The tenth membrane region of band 3 is initially exposed to the luminal side of the endoplasmic reticulum and then integrated into a partially folded band 3 intermediate.
Band 3 is a typical polytopic membrane protein that mediates anion exchange activity [anion exchanger 1 (AE1)]. Although the topology and topogenesis of approximately 40 residues just after transmembrane (TM) 9 have been extensively studied, the topogenesis of this region [tenth region (10thR)] has been unclear. Glycosylation sites created in the 10thR were efficiently glycosylated in a cell-free transcription/translation system, whereas the glycosylation efficiencies were quite low in a cultured cell system. When TM12-14 was deleted or when cycloheximide was added to the culture medium, however, the glycosylation efficiency in the cultured cells increased to the same level as in the cell-free system, indicating that TM12 is essential for the sequestration from oligosaccharyl transferase into membrane and that cycloheximide treatment of the cells can mimic the cell-free system by reducing the rate of chain elongation. The glycosylation efficiency in cultured cells also increased with deletion of TM1-3. These results suggest that the 10thR is transiently extruded into the lumen and then inserted into the membrane. Both TM12 and the distant TM1-3 affect the membrane insertion of the 10thR. This indicates that during the folding of the protein, the 10thR is inserted into the membrane after the TM1-12 segments are properly assembled.
Kanki
Tomotake
T
Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Sakaguchi
Masao
M
Kitamura
Akiko
A
Sato
Takashi
T
Mihara
Katsuyoshi
K
Hamasaki
Naotaka
N
D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D001457:Anion Exchange Protein 1, Erythrocyte; D010446:Peptide Fragments; D011994:Recombinant Proteins