A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum

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The segregation of secretory and membrane proteins to the mammalian endoplasmic reticulum is mediated by remarkably diverse signal sequences that have little or no homology with each other1,2. Despite such sequence diversity, these signals are all recognized and interpreted by a highly conserved protein-conducting channel composed of the Sec61 complex3,4. Signal recognition by Sec61 is essential for productive insertion of the nascent polypeptide into the translocation site5, channel gating6 and initiation of transport. Although subtle differences in these steps can be detected between different substrates7,8, it is not known whether they can be exploited to modulate protein translocation selectively. Here we describe cotransin, a small molecule that inhibits protein translocation into the endoplasmic reticulum. Cotransin acts in a signal-sequence-discriminatory manner to prevent the stable insertion of select nascent chains into the Sec61 translocation channel. Thus, the range of substrates accommodated by the channel can be specifically and reversibly modulated by a cell-permeable small molecule that alters the interaction between signal sequences and the Sec61 complex.

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Figure 1: Cotransin inhibits expression of a subset of secreted and membrane proteins.
Figure 2: Cotransin inhibits cotranslational translocation.
Figure 3: Cotransin inhibits insertion of VCAM1 into the translocation channel.
Figure 4: Cotransin prevents signal sequence recognition by the Sec61 complex.


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E.J.K. thanks E. Rosler, S. Privat, D. Nguyen, J. Melrose, M. Fischer and M. Dea for technical assistance. R.S.H. thanks A. Sharma and M. Alken for technical assistance, and N. Rane and E. Snapp for discussions. J.L.G. thanks Z. Knight for discussions and critical reading of the manuscript. J.L.G. is supported by an NSF graduate fellowship. J.T. is a Searle Scholar and a fellow of the Alfred P. Sloan Foundation.

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Correspondence to Ramanujan S. Hegde or Jack Taunton.

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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure S1

Effect of cotransin and nor-cotransin on protein expression in stimulated primary human cells. (PDF 19 kb)

Supplementary Figure S2

Effect of cotransin and nor-cotransin on cellular glycoprotein expression. (PDF 1888 kb)

Supplementary Figure S3

Cotransin does not influence the translocation, glycosylation, membrane insertion, or topology of model membrane proteins synthesized in vitro. (PDF 1174 kb)

Supplementary Figure S4

Cotransin's effects are independent of ER lumenal proteins. (PDF 1266 kb)

Supplementary Figure S5

Analysis of VCAM1 and pPrl translocation in fractionated proteoliposomes. (PDF 256 kb)

Supplementary Figure S6

Purification of Sec61 complex / SR and preparation of minimal proteoliposome. (PDF 381 kb)

Supplementary Table 1

Additional full length proteins assayed for translocation in the presence of cotransin in vitro. (PDF 11 kb)

Supplementary Figure Legends

Text to accompany the above Supplementary Figures. (DOC 46 kb)

Supplementary Methods

Methods and References to accompany Supplementary Figures. (DOC 27 kb)

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Garrison, J., Kunkel, E., Hegde, R. et al. A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum. Nature 436, 285–289 (2005) doi:10.1038/nature03821

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