The tenth membrane region of band 3 is initially exposed to the luminal side of the endoplasmic reticulum and then integrated into a partially folded band 3 intermediate
The tenth membrane region of band 3 is initially exposed to the luminal side of the endoplasmic reticulum and then integrated into a partially folded band 3 intermediate
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DOI：
10.1021/bi026619q
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发表时间：
2002-11-26
期刊：
影响因子：
2.9
通讯作者：
Hamasaki, N
中科院分区：
文献类型：
--
作者：
Kanki, T;Sakaguchi, M;Hamasaki, N
带3是介导阴离子交换活性[阴离子交换剂1（AE 1）]的典型多位膜蛋白。尽管跨膜区（TM）9后40个残基的拓扑和拓扑发生已被广泛研究，但该区域[第10区（10 thR）]的拓扑发生尚不清楚。在10 thR中产生的糖基化位点在无细胞转录/翻译系统中被有效地糖基化，而在培养的细胞系统中糖基化效率相当低。然而，当TM 12 -14被删除或当放线菌酮被添加到培养基中时，培养细胞中的糖基化效率增加到与无细胞系统中相同的水平，表明TM 12对于寡糖基转移酶到膜中的螯合是必需的，并且放线菌酮处理细胞可以通过降低链延伸速率来模拟无细胞系统。培养细胞中的糖基化效率也随着TM 1 -3的缺失而增加。这些结果表明，10 thR被短暂地挤出到管腔中，然后插入到膜中。TM 12和远处的TM 1 -3都影响第10 thR的膜插入。这表明在蛋白质折叠过程中，在TM 1 -12片段正确组装后，10 thR插入膜中。
Band 3 is a typical polytopic membrane protein that mediates anion exchange activity [anion exchanger 1 (AE1)]. Although the topology and topogenesis of similar to40 residues just after transmembrane (TM) 9 have been extensively studied, the topogenesis of this region [tenth region (10thR)] has been unclear. Glycosylation sites created in the 10thR were efficiently glycosylated in a cell-free transcription/ translation system, whereas the glycosylation efficiencies were quite low in a cultured cell system. When TM12-14 was deleted or when cycloheximide was added to the culture medium, however, the glycosylation efficiency in the cultured cells increased to the same level as in the cell-free system, indicating that TM12 is essential for the sequestration from oligosaccharyl transferase into membrane and that cycloheximide treatment of the cells can mimic the cell-free system by reducing the rate of chain elongation. The glycosylation efficiency in cultured cells also increased with deletion of TM1-3. These results suggest that the 10thR is transiently extruded into the lumen and then inserted into the membrane. Both TM12 and the distant TM1-3 affect the membrane insertion of the 10thR. This indicates that during the folding of the protein, the 10thR is inserted into the membrane after the TM1-12 segments are properly assembled.