Mutational analysis of the role of N-glycosylation in α-factor receptor function
Mutational analysis of the role of N-glycosylation in α-factor receptor function
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DOI：
10.1021/bi0108507
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发表时间：
2001-08-14
期刊：
影响因子：
2.9
通讯作者：
Konopka, JB
中科院分区：
文献类型：
--
作者：
Mentesana, PE;Konopka, JB
α 因子交配信息素受体（由 STE2 编码）激活 G 蛋白信号传导通路，刺激酿酒酵母细胞的接合。已知α因子受体经历多种形式的翻译后修饰，包括磷酸化、单泛素化和N-连接糖基化。由于磷酸化和单泛素化先前已被证明在调节 α 因子受体的信号传导活性和膜运输中发挥关键作用，因此本研究研究了 N 连接糖基化的作用。受体蛋白胞外区域中存在的 N 连接糖基化的五个共有位点中的 Asn 残基发生突变，以防止碳水化合物在这些位点附着。受体 N 末端附近的两个位点（N25Q 和 N32Q）的突变降低了受体糖基化程度，并且相应的双突变体未检测到 N-糖基化。非糖基化受体显示出正常功能和亚细胞定位，表明糖基化对于野生型受体活性并不重要。然而，糖基化位点的突变导致 Ste2-3 突变受体的质膜定位得到改善，这些受体通常在高温下保留在细胞内。这些结果表明，N-糖基化可能参与错误折叠的 Ste2 蛋白的分选过程，并且可能类似地影响某些突变受体，这些受体的运输改变与人类疾病有关。
The alpha -factor mating pheromone receptor (encoded by STE2) activates a G protein signaling pathway that stimulates the conjugation of Saccharomyces cerevisiae yeast cells. The alpha -factor receptor is known to undergo several forms of post-translational modification, including phosphorylation, mono-ubiquitination, and N-linked glycosylation. Since phosphorylation and mono-ubiquitination have been shown previously to play key roles in regulating the signaling activity and membrane trafficking of the alpha -factor receptors, the role of N-linked glycosylation was investigated in this study. The Asn residues in the five consensus sites for N-linked glycosylation present in the extracellular regions of the receptor protein were mutated to prevent carbohydrate attachment at these sites. Mutation of two sites near the receptor N-terminus (N25Q and N32Q) diminished the degree of receptor glycosylation, and the corresponding double mutant was not detectably N-glycosylated. The nonglycosylated receptors displayed normal function and subcellular localization, indicating that glycosylation is not important for wild-type receptor activity. However, mutation of the glycosylation sites resulted in improved plasma membrane localization for the Ste2-3 mutant receptors that are normally retained intracellularly at elevated temperatures. These results suggest that N-glycosylation may be involved in the sorting process for misfolded Ste2 proteins, and may similarly affect certain mutant receptors whose altered trafficking is implicated in human diseases.