Localization of an alpha 1,2 galactosyltransferase activity to the Golgi apparatus of Schizosaccharomyces pombe.
Localization of an alpha 1,2 galactosyltransferase activity to the Golgi apparatus of Schizosaccharomyces pombe.
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裂殖酵母高尔基体的 α1,2 半乳糖基转移酶活性定位。
DOI：
10.1091/mbc.5.5.519
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发表时间：
1994
影响因子：
3.3
通讯作者：
Warren,G
中科院分区：
文献类型：
--
作者：
Chappell,TG;Hajibagheri,MA;Ayscough,K;Pierce,M;Warren,G
我们从裂殖酵母中克隆了一个编码α1，2半乳糖基转移酶活性的基因。该基因的开放阅读框(GMA12代表半乳甘露聚糖，α1，2)，结合先前的蛋白质纯化(查佩尔和沃伦，)，预测了一种具有II型跨膜拓扑结构的O-连接糖蛋白。通过同源基因的破坏，我们已经证明了GMA12基因产物(Gma12p)是非必需的。缺失菌株(GMA12-D10：：ura4)的半乳糖基转移酶活性显著低于亲本菌株，但凝集素原位结合和体外生化分析表明，除gma12p外，还存在进一步的半乳糖基转移酶活性。虽然gma12p不是唯一的半乳糖基转移酶，但它能在酵母细胞表面产生一种独特的碳水化合物结构。我们已经制备了针对该碳水化合物表位的多克隆抗血清，并表明gma12p能够在体外和体内合成该表位。GMA12+细胞中GMA12+特异性表位的电子显微镜定位表明，gma12p合成高尔基体中的碳水化合物结构，随后的细胞内转运将表位分配到分泌途径的后期阶段。免疫定位研究证实了裂殖酵母高尔基体中存在一种或多种半乳糖基转移酶活性。
We have cloned a gene encoding an alpha 1,2 galactosyltransferase activity from Schizosaccharomyces pombe. The open reading frame of the gene (gma12 for galactomannan, alpha 1,2), combined with the previous protein purification (Chappell and Warren, 1989), predicts an O-linked glycoprotein with type II transmembrane topology. By homologous gene disruption, we have demonstrated that the gma12 gene product (gma12p) is nonessential. The deletion strain (gma12-D10::ura4) has a significantly reduced level of galactosyltransferase activity relative to the parental strain, but both in situ lectin binding and in vitro biochemical assays demonstrate the presence of further galactosyltransferase activity in addition to gma12p. Although gma12p is not the only galactosyltransferase in S. pombe, it produces a unique carbohydrate structure on the surface of the yeast cells. We have generated a polyclonal antiserum against this carbohydrate epitope and shown that gma12p is capable of synthesizing the epitope both in vitro and in vivo. Electron microscopic localization of the gma12+ specific epitope in gma12+ cells revealed that gma12p synthesizes the carbohydrate structure in the Golgi apparatus, and subsequent intracellular transport distributes the epitope to later stages of the secretory pathway. The immunolocalization studies confirm the presence of one or more galactosyltransferase activities in the Golgi apparatus in fission yeast.