Study On The Regulation Of Sphingolipid LCB During Exocytosis N Budding Yeast

Objective:Exocytosis is a form of vesicle transport that is often limited to specific areas of the plasma membrane.The vesicles enveloping the secretions detach from the donor membrane,pass through the cytoplasm to reach the vicinity of the receptor membrane,are bound and fuse to the receptor membrane,and the transported secretions are released into the extracellular matrix,and the membrane of the vesicles is incorporated into the plasma membrane.The plasma membrane is replenished.As a signal molecule,sphingolipid LCB is also a component of the plasma membrane,and the synthetic transport pathway of sphingolipids coincides with the secretory vesicle transport pathway.And it has been reported in the literature that sphingolipid LCB can inhibit the aggregation of an artificial protein?Fus-Mid-GFP?localized on the cell membrane.Through the above series of studies,we speculate that sphingolipid LCB may affect the cell's exocytosis.In this study,a series of experimental methods such as exocyst subcellular localization,Bgl2 secretion assay,Invertase secretion assay and thin-film electron microscopy were used to systematically study the regulation of sphingolipid LCB during exocytosis.Methods:1.The plasmids of exocyst with green fluorescent protein?GFP?,SEC3-GFP,SEC5-GFP,SEC8-GFP and EXO70-GFP,were transformed into wild-type and temperature-sensitive mutants,respectively,at the limiting temperature?39??condition.After the treatment,the subcellular localization of exocyst in the cells was observed under the fluorescence microscope.2.Exocytosis in wild-type and temperature-sensitive mutants under limiting temperature?39°C?condition was detected by classical exocytosis assays such as Bgl2 secretion assay and Invertase secretion assay.3.Using the thin-film electron microscopy technique to analyze the submicroscopic structure of wild-type and lcb1-100 temperature-sensitive mutant cells and analyze the stage of the regulation of sphingolipid LCB on exocytosis.Results:At the gene level,the temperature-sensitive mutant?lcb1-100?of the translation initiation gene LCB1 of sphingolipid LCB and its temperature-sensitive mutants(pkh1^tspkh2?,pkc1^ts,ypk1^tsypk2?,sch9?)of each kinase are transferred into the exocyst plasmid carring the green fluorescent protein?GFP?was used to observe the subcellular localization of exocyst in cells at 25°C and limiting temperature?39°C?by fluorescence microscopy,and to detect Bgl2 and invertase secretion assay.Classical exocytosis studies have demonstrated that the expression of sphingolipid LCB plays an important role in exocytosis.At the molecular level,it was confirmed by the action of myriocin,a specific inhibitor of sphingolipid LCB,that sphingolipid LCB has regulation of yeast exocytosis at both the gene and molecular levels.At the same time,a thin-film electron microscope is used to clearly show the period during which sphingolipid LCB regulates the secretory vesicle transport pathway.In addition,by studying the regulation of exocytosis by the sphingolipid LCB downstream kinase,it is speculated that sphingolipid LCB may regulate the exocytosis of yeast through other pathways and may directly regulate exocytosis.Conclusion:1.Sphingolipid LCB can affect the subcellular localization of exocyst in yeast cells.2.Sphingolipid LCB is involved in the regulation of exocytosis.3.The regulation of sphingolipid LCB on exocytosis occurs during the separation of secretory vesicles from the endoplasmic reticulum into the cytoplasm.4.Sphingolipid LCB may not regulate exocytosis through Pkh1/2 and its downstream kinase,or may directly regulate exocytosis.