Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1).
Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1).
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人红细胞阴离子交换器跨膜跨度组织的结构模型（带 3；AE1）。
DOI：
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发表时间：
1999
影响因子：
4.1
通讯作者：
Michael J. A. Tanner
中科院分区：
文献类型：
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作者：
J. D. Groves;Michael J. A. Tanner
我们研究了人红细胞带3的阴离子转运结构域（b3 β）的N-和C-末端多肽片段的非互补对的功能性共组装。cDNA克隆编码非连续的片段对与一个跨膜（TM）区域省略，或重叠的片段对之间的一个和十个TM区域重复，在非洲爪蟾卵母细胞和无细胞翻译系统中共表达。二苯乙烯二磺酸盐敏感的氯离子摄取试验在卵母细胞中发现，省略任何单一的TM区域的b3除了跨度6和7引起的功能表达的完全丧失。相反，与单个TM区域5、6、7、8、9-10或11-12重叠的片段的共表达对保留了高水平的功能性，而与TM区域2-5、4-5、5-8和8-10的簇重叠的片段也介导了一些二苯乙烯二磺酸盐敏感性摄取。通过在非变性洗涤剂溶液中使用针对b3 β末端的单克隆抗体，通过免疫共沉淀来检查N-或C-末端片段与完整条带3、b3 β或其他片段的共组装。除TM跨度13-14外，所有片段均与b3 β共免疫沉淀。包含跨度1-6、1-7或1-8的中等大小的N-末端片段与包含跨度8-14或9-14的C-末端片段特别强烈地共免疫沉淀。包含跨度1-4或1-12的片段与其他N-末端片段相比，与b3-末端或C-末端片段的共免疫沉淀程度较低。在b3 bits的结构中有足够的灵活性，以允许包含至少一个重复的TM跨度而不丧失功能。我们提出了一个工作模型的组织TM跨度的二聚体带3根据目前的证据。
We have examined the functional co-assembly of non-complementary pairs of N- and C-terminal polypeptide fragments of the anion transport domain (b3mem) of human red-cell band 3. cDNA clones encoding non-contiguous pairs of fragments with one transmembrane (TM) region omitted, or overlapping pairs of fragments with between one and ten TM regions duplicated, were co-expressed in Xenopus oocytes and a cell-free translation system. Stilbene disulphonate-sensitive chloride uptake assays in oocytes revealed that the omission of any single TM region of b3mem except spans 6 and 7 caused a complete loss of functional expression. In contrast, co-expressed pairs of fragments overlapping a single TM region 5, 6, 7, 8, 9-10 or 11-12 retained a high level of functionality, whereas fragments overlapping the clusters of TM regions 2-5, 4-5, 5-8 and 8-10 also mediated some stilbene disulphonate-sensitive uptake. The co-assembly of N- or C-terminal fragments with intact band 3, b3mem or other fragments was examined by co-immunoprecipitation in non-denaturing detergent solutions by using monoclonal antibodies against the termini of b3mem. All the fragments, except for TM spans 13-14, co-immunoprecipitated with b3mem. The medium-sized N-terminal fragments comprising spans 1-6, 1-7 or 1-8 co-immunoprecipitated particularly strongly with the C-terminal fragments containing spans 8-14 or 9-14. The fragments comprising spans 1-4 or 1-12 co-immunoprecipitated less extensively than the other N-terminal fragments with either b3mem or C-terminal fragments. There is sufficient flexibility in the structure of b3mem to allow the inclusion of at least one duplicated TM span without a loss of function. We propose a working model for the organization of TM spans of dimeric band 3 based on current evidence.