Two distinct domains of the β-subunit of glucosidase II interact with the catalytic α-subunit
Two distinct domains of the β-subunit of glucosidase II interact with the catalytic α-subunit
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DOI：
10.1093/glycob/10.5.487
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发表时间：
2000-05-01
期刊：
影响因子：
4.3
通讯作者：
Ostergaard, HL
中科院分区：
文献类型：
--
作者：
Arendt, CW;Ostergaard, HL
最近对内质网加工酶葡萄糖苷酶II进行了纯化和cDNA克隆，发现它由两个在哺乳动物中高度保守的可溶性蛋白质组成：一个催化α亚基和一个未知功能的P亚基。由于P-亚基包含一个C-末端的His-Asp-Glu-Leu(HDEL)基序，可能起到将催化亚基与KDEL受体连接作为修复机制的功能，我们试图定位小鼠β-亚基蛋白负责介导与α-亚基的联系的区域，通过筛选一组重组的β-亚基谷胱甘肽S-转移酶融合蛋白来沉淀葡萄糖苷酶II的活性，我们在β-亚基中发现了两个不重叠的相互作用结构域(ID1和ID2)，ID1在成熟多肽的N端包含118个氨基酸，跨越该区域的富含半胱氨酸的元件。ID2位于C-末端附近，包含在氨基酸273-400中，该区域部分由一段酸性残基占据。ID2内7个选择性剪接氨基酸的可变用法不影响两个亚基的结合，我们推测葡萄糖苷酶II的催化亚基与ID1和ID2协同结合，解释了酶复合体的高结合稳定性。
Recent purification and cDNA cloning of the endoplasmic reticulum processing enzyme glucosidase II have revealed that it is composed of two soluble proteins: a catalytic a-subunit and a P-subunit of unknown function, both of which are highly conserved in mammals. Since the P-subunit, which contains a C-terminal His-Asp-Glu-Leu (HDEL) motif, may function to link the catalytic subunit to the KDEL receptor as a retrieval mechanism, we sought to map the regions of the mouse beta-subunit protein responsible for mediating the association with the alpha-subunit, By screening a panel of recombinant beta-subunit glutathione S-transferase fusion proteins for the ability to precipitate glucosidase II activity, we have identified two nonoverlapping interaction domains (IDI and ID2) within the beta-subunit, ID1 encompasses 118 amino acids at the N-terminus of the mature polypeptide, spanning the cysteine-rich element in this region. ID2, located near the C-terminus, is contained within amino acids 273-400, a region occupied in part by a stretch of acidic residues. Variable usage of 7 alternatively spliced amino acids within ID2 was found not to influence the association of the two subunits, We theorize that the catalytic subunit of glucosidase II binds synergistically to ID1 and ID2, explaining the high associative stability of the enzyme complex.