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J. Biol. Chem., Vol. 283, Issue 30, 20864-20873, July 25, 2008

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Control of Translocation through the Sec61 Translocon by Nascent Polypeptide Structure within the Ribosome^*

Colin J. Daniel, Brian Conti, Arthur E. Johnson, and William R. Skach¹

From the Department of Biochemistry & Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239 and the Department of Molecular and Cellular Medicine and the Departments of Chemistry and of Biochemistry and Biophysics, Texas A&M University System Health Science Center, College Station, Texas 77843-1114

During polytopic protein biogenesis, multiple transmembrane segments (TMs) must pass through the ribosome exit tunnel and into the Sec61 translocon prior to insertion into the endoplasmic reticulum membrane. To investigate how movement of a newly synthesized TM along this integration pathway might be influenced by synthesis of a second TM, we used photocross-linking probes to detect the proximity of ribosome-bound nascent polypeptides to Sec61. Probes were inserted at sequential sites within TM2 of the aquaporin-1 water channel by in vitro translation of truncated mRNAs. TM2 first contacted Sec61 when the probe was positioned 38 residues from the ribosome peptidyltransferase center, and TM2-Sec61 photoadducts decreased markedly when the probe was >80 residues from the peptidyltransferase center. Unexpectedly, as nascent chain length was gradually extended, photocross-linking at multiple sites within TM2 abruptly and transiently decreased, indicating that TM2 initially entered, withdrew, and then re-entered Sec61. This brief reduction in TM2 photocross-linking coincided with TM3 synthesis. Replacement of TM3 with a secretory reporter domain or introduction of proline residues into TM3 changed the TM2 cross-linking profile and this biphasic behavior. These findings demonstrate that the primary and likely secondary structure of the nascent polypeptide within the ribosome exit tunnel can influence the timing with which topogenic determinants contact, enter, and pass through the translocon.

Received for publication, May 8, 2008

^* This work was supported, in whole or in part, by National Institutes of Health Grants DK51818 and GM53457 (to W. R. S.) and GM26494 (to A. E. J.). This work was also supported by American Heart Association Established Investigator Grant and Grant-in-aid 0755832Z (to W. R. S.) and Robert A. Welch Foundation Chair Grant BE-0017 (to A. E. J.).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

This article was selected as a Paper of the Week.

¹ To whom correspondence should be addressed: Dept. of Biochemistry & Molecular Biology, Oregon Health & Science University, L-224, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97239. Fax: 503-494-8393; E-mail: skachw{at}ohsu.edu.

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