Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis.
During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61alpha. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.
Kida
Yuichiro
Y
Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan.
Morimoto
Fumiko
F
Sakaguchi
Masao
M
D000818:Animals; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D014176:Protein Biosynthesis / Q000502:physiology; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D000069816:SEC Translocation Channels
D008565:Membrane Proteins; D018832:Molecular Chaperones; D000069816:SEC Translocation Channels; C000613244:SEC61A1 protein, human