Zatorska Ewa, Gal Lihi, Schmitt Jaro, Bausewein Daniela, Schuldiner Maya, Strahl Sabine
Centre for Organismal Studies (COS), Heidelberg University, 69120 Heidelberg, Germany.
Department of Molecular Genetics, Weizmann Institute of Science, 7610001 Rehovot, Israel.
Int J Mol Sci. 2017 Jun 9;18(6):1226. doi: 10.3390/ijms18061226.
-Mannosylation is a type of protein glycosylation initiated in the endoplasmic reticulum (ER) by the protein -mannosyltransferase (PMT) family. Despite the vital role of -mannosylation, its molecular functions and regulation are not fully characterized. To further explore the cellular impact of protein -mannosylation, we performed a genome-wide screen to identify mutants with increased sensitivity towards the PMT-specific inhibitor compound R3A-5a. We identified the cell wall and the ER as the cell compartments affected most upon PMT inhibition. Especially mutants with defects in -glycosylation, biosynthesis of glycosylphosphatidylinositol-anchored proteins and cell wall β-1,6-glucan showed impaired growth when -mannosylation became limiting. Signaling pathways that counteract cell wall defects and unbalanced ER homeostasis, namely the cell wall integrity pathway and the unfolded protein response, were highly crucial for the cell growth. Moreover, among the most affected mutants, we identified Ost3, one of two homologous subunits of the oligosaccharyltransferase complexes involved in -glycosylation, suggesting a functional link between the two pathways. Indeed, we identified Pmt2 as a substrate for Ost3 suggesting that the reduced function of Pmt2 in the absence of -glycosylation promoted sensitivity to the drug. Interestingly, even though Pmt1 and Pmt2 proteins are highly similar on the sequence, as well as the structural level and act as a complex, we identified only Pmt2, but not Pmt1, as an Ost3-specific substrate protein.
甘露糖基化是一种蛋白质糖基化类型，由内质网（ER）中的蛋白质甘露糖基转移酶（PMT）家族起始。尽管甘露糖基化起着至关重要的作用，但其分子功能和调控尚未完全明确。为了进一步探究蛋白质甘露糖基化对细胞的影响，我们进行了全基因组筛选，以鉴定对PMT特异性抑制剂化合物R3A - 5a敏感性增加的突变体。我们发现细胞壁和内质网是PMT抑制后受影响最大的细胞区室。特别是在甘露糖基化受限的情况下，糖基化、糖基磷脂酰肌醇锚定蛋白生物合成以及细胞壁β-1,6-葡聚糖存在缺陷的突变体，其生长受到损害。对抗细胞壁缺陷和内质网稳态失衡的信号通路，即细胞壁完整性通路和未折叠蛋白反应，对细胞生长至关重要。此外，在受影响最严重的突变体中，我们鉴定出Ost3，它是参与糖基化的寡糖基转移酶复合物的两个同源亚基之一，这表明两条通路之间存在功能联系。事实上，我们确定Pmt2是Ost3的底物，这表明在缺乏糖基化时Pmt2功能的降低促进了对该药物的敏感性。有趣的是，尽管Pmt1和Pmt2蛋白在序列以及结构水平上高度相似且作为一个复合物发挥作用，但我们仅确定Pmt2而非Pmt1是Ost3特异性底物蛋白。
