Environmental transition of signal-anchor sequences during membrane insertion via the endoplasmic reticulum translocon.
In biogenesis of membrane proteins on the endoplasmic reticulum, a protein-conducting channel called the translocon functions in both the membrane translocation of lumenal domains and the integration of transmembrane segments. Here we analyzed the environments of polypeptide chains during the processes by water-dependent alkylation of N-ethylmaleimide at site-directed Cys residues. Using the technique, the region embedded in the hydrophobic portion of the membrane within a signal-anchor sequence and its shortening by insertion of a Pro residue could be detected. When translocation of the N-terminal domain of the signal-anchor was arrested by trapping an N-terminally fused affinity tag sequence, the signal-anchor was susceptible to alkylation, indicating that its migration into the hydrophobic environment was also arrested. Furthermore, when the tag sequence was separated from the signal-anchor by insertion of a hydrophilic sequence, the signal-anchor became inaccessible to alkylation even in the N-terminally trapped state. This suggests that membrane integration of the signal-anchor synchronizes with partial translocation of its N-terminal domain. Additionally, in an integration intermediate of a membrane protein, both of the two translocation-arrested hydrophilic chains were in an aqueous environment flanking the translocon, suggesting that the translocon provides the hydrophilic pathway capable of at least two translocating chains.
Kida
Yuichiro
Y
Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan. ykida@sci.u-hyogo.ac.jp
Kume
Chisato
C
Hirano
Maki
M
Sakaguchi
Masao
M
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport / Q000502:physiology
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011392:Proline; D003545:Cysteine