Membrane topogenesis of the three amino-terminal transmembrane segments of glucose-6-phosphatase on endoplasmic reticulum.
We investigated the membrane topogenesis of glucose-6-phosphatase (G6Pase), a multispanning membrane protein, on the endoplasmic reticulum. In COS-7 cells, the first transmembrane segment (TM1) with weak hydrophobicity is inserted into the membrane in the N-terminus-out/C-terminus-cytoplasm orientation. The following TM2 is inserted depending on TM3. TM3 has the same orientation as TM1. In contrast to data from living cells, the full-length molecule and N-terminal fusion constructs were not inserted into the membrane in a cell-free system. Addition of a signal recognition particle did not improve G6Pase insertion. When the 37-residue N-terminal segment was deleted, however, TM2 and TM3 were correctly inserted. We concluded that the three N-terminal TM segments are inserted into the membrane dependent on the two signal-anchor sequences of TM1 and TM3. TM1 is likely to be an unconventional signal sequence that barely functions in vitro. The 37-residue N-terminal segment inhibits the signal function of the following TM3 in cell-free systems.
Ukaji
Koutarou
K
Department of Molecular Biology, Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Science, Kyushu University, Maidashi 3-1-1, Higashiku, Fukuoka 812-8582, Japan.
Ariyoshi
Naoko
N
Sakaguchi
Masao
M
Hamasaki
Naotaka
N
Mihara
Katsuyoshi
K
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005952:Glucose-6-Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000032:analysis; D007425:Intracellular Membranes / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005952:Glucose-6-Phosphatase