Manipulation of membrane protein topology on the endoplasmic reticulum by a specific ligand in living cells.
Almost all integral membrane proteins in the secretory pathway are cotranslationally inserted into the endoplasmic reticulum membrane. Their membrane topology is determined by their amino acid sequences. Here we show that the topology can be manipulated by a factor other than the amino acid sequence. A dihydrofolate reductase (DHFR) domain was fused to the N-terminus of the type I signal-anchor sequence of synaptotagmin II, which mediates translocation of the preceding portion. The DHFR domain was translocated through the membrane in COS7 cells and a transmembrane (TM) topology was achieved. When a DHFR ligand, methotrexate, was added to the culture medium, translocation of the DHFR domain was suppressed and both ends of the signal-anchor sequence remained on the cytoplasmic side. In contrast, translocation of the DHFR domain fused after the signal peptide, which translocates the following region, was not affected by the ligand. The topology-altered fusion protein was anchored to the membrane in a high salt-resistant state, and partially extracted from the membrane under alkali conditions. We concluded that the topology of membrane proteins can be manipulated by a trans-acting factor, even in living cells.
Ikeda
Motoyasu
M
Graduate School of Life Science, University of Hyogo, Ako, Hyogo 678-1297.
Kida
Yuichiro
Y
Ikushiro
Shin-ichi
S
Sakaguchi
Masao
M
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008727:Methotrexate / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014162:Transfection; D014178:Translocation, Genetic
D008024:Ligands; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate