Transmembrane topology of the mammalian Slc11a2 iron transporter.
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).
Czachorowski
Maciej
M
Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G-0B1.
Lam-Yuk-Tseung
Steven
S
Cellier
Mathieu
M
Gros
Philippe
P
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D008565:Membrane Proteins; D026901:Membrane Transport Proteins