Control of translocation through the Sec61 translocon by nascent polypeptide structure within the ribosome

被引:31
作者
Daniel, Colin J. [1 ]
Conti, Brian [1 ]
Johnson, Arthur E. [2 ,3 ,4 ]
Skach, William R. [1 ]
机构
[1] Oregon Hlth & Sci Univ, Dept Biochem & Mol Biol, Portland, OR 97239 USA
[2] Texas A&M Univ, Syst Hlth Sci Ctr, Dept Mol & Cellular Med, College Stn, TX 77843 USA
[3] Texas A&M Univ, Syst Hlth Sci Ctr, Dept Chem, College Stn, TX 77843 USA
[4] Texas A&M Univ, Syst Hlth Sci Ctr, Dept Biochem & Biophys, College Stn, TX 77843 USA
关键词
D O I
10.1074/jbc.M803517200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 alpha. 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 alpha when the probe was positioned similar to 38 residues from the ribosome peptidyltransferase center, and TM2-Sec61 alpha photo-adducts 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 alpha. 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.
引用
收藏
页码:20864 / 20873
页数:10
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