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Published online 21 August 2000. doi:10.1083/jcb.150.4.719
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© The Rockefeller University Press, 0021-9525/2000//719 $5.00
The Journal of Cell Biology, Volume 150, Number 4, , 2000 719-730

Original Article

Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse Synaptotagmin Ii, on the Endoplasmic Reticulum



Yuichiro Kidaa, Masao Sakaguchia, Mitsunori Fukudab, Katsuhiko Mikoshibab, and Katsuyoshi Miharaa

a Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
b Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.+81-92-642-6183+81-92-642-6178

sakag{at}cell.med.kyushu-u.ac.jp

Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.

Key Words: membrane topology • membrane protein • signal-anchor sequence • protein translocation • translocon

© 2000 The Rockefeller University Press

Abbreviations used in this paper: C-domain, COOH-terminal domain; EndoH, endoglycosidase H; H-region, hydrophobic region; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; N-domain, NH2-terminal domain; RM, rough microsomal membrane; SA-I, type I signal-anchor; SA-II, type II signal-anchor; SP, signal peptide; SRP, signal recognition particle; Syt, synaptotagmin.


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