The molecular system of Golgi retentior of glycosyltransferases

蛋白质和脂质的糖基化是通过糖基转移酶在高尔基体中进行的。它们是ii型膜蛋白，由n端细胞质区、跨膜区、茎区和面向高尔基体管腔的c端催化区组成。尽管大量的蛋白质和脂质流向细胞内的其他细胞器，但驻留的糖基转移酶和分选组分必须保留在高尔基体中。本项目的目的是阐明糖基转移酶高尔基保留的分子机制。双杂交系统是一种基于酵母的体内系统，通过重组活性转录因子二聚体来识别两种蛋白质之间的相互作用。从手术标本中获得的人非癌性右半结肠组织中制备cDNA，并在GAL4激活域载体pPC86中构建cDNA文库。我们报道了H酶（FUT1）、Se酶（FUT2）、Lewis型α1,3/1,4酰基转移酶（FUT3）、α2,3唾液转移酶（ST3GaII和ST3GaIIV）和β1,4乳糖基转移酶1 （β1,4 gait1）在右半结肠表达。以编码DNA结合域的GAL4序列为诱饵，在框架内克隆每个基因。我们通过筛选获得了与FUT1和FUT2相互作用的候选分子ε-COP蛋白、gp25L2蛋白和β-微管蛋白。

Glycosylation of proteins and lipids is performed in the Golgi apparatus by glycosyl-transferases. They are type-II membrane proteins which consist of an N-terminal cytoplasmic region, a trans membrane region, a stem region and a c-terminal catalytic region facing to lumen of Golgi. The population of resident glycosyltransferases and sorting components must be retained in the Golgi apparatus in spite of the large flow of proteins and lipids through to other organelles in the cell. The aim of this project is to elucidate the molecular mechanisms underlying Golgi retention of glycosyltransferases.The two-hybrid system is an in vivo yeast-based system that identifies interaction between two proteins by reconstituting active transcription factor dimmers. cDNAs were prepared from human non-cancerous right hemi-colon tissues obtained as a surgical sample and a cDNA library constructed in the GAL4 activation domain vector, pPC86. We have reported that H enzyme (FUT1), Se enzyme (FUT2), Lewis type α1,3/1,4 fucosyltransferase (FUT3), α2,3 sialyl-transferases (ST3GaII and ST3GaIIV) and β1,4gaqlactosyltransferase 1 (β1,4GaIT1) express in the right hemicolon. Each gene was cloned in-frame with the GAL4 sequence encoding the DNA binding domain into pDBLeu as the bait. We carried out screening and obtained ε-COP protein, gp25L2 protein and β-tubulin as the candidate molecules interacting with FUT1 and FUT2.