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Published August 13, 2007 | Supplemental Material + Published
Journal Article Open

Conformational changes in the GTPase modules of the signal reception particle and its receptor drive initiation of protein translocation

Abstract

During cotranslational protein targeting, two guanosine triphosphatase (GTPase) in the signal recognition particle (SRP) and its receptor (SR) form a unique complex in which hydrolyses of both guanosine triphosphates (GTP) are activated in a shared active site. It was thought that GTP hydrolysis drives the recycling of SRP and SR, but is not crucial for protein targeting. Here, we examined the translocation efficiency of mutant GTPases that block the interaction between SRP and SR at specific stages. Surprisingly, mutants that allow SRP–SR complex assembly but block GTPase activation severely compromise protein translocation. These mutations map to the highly conserved insertion box domain loops that rearrange upon complex formation to form multiple catalytic interactions with the two GTPs. Thus, although GTP hydrolysis is not required, the molecular rearrangements that lead to GTPase activation are essential for protein targeting. Most importantly, our results show that an elaborate rearrangement within the SRP–SR GTPase complex is required to drive the unloading and initiate translocation of cargo proteins.

Additional Information

© The Rockefeller University Press, 2007. Submitted: 5 February 2007. Accepted: 1 July 2007. Published online 6 August 2007. We thank Ted Powers for help in reconstituting the heterologous protein targeting reaction. This work was supported by National Institutes of Health grant GM 32384 to P. Walter and career awards from the Burroughs Wellcome Fund and the Camille and Henry Dreyfus Foundation to S. Shan. P. Walter is an investigator at the Howard Hughes Medical Institute. Fig. S1 shows that the targeting and translocation of pPL occurs on a faster time scale than completion of protein synthesis. Fig. S2 shows that FtsY(47–497) is reduced by about half in translocation of pPL compared with full-length FtsY. Fig. S3 and Tables S1–S7 show additional data for repetitions of the experiments shown in Figs. 2–5. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200702018/DC1.

Attached Files

Published - SHAjcb07.pdf

Published - SHAjcb07figS1.jpg

Supplemental Material - SHAjcb07figS2.jpg

Supplemental Material - SHAjcb07figS3.jpg

Supplemental Material - SHAjcb07tables.pdf

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Created:
August 22, 2023
Modified:
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