Liu Tiemin, Speight Pam, Silverman Mel
Department of Medicine, University of Toronto, Medical Sciences Building, Room 7336, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8.
Biochem Biophys Res Commun. 2009 Jan 2;378(1):133-8. doi: 10.1016/j.bbrc.2008.11.015. Epub 2008 Nov 20.
The predicted topology of the mammalian high-affinity sodium/glucose cotransporter (SGLT1), in the region surrounding transmembrane segments 4 and 5, disagrees with the recent published crystal structure of bacterial SGLT from Vibrio parahaemolyticus (vSGLT). To investigate this issue further, 38 residues from I143 to A180 in the N-terminal half of rabbit SGLT1 were each replaced with cysteine and then expressed in COS-7 cells or Xenopus laevis oocytes. The membrane orientations of the substituted cysteines were determined by treatment with the thiol-specific reagent N-Biotinoylaminoethyl methanethiosulfonate (biotin-MTSEA), combined with the membrane impermeant thiol-specific reagent sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES). The present results combined with previous structure/function studies of SGLT1, suggest that transmembrane domain (TM) 4 of mammalian SGLT1 extends from residue 143-171 and support the topology observed in the crystal structure of vSGLT.
哺乳动物高亲和力钠/葡萄糖共转运蛋白（SGLT1）在跨膜片段4和5周围区域的预测拓扑结构与最近发表的副溶血性弧菌细菌SGLT（vSGLT）晶体结构不一致。为了进一步研究这个问题，将兔SGLT1 N端一半中从I143到A180的38个残基分别替换为半胱氨酸，然后在COS-7细胞或非洲爪蟾卵母细胞中表达。通过用硫醇特异性试剂N-生物素酰基氨基乙基甲硫基磺酸盐（生物素-MTSEA）处理，并结合膜不透性硫醇特异性试剂（2-磺基乙基）甲硫基磺酸钠（MTSES），确定取代半胱氨酸的膜取向。目前的结果与先前对SGLT1的结构/功能研究相结合，表明哺乳动物SGLT1的跨膜结构域（TM）4从残基143延伸至171，并支持在vSGLT晶体结构中观察到的拓扑结构。
