Topogenesis of NHE1: direct insertion of the membrane loop and sequestration of cryptic glycosylation and processing sites just after TM9.
Multispanning membrane proteins are synthesized by membrane-bound ribosomes and integrated into the endoplasmic reticulum membrane cotranslationally. To uncover the topogenic process of membrane loop, of which both ends are in the same side of the membrane, we examined topogenesis of a relatively hydrophobic lumenal loop segment (H10 segment) between TM9 and TM10 of human Na(+)/H(+) exchanger isoform 1 using an in vitro expression system. The H10 segment was translocated through the membrane. Any potential sites created within the H10 segment were not glycosylated. Just after TM9, there are potential glycosylation and signal peptidase processing sites. When the reporter domain of prolactin was fused at the position preceding the H10 segment, these sites were modified by the enzymes, while they were not modified in the original molecule. Thus, we concluded that the H10 segment was translocated through the membrane and directly inserted into the membrane and that its membrane insertion caused sequestration of the preceding processing and glycosylation sites from the lumenal modifying enzymes. This topogenic process shows clear contrast to that of pore loops of K(+) channels, which are once exposed in the lumen and accessible to glycosylation enzyme.
Sato
Yoko
Y
Graduate School of Life Science, University of Hyogo, Ako, Hyogo 678-1297, Japan.
Ariyoshi
Naoko
N
Mihara
Katsuyoshi
K
Sakaguchi
Masao
M
D000595:Amino Acid Sequence; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D027682:Cation Transport Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers