Membrane integration of Na,K-ATPase α-subunits and β-subunit assembly
Membrane integration of Na,K-ATPase α-subunits and β-subunit assembly
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DOI：
10.1074/jbc.273.38.24921
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发表时间：
1998-09-18
影响因子：
4.8
通讯作者：
Geering, K
中科院分区：
文献类型：
--
作者：
Béguin, P;Hasler, U;Geering, K
我们在非洲爪蟾卵母细胞中进行了体内翻译/插入实验，结合了异源二聚体Na，K-ATP酶α-亚基的野生型或突变型膜片段与糖基化报告序列连接。我们证实，α-亚基的四个N-末端疏水片段作为交替的信号锚/停止传递基序的两个脂质插入膜对，然而，对于六个C-末端膜片段，适当的包装取决于特定的序列信息和与β-亚基的关联，M5是一个非常低效的信号锚序列，由于脯氨酸和极性氨基酸的存在。其正确的膜插入可能是通过与M6的翻译后发夹形成介导的，这受到连接环中的脯氨酸对的促进，M7具有部分信号锚功能，这可能是通过甘氨酸和谷氨酰胺残基的存在介导的，跨膜M7/M8对的形成需要β亚基的缔合，其诱导了有利于M7/M8包装的连接胞质外环的构象变化。M9/M10对的形成似乎主要由M10的有效终止转移功能介导。为M5、M7和M9提供信号锚功能的突变消除或阻碍了酶的转运活性。这些数据说明了疏水区附近或疏水区内的特定氨基酸以及亚基寡聚化对于正确的拓扑对齐的重要性，这对于寡聚膜蛋白的正确折叠和/或活性是必要的。
The control of membrane insertion of polytopic proteins is still poorly understood, We carried out in vivo translation/insertion experiments in Xenopus oocytes with combined wild type or mutant membrane segments of the alpha-subunit of the heterodimeric Na,K-ATPase linked to a glycosylation reporter sequence. We confirm that the four N-terminaI hydrophobic segments of the alpha-subunit behave as alternating signal anchor/stop transfer motifs necessary for two lipid-inserted membrane pairs, For the six C-terminal membrane segments, however, proper packing depends on specific sequence information and association with the beta-subunit, M5 is a very inefficient signal anchor sequence due to the presence of prolines and polar amino acids. Its correct membrane insertion is probably mediated by posttranslational hairpin formation with M6, which is favored by a proline pair in the connecting loop, M7 has partial signal anchor function, which may be mediated by the presence of glycine and glutamine residues, The formation of a transmembrane M7/M8 pair requires the association of the beta-subunit, which induces a conformational change in the connecting extracytoplasmic loop that favors M7/M8 packing, The formation of the M9/M10 pair appears to be predominantly mediated by the efficient stop transfer function of M10. Mutations that provide signal anchor function to M5, M7, and M9 abolish or impede the transport activity of the enzyme, These data illustrate the importance of specific amino acids near or within hydrophobic regions as well as of subunit oligomerization for correct topographical alignment that is necessary for proper folding and/or activity of oligomeric membrane proteins.