Free Oligosaccharides to Monitor Glycoprotein Endoplasmic Reticulum-associated Degradation in Saccharomyces cerevisiae
Free Oligosaccharides to Monitor Glycoprotein Endoplasmic Reticulum-associated Degradation in Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m109.082081
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发表时间：
2010-04-16
影响因子：
4.8
通讯作者：
Suzuki, Tadashi
中科院分区：
文献类型：
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作者：
Hirayama, Hiroto;Seino, Junichi;Suzuki, Tadashi
在真核细胞中，N-糖基化被认为是蛋白质翻译过程中最常见、最重要的修饰方式之一。“游离”形式的N-聚糖在哺乳动物细胞的胞质溶胶中积累，但其形成和降解的精确机制仍然未知。在这里，我们报告了一种使用内切β-1，6-葡聚糖酶消化分离酵母游离寡糖（fOS）的方法。在缺乏编码胞质肽：N-聚糖酶基因的PNG 1的细胞中检测不到fOS，这表明几乎所有的fOS都是由Png 1 p错误折叠的糖蛋白形成的。结构研究表明，最丰富的fOS是M8 B，这是不能很好地识别内质网相关降解（ERAD）相关的凝集素，Yos 9 p。此外，我们提供的证据表明，一些ERAD基板到达高尔基体前retrotranslocation到胞质溶胶。在缺乏胞质溶胶/液泡α-甘露糖苷酶Ams 1 p的细胞中，错误折叠的糖蛋白上的N-聚糖结构仍然相当多样化，这表明错误折叠的糖蛋白上的N-聚糖的加工比目前设想的更复杂。在ER应激下，观察到fOS增加，而Yos 9 p识别的关键聚糖结构M7 C的水平不变。因此，我们的方法可以提供有价值的信息糖蛋白ERAD在酿酒酵母的分子机制。
In eukaryotic cells, N-glycosylation has been recognized as one of the mostcommonand functionally important co-or post-translational modifications of proteins. "Free" forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-beta-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide: N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole alpha-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae.