Study Of An Evolutionary Conserved Dop 1-Neo 1-Mon 2 Complex In Glycosylation And Vesicular Trafficking

Glycosylation is a complex posttranslational modification that is essential for a variety of cellular physiological activities.Glycosylation of proteins and lipids is catalyzed by glycosyltransferase or glycosidases localized at the specific compartments of the Golgi apparatus.Vesicular retrograde transport within the Golgi apparatus is essential for maintaining the correct localization of those enzymes.Therefore,it is of great academic significance to study mechanisms for trafficking of proteins at intra-and inter-Golgi network.In this study,a conserved Dop1-Neo1-Mon2 complex was focused on the researches in budding yeast,Saccharomyces cerevisiae,and human embryonic kidney 293(HEK293)cells.Using molecular and cell biological techniques,the subcellular localization of the proteins was analyzed,and functions of the protein complex in protein glycosylation and vesicular transport were revealed.We clarified that Dop1-Neo1-Mon2 complex is essential for the late stage of membrane trafficking in both budding yeast and mammalian cells.The main conclusions are as follows:1.By bioinformatics analysis in the Yeast Genome Database,essential proteins that localize at the Golgi apparatus and the endosomes were selected.Among these candidate proteins,Dop1,an essential protein with unknown function,was focused on the analysis.Dop1 is known to interact with a P4-ATPase Neo1,which functions in phospholipid translocation.The fluorescence microscopic analysis revealed that Dop1 was mainly localized in the early trans-Golgi network(TGN),and Neo1 was mainly localized in the late TGN.2.Depletion of Dop1 or Neo1 caused defects in N-glycosylation of invertase and O-glycosylation of Gas1.In Dop1-depleted yeast cells,membrane transport from TGN was impaired.Localization of a Golgi-resident glycosyltransferase Och1 was changed from cis-Golgi to TGN after Dop1-depleted.The results suggest that Dop1 is required for the retrograde transport of glycosyltransferase from TGN to Golgi apparatus.3.Dop1-Neo1-Mon2 complex is conserved among eukaryotes.To better understand the function of the complex in vesicular transport in late stage of membrane trafficking,mammalian homologs of Dop1 and Mon2 were analyzed.Interaction of mammalian MON2 and Dop1 homologs,DOPEY1 and DOPEY2 was confirmed in HEK293 cells.By confocal fluorescence microscopic analysis,MON2 was mainly localized in the recycling endosomes.MON2-DOPEY complex has the ability to bind to motor proteins.MON2-localized recycling endosomes approached,interacted with,and separated from early endosomes.Knockout of MON2 in HEK293 cells resulted in clustering of recycling endosomes around early endosomes.4.Knockout of MON2 impaired intracellular transferrin recycling from endosomes to plasma membrane.In addition,the retrograde transport of Wntless protein from plasma membrane to Golgi was prevented in MON2-knockout cells.Wntless-EGFP was mis-transported to lysosomes due to the blockade of recycling pathway in MON2-KO cells.These results suggest that the MON2 is involved in the transport of transferrin receptor and Wntless by regulating the separation of recycling endosomes from early endosomes.