In Saccharomyces cerevisiae a Golgi lumenal GDPase (ScGda1p) generates GMP, the antiporter required for entry of GDP-mannose, from the cytosol, into the Golgi lumen. Scgda1 deletion strains have severe defects in N- and O-mannosylation of proteins and glycosphingolipids. ScGda1p has also significant UDPase activity even though S. cerevisiae does not utilize uridine nucleotide sugars in its Golgi lumen. Kluyveromyces lactis, a species closely related to S. cerevisiae, transports UDP-N-acetylglucosamine into its Golgi lumen, where it is the sugar donor for terminal N-acetylglucosamine of the mannan chains. We have identified and cloned a K. lactis orthologue of ScGda1p. KlGda1p is 65% identical to ScGda1p and shares four apyrase conserved regions with other nucleoside diphosphatases. KlGda1p has UDPase activity as ScGda1p. Transport of both GDP-mannose, and UDP-GlcNAc was decreased into Golgi vesicles from Klgda1 null mutants, demonstrating that KlGda1p generates both GMP and UMP required as antiporters for guanosine and uridine nucleotide sugar transport into the Golgi lumen. Membranes from Klgda1 null mutants showed inhibition of glycosyltransferases utilizing uridine- and guanosine-nucleotide sugars, presumably due to accumulation of nucleoside diphosphates because the inhibition could be relieved by addition of apyrase to the incubations. KlGDA1 and ScGDA1 restore the wild-type phenotype of the other yeast gda1 deletion mutant. Surprisingly, KlGDA1 has only a role in O-glycosylation in K. lactis but also complements N-glycosylation defects in S. cerevisiae. Deletion mutants of both genes have altered cell wall stability and composition, demonstrating a broader role for the above enzymes.
在酿酒酵母(Saccharomyces cerevisiae)中,高尔基体腔内 GDP 酶(ScG
DA1p)生成 GMP,这是 GDP-
甘露糖从细胞质进入高尔基体腔所需的反转运体。Scg
DA1
基因缺失株在蛋白质和糖
磷脂的 N- 和 O-mannosyl 化方面存在严重缺陷。尽管 S. cerevisiae 在高尔基体腔内不利用
尿苷核苷酸糖,但 ScG
DA1p 也具有显著的
尿苷核苷酸酶活性。
乳酸克鲁维酵母菌(Kluyveromyces lactis)是一种与麦角菌(S. cerevisiae)亲缘关系很近的菌种,它将
UDP-N-乙酰
葡糖胺转运到高尔基管腔中,作为
甘露聚糖链末端 N-乙酰
葡糖胺的供糖体。我们发现并克隆了 K. lactis 的 ScG
DA1p 同源物。KlG
DA1p与ScG
DA1p有65%的相同之处,并与其他核苷二
磷酸酶共享四个apyrase保守区。KlG
DA1p 与 ScG
DA1p 一样具有
UDP 酶活性。在 Klg
DA1 空缺突变体的高尔基体囊泡中,GDP-
甘露糖和
UDP-GlcNAc 的转运都有所减少,这表明 KlG
DA1p 产生的 GMP 和
UMP 都是
鸟苷酸和
尿苷核苷酸糖转运到高尔基体内腔所需的反转运体。来自 Klg
DA1 空缺突变体的膜显示出对利用
尿苷和
鸟苷酸核苷酸糖的糖基转移酶的抑制,这可能是由于核苷二
磷酸盐的积累,因为在培养液中加入 apyrase 可以缓解抑制作用。KlG
DA1 和 ScG
DA1 恢复了另一种酵母 g
DA1 缺失突变体的野生型表型。令人惊讶的是,KlG
DA1在
乳酸酵母中只起O-糖基化作用,但在酿酒酵母中却能补充N-糖基化缺陷。这两个
基因的缺失突变体都改变了细胞壁的稳定性和组成,表明上述酶具有更广泛的作用。