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Published June 12, 2018 | Published + Supplemental Material
Journal Article Open

Sequential activation of human signal recognition particle by the ribosome and signal sequence drives efficient protein targeting


Signal recognition particle (SRP) is a universally conserved targeting machine that mediates the targeted delivery of ∼30% of the proteome. The molecular mechanism by which eukaryotic SRP achieves efficient and selective protein targeting remains elusive. Here, we describe quantitative analyses of completely reconstituted human SRP (hSRP) and SRP receptor (SR). Enzymatic and fluorescence analyses showed that the ribosome, together with a functional signal sequence on the nascent polypeptide, are required to activate SRP for rapid recruitment of the SR, thereby delivering translating ribosomes to the endoplasmic reticulum. Single-molecule fluorescence spectroscopy combined with cross-complementation analyses reveal a sequential mechanism of activation whereby the ribosome unlocks the hSRP from an autoinhibited state and primes SRP to sample a variety of conformations. The signal sequence further preorganizes the mammalian SRP into the optimal conformation for efficient recruitment of the SR. Finally, the use of a signal sequence to activate SRP for receptor recruitment is a universally conserved feature to enable efficient and selective protein targeting, and the eukaryote-specific components confer upon the mammalian SRP the ability to sense and respond to ribosomes.

Additional Information

© 2018 National Academy of Sciences. Published under the PNAS license. Edited by Joseph D. Puglisi, Stanford University School of Medicine, Stanford, CA, and approved May 2, 2018 (received for review February 6, 2018). Published ahead of print May 30, 2018. We thank E. Menichelli, K. Nagai, E. Mandon, R. Gilmore, K. Strub, and C. Zwieb for the expression constructs and purification protocols on SRP proteins and SRP RNA; A. Sharma for advice on RRL reagents and protocols; H. Bernstein for sharing canine pancreatic microsomes; the laboratory of D. Rees for the use of MST; the laboratory of D. Dougherty for the use of HPLC; and K. Strub for advice on SRP assembly and purification procedures. This work was supported by National Institutes of Health Grant GM078024, Gordon and Betty Moore Foundation Grant GBMF2939 (to S.-o.S.), and Dean Willard Chair funds (to S.W.). Author contributions: J.H.L., S.W., and S.-o.S. designed research; J.H.L., S. Chandrasekar, S. Chung, Y.-H.H.F., D.L., and S.-o.S. performed research; J.H.L., S. Chandrasekar, S. Chung, Y.-H.H.F., D.L., and S.W. contributed new reagents/analytic tools; J.H.L., S. Chandrasekar, S. Chung, Y.-H.H.F., D.L., S.W., and S.-o.S. analyzed data; J.H.L. and S.-o.S. wrote the paper; S.W. led supervision on single molecule spectroscopy experiments; and S.-o.S. supervised the project. The authors declare no conflict of interest. This article is a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1802252115/-/DCSupplemental.

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Published - E5487.full.pdf

Supplemental Material - pnas.1802252115.sapp.pdf


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