD36 was significantly reduced in HeLa cells,whether it was knockdown of RAB43 or ARF6.In terms of fatty acid uptake,we found that after knocking down RAB43 or ARF6 in 3T3-L1 cells,the fatty acids uptake by the cells also decreased.We will further explore the mechanisms by which RAB43 and ARF6 regulate the membrane of CD36.This experiment provides a theoretical basis for further revealing the transport mechanism of CD36 and protein palmitoylation for metabolic regulation.