Glucose transporter function is controlled by transporter oligomeric structure. A single, intramolecular disulfide promotes GLUT1 tetramerization.
Glucose transporter function is controlled by transporter oligomeric structure. A single, intramolecular disulfide promotes GLUT1 tetramerization.
复制标题
葡萄糖转运蛋白功能由转运蛋白寡聚结构控制。
DOI：
10.1021/bi00030a011
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Carruthers,A
中科院分区：
文献类型：
--
作者：
Zottola,RJ;Cloherty,EK;Coderre,PE;Hansen,A;Hebert,DN;Carruthers,A
摘要：人红细胞葡萄糖转运蛋白是四种GLUT 1蛋白的变构复合物，其结构和底物结合特性通过还原敏感的非共价亚基相互作用而稳定[Hebert，D. N.，& Carruthers，A.（1992）J.Biol.Chem.267，23829-23838]。在本研究中，我们使用生物化学和分子生物学的方法来分离转运蛋白寡聚体结构和转运功能的特定决定因素。当在变性剂中展开时，转运蛋白复合物的每个亚基（GLUT 1蛋白）暴露两个巯基。亚基暴露于还原剂后，可获得四个额外的巯基。亚基二硫键含量的测定表明，两个不可接近的巯基形成了一个内部二硫键。差异烷基化/肽图谱/N-末端序列分析表明，GLUT 1羧基末端肽（残基232-492）含有三个不可接近的巯基，N-末端GLUT 1肽（残基147-261/299）含有两个可接近的巯基。羧基末端肽很可能含有分子内二硫键，因为它的产率和电泳迁移率都不会因添加还原剂而改变。每个GLUT 1半胱氨酸被改变为丝氨酸的阿托伐他汀定向，在体外诱变。将所得转运蛋白在CHO细胞中表达，并通过免疫荧光显微术筛选其暴露四聚体GLUT 1特异性表位的能力。在半胱氨酸残基133、201、207和429处的丝氨酸取代不抑制四聚体GLUT 1特异性表位的暴露。在半胱氨酸347或421处的丝氨酸取代防止四聚体GLUT 1特异性表位的暴露。在CHO细胞中表达并随后从CHO细胞中溶解的GLUT 1/GLUT 4嵌合体的流体动力学分析表明，GLUT 1残基1- 199促进嵌合体二聚化并允许GLUT 1/嵌合体异源四聚化。该GLUT 1 N-末端结构域不足以用于嵌合体四聚化，嵌合体四聚化另外需要GLUT 1残基200-463。细胞外还原剂（二硫苏糖醇、β-巯基乙醇或谷胱甘肽）使红细胞3-O-甲基葡萄糖摄取减少多达15倍。这种糖摄取的非竞争性抑制被不渗透细胞的氧化型谷胱甘肽逆转。还原剂对糖从红细胞排出无影响。二硫苏糖醇使红细胞驻留葡萄糖转运蛋白的细胞松弛素B结合能力加倍，消除相邻亚基上底物结合位点之间的变构相互作用，并原位封闭四聚体GLUT 1特异性GLUT 1表位。CHO细胞内GLUT 1的结构和转运功能同样受到胞外还原剂的影响。我们的结论是，每个亚基的葡萄糖转运蛋白含有一个胞外二硫键（Cys 347和Cys 421），稳定转运蛋白寡聚体结构，从而加速运输功能。
Revised Manuscript Received April 25, 1995® abstract: The human erythrocyte glucose transporter is an allosteric complex of four GLUT1 proteins whose structure and substrate binding properties are stabilized by reductant-sensitive, noncovalent subunit interactions [Hebert, D. N., & Carruthers, A.(1992) J. Biol. Chem. 267, 23829—23838]. In the present study, we use biochemical and molecular approaches to isolate specific determinants of transporter oligomeric structure and transport function. When unfoldedin denaturant, each subunit (GLUT1 protein) of the transporter complex exposes two sulfhydryl groups. Four additional thiol groups are accessible following subunit exposure to reductant. Assays of subunit disulfide bridge contentsuggest that two inaccessible sulfhydryl groups form an internal disulfide bridge. Differential alkylation/peptide mapping/N-terminal sequence analyses show that a GLUT1 carboxyl-terminal peptide (residues 232—492) contains three inaccessible sulfhydryl groups and that an N-terminal GLUT1 peptide (residues 147—261/299) contains two accessible thiols. The carboxyl-terminal peptide most likely contains the intramolecular disulfide bridge since neither its yield nor its electrophoretic mobility is alteredby addition of reductant. Each GLUT1 cysteine was changed to serine by oligonucleotide-directed, in vitro mutagenesis. The resulting transport proteins were expressed in CHO cells and screened by immunofluorescence microscopy for their ability to expose tetrameric GLUTl-specific epitopes. Serine substitution at cysteine residues133, 201, 207, and 429 doesnot inhibit exposure of tetrameric GLUTl-specific epitopes. Serine substitution at cysteines 347 or 421 prevents exposure of tetrameric GLUTl-specific epitopes. Hydrodynamic analysis of GLUT1/GLUT4 chimeras expressed in and subsequently solubilized from CHO cells indicates that GLUT1 residues 1— 199 promote chimera dimerization and permit GLUTl/chimera heterotetramerization. This GLUT1 N-terminal domain is insufficient for chimera tetramerization which additionally requires GLUT1 residues 200—463. Extracellular reductants (dithiothreitol,/3-mercaptoethanol, or glutathione) reduce erythrocyte 3-O-methylglucose uptake by up to 15-fold. This noncompetitive inhibition of sugar uptake is reversed by the cell-impermeant, oxidized glutathione. Reductant is without effect on sugar exit from erythrocytes. Dithiothreitol doubles the cytochalasin B binding capacity of erythrocyte-resident glucose transporter, abolishes allosteric interactions between substrate binding sites on adjacent subunits, and occludes tetrameric GLUTl-specific GLUT1 epitopes in situ. CHO cell-residentGLUT1 structure and transportfunction are similarly affected by extracellular reductant. We conclude that each subunit of the glucose transporter contains an extracellular disulfide bridge (Cys347and Cys421) that stabilizes transporter oligomeric structure and thereby accelerates transport function.