Transmembrane topology of the mammalian Slc11a2 iron transporter.
Transmembrane topology of the mammalian Slc11a2 iron transporter.
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DOI：
10.1021/bi900606y
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发表时间：
2009-09-08
期刊：
影响因子：
2.9
通讯作者：
Gros, Philippe
中科院分区：
文献类型：
--
作者：
Czachorowski, Maciej;Lam-Yuk-Tseung, Steven;Cellier, Mathieu;Gros, Philippe
哺乳动物Slc 11 a1和Slc 11 a2蛋白定义了一个大家族的二级金属转运蛋白。Slc 11 a1和Slc 11 a2作为pH依赖性二价阳离子转运蛋白发挥功能，分别在宿主防御感染和Fe 2+稳态中发挥关键作用。Slc 11 a2的跨膜结构域（TMD）的位置和极性进行了研究的基础上插入的小抗原性血凝素A（HA）肽（YPYDVPDYAS）在预测的蛋白质内或胞外环的表位标记方法。标记的蛋白质在转染的LLC-PK 1肾细胞中表达并测试转运活性，并通过完整和透化细胞中的免疫荧光确定插入的标签相对于质膜的极性。在位置1、98、131、175、201、243、284、344、403、432、468、504和561处插入HA表位标签。在位置98，131，175，403和432处的插入废除了Slc 11 a2的金属转运，而在位置1，201，243，284，344，468，504和561处的插入产生功能蛋白。功能性HA标记的Slc 11 a2蛋白的拓扑图表明，N端（1）以及由TMD 4 −5（201），TMD 6 −7（284）和TMD 10 −11（468）和C端（561）描绘的环是细胞内的，而分隔TMD 5 −6（243），TMD 7 −8（344）和TMD 11 −12（504）的环是细胞外的。这些结果与具有细胞内氨基和羧基末端的12个跨膜结构域的拓扑结构相容。通过同源穿线构建的结构模型支持这种12 TMD拓扑结构，并在TM 1 −5和TM 6 −10（保守的Slc 11疏水核心）的膜螺旋排列中显示出2重结构对称性。
The mammalian Slc11a1 and Slc11a2 proteins define a large family of secondary metal transporters. Slc11a1 and Slc11a2 function as pH-dependent divalent cation transporters that play a critical role in host defenses against infections and in Fe2+ homeostasis, respectively. The position and polarity of individual transmembrane domains (TMD) of Slc11a2 were studied by an epitope tagging method based on the insertion of small antigenic hemagglutinin A (HA) peptides (YPYDVPDYAS) in predicted intra- or extracellular loops of the protein. The tagged proteins were expressed in transfected LLC-PK1 kidney cells and tested for transport activity, and the polarity of inserted tags with respect to the plasma membrane was determined by immunofluorescence in intact and permeabilized cells. HA epitope tags were inserted at positions 1, 98, 131, 175, 201, 243, 284, 344, 403, 432, 468, 504, and 561. Insertions at positions 98, 131, 175, 403, and 432 abrogated metal transport by Slc11a2, while insertions at positions 1, 201, 243, 284, 344, 468, 504, and 561 resulted in functional proteins. Topology mapping in functional HA-tagged Slc11a2 proteins indicated that the N-terminus (1), as well as loops delineated by TMD4−5 (201), TMD6−7 (284), and TMD10−11 (468), and C-terminus (561) are intracellular, while loops separating TMD5−6 (243), TMD7−8 (344), and TMD11−12 (504) are extracellular. These results are compatible with a topology of 12 transmembrane domains, with intracellular amino and carboxy termini. Structural models constructed by homology threading support this 12TMD topology and show 2-fold structural symmetry in the arrangement of membrane helices for TM1−5 and TM6−10 (conserved Slc11 hydrophobic core).
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影响因子：
2.9
作者：
Indarte, Martin;Madura, Jeffry D.;Surratt, Christopher K.
通讯作者：
Surratt, Christopher K.
DOI：
10.1073/pnas.0937287100
发表时间：
2003-05-13
影响因子：
11.1
作者：
Jabado, N;Cuellar-Mata, P;Gros, P
通讯作者：
Gros, P
影响因子：
4.8
作者：
Kast, C;Canfield, V;Gros, P
通讯作者：
Gros, P
影响因子：
30.8
作者：
Fleming, MD;Trenor, CC;Andrews, NC
通讯作者：
Andrews, NC
影响因子：
20.3
作者：
Forbes, JR;Gros, P
通讯作者：
Gros, P