BASIS FOR GOLGI LOCALIZATION OF GLYCOSYLTRANSFERASES

点击翻译按钮获取中文摘要

The terminal Golgi glycosyltransferases are largely responsible for the wide array of oligosaccharide structures which modify proteins and lipids. Specific oligosaccharide structures are frequently required for the folding, stability and biologic activity of proteins and a variety of cell- cell interactions involved in development and disease. The types of oligosaccharide structures which modify proteins and lipids depend on both the presence of specific glycosyltransferases and the precise compartmentation of these enzymes in the cisternae of the Golgi apparatus. The molecular basis for the precise localization of these Golgi glycosyltransferases and Golgi proteins, in general, is not known. The long term goal of this proposal is to elucidate the mechanisms underlying the Golgi apparatus localization of Golgi terminal glycosyltransferases and other Golgi proteins. It is expected that general principles of Golgi apparatus protein localization will emerge from the investigation of the signals and mechanisms underlying the Golgi localization of a unique glycosyltransferase, the alpha2, 6-sialyltransferase (ST), which has a dual localization in the trans cisternae of the Golgi apparatus and trans Golgi network. Preliminary evidence suggests that the ST possesses Golgi localization signals in both the signal anchor domain and the stem region and that these regions also mediate the oligomerization of the enzyme. The focus of this research proposal will be 1) to determine which sequences in the signal anchor domain and stem region are necessary and sufficient for the localization of the ST and other Golgi proteins to the Golgi apparatus, 2) to investigate the role of oligomerization in the transport and sorting of the ST, and 3) to determine what protein and/or lipid components of the Golgi apparatus participate in ST Golgi localization. These aims will be accomplished by the biochemical analysis and immunofluorescence and immunoelectron microscopic localization of mutant and chimeric ST proteins constructed by in vitro oligonucleotide-directed mutagenesis and PCR techniques. The results of these studies will be fundamentally important for the understanding protein transport and sorting in the secretory pathway, and more specifically for the understanding of Golgi protein localization and the assembly of the Golgi apparatus. In addition, these studies will begin to evaluate the molecular basis for the localization of terminal glycosyltransferases across the Golgi stack and how this strict compartmentation of glycosyltransferases ultimately controls the structure of oligosaccharides on glycoproteins and glycolipids.