FUNCTION OF THE TRANSMEMBRANE DOMAIN OF GLYCOSYLTRANSFERASES

糖基转移酶的cDNA序列分析表明， 这些酶具有由短的 氨基末端胞质尾，疏水性跨膜锚结构域 和羧基末端催化结构域。为了研究 跨膜结构域的一系列突变体和嵌合cDNA的（β-1，4- 用PCR方法构建了β-1，4-GT基因的瞬时表达载体 在COS-7细胞中表达，测量酶活性并检测蛋白质 通过亚细胞分级分离或间接的 免疫荧光显微术。氨基末端缺失分析 区域显示β-1，4-GT的前21个氨基酸不是必需的 稳定生产蛋白质，并始终定位于 高尔基体然而，如前所述， 跨膜结构域消除了这种蛋白在细胞中的稳定表达， 哺乳动物细胞此外，杂交构建体的分析显示， α-1，3-GT的残基1-25可以在功能上取代β-1，4-GT 氨基末端胞质和跨膜结构域（残基1-43）。这 融合蛋白也显示出高尔基体定位。另一方面，蛋白质 融合到（α-2，6-唾液酸转移酶（α- 2，6-ST）需要额外的COOH-末端序列侧翼的结构域， 稳定性和高尔基体定位。Arg 24、Leu 25、Leu 26和Leu 27的取代 β-l，4-GT跨膜的His 33被IIe取代或Tyr被IIe取代 在位置40和41处， 43从高尔基体释放突变蛋白，并在 细胞表面我们的研究结果表明：a）（β- 1，4-GT、α-1，3-GT和α-2，6-ST沿着及其茎区的所有 在高尔基体定位中发挥作用，并参与一种共同的机制， 允许蛋白质被适当地加工并且在体内不被降解; B）增加跨膜结构域的长度覆盖高尔基体 保留信号并将酶引导至质膜;和 跨膜结构域的疏水区的长度是 重要参数，但本身不足以保持高尔基体。

Analyses of the cDNA sequences of glycosyltransferases have shown that these enzymes have inverted membrane topology that consists of a short amino-terminal cytoplasmic tail, a hydrophobic transmembrane anchor domain and the carboxyl-terminal catalytic domain. To examine the role of the transmembrane domain a series of mutants and chimeric cDNA of (beta-1,4- galactosyltransferase (beta-1,4-GT) were constructed by PCR, transiently expressed in COS-7 cells, enzyme activities measured and the protein localized in the cells by subcellular fractionation or indirect immunofluorescence microscopy. Deletion analyses of the amino-terminal region show that the first 21 amino acids of beta-1,4-GT are not essential for the stable production of the protein and are consistently localized in the Golgi apparatus. However, as reported earlier, the deletion of the transmembrane domain abolishes the stable expression of this protein in mammalian cells. In addition, analysis of hybrid constructs showed that residue 1-25 of alpha-1,3-GT can functionally replace the beta-1,4-GT amino-terminal cytoplasmic and transmembrane domain (residues 1-43). This fusion protein also showed Golgi localization. On the other hand, protein fused to the transmembrane domain of (alpha-2,6-sialyltransferase (alpha- 2,6-ST) needed additional COOH-terminal sequences flanking the domain for stability and Golgi localization. Substitution of Arg24, Leu25, Leu26 and His33 of (beta-l,4-GT transmembrane by lIe or substitution of Tyr by Ile at positions 40 and 41 coupled with the insertion of four lIe at position 43 released the mutant proteins from the Golgi and were detected on the cell surface. Our results show that a) the transmembrane domains of (beta- l,4-GT, alpha-1,3-GT, and of alpha-2,6-ST along with its stem region all play a role in Golgi targeting, and participate in a common mechanism that allows the protein to be processed properly and not be degraded in vivo; b) increasing the length of the transmembrane domain overrides the Golgi retention signal and directs the enzyme to the plasma membrane; and c) the length of the hydrophobic region of the transmembrane domain is an important parameter, but is not sufficient by itself for Golgi retention.