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Published online December 31, 2007
doi:10.1083/jcb.200707050
The Journal of Cell Biology, Vol. 179, No. 7, 1441-1452
The Rockefeller University Press, 0021-9525 $30.00
© 2007 Kida et al.
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Article

 Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

Yuichiro Kida1,2, Fumiko Morimoto1, and Masao Sakaguchi1,2

1 Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan
2 CREST of the Japan Science and Technology Agency, Hyogo 678-1297, Japan

Correspondence to Masao Sakaguchi: sakag{at}sci.u-hyogo.ac.jp

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, Sec61{alpha}. 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.

Abbreviations used in this paper: BMB, 1,4-bismaleimidobutane; BMH, 1,6-bis-maleimidohexane; BMOE, bismaleimidoethane; BM(PEO)2, N,N-(methylene- 4-1-phenylene)bismaleimide; CHX, cycloheximide; EndoH, endoglycosidase H; H-segment, hydrophobic segment; N-domain, N-terminal domain; Puro, puromycin; RM, rough microsomal membrane; SA-I, type I signal-anchor sequence; SAv, streptavidin; SytII, synaptotagmin II; TM, transmembrane; TRAM, translocating chain-associating membrane protein.


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