Proper localization of proteins is crucial to all cells. The signal recognition particle (SRP) and its receptor (SR) constitute the major cellular machinery that delivers newly synthesized proteins to the eukaryotic endoplasmic reticulum membrane, or the bacterial plasma membrane. This process is regulated by two homologous GTPases in the SRP and SR that directly interact with one another, making it particularly exciting for mechanistic investigations. Though past work has defined the components of the targeting pathway, the molecular mechanism of this process remains unclear. Our general goal is to decipher, at a biochemical and biophysical level, the intricate inner workings of this universally conserved targeting machine. Our specific goal is to understand the mechanism by which the SRP and SR GTPases use their cycles of GTP binding and hydrolysis to provide spatial and temporal coordination of the protein targeting reaction. To this end, three specific aims are envisioned: (1) We will define and characterize the dynamics and conformational intermediates during SRP-SR complex formation and their reciprocal GTPase activation;(2) We will define whether, when and how other components of the protein targeting pathway - the ribosome, the signal sequence, and the membrane translocation channel - modulate the conformational changes of SRP and SR during their binding and activation cycle;(3) We will use the array of mutant GTPases and GTP analogues to perturb specific conformational steps in the GTPase cycle of SRP and SR, and test how these perturbations affect the recognition, delivery and unloading of cargo protein during the protein targeting reaction. These experiments will allow us to define the precise role of each GTP binding and hydrolysis event in providing the driving force or improving the fidelity of the protein targeting reaction. Ultimately, these studies will not only advance our understanding of the process of protein localization within the cell, but also provide new insights into the general principles of molecular recognition and regulation at a very fundamental level. The proposed research is of a most basic nature, and will contribute profoundly to our general understanding of physiology and pathology of all living cells at the molecular level.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM078024-04
Application #
7923876
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2007-09-28
Project End
2012-08-31
Budget Start
2010-09-01
Budget End
2011-08-31
Support Year
4
Fiscal Year
2010
Total Cost
$309,417
Indirect Cost
Name
California Institute of Technology
Department
Chemistry
Type
Schools of Engineering
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Lee, Jae Ho; Chandrasekar, Sowmya; Chung, SangYoon et al. (2018) Sequential activation of human signal recognition particle by the ribosome and signal sequence drives efficient protein targeting. Proc Natl Acad Sci U S A 115:E5487-E5496
Kobayashi, Kan; Jomaa, Ahmad; Lee, Jae Ho et al. (2018) Structure of a prehandover mammalian ribosomal SRP·SRP receptor targeting complex. Science 360:323-327
Hwang Fu, Yu-Hsien; Huang, William Y C; Shen, Kuang et al. (2017) Two-step membrane binding by the bacterial SRP receptor enable efficient and accurate Co-translational protein targeting. Elife 6:
Wang, Shuai; Yang, Chien-I; Shan, Shu-Ou (2017) SecA mediates cotranslational targeting and translocation of an inner membrane protein. J Cell Biol 216:3639-3653
Jomaa, Ahmad; Fu, Yu-Hsien Hwang; Boehringer, Daniel et al. (2017) Structure of the quaternary complex between SRP, SR, and translocon bound to the translating ribosome. Nat Commun 8:15470
Chen, Yang; Shen, Kuang; Shan, Shu-Ou et al. (2016) Analyzing Single-Molecule Protein Transportation Experiments via Hierarchical Hidden Markov Models. J Am Stat Assoc 111:951-966
Shan, Shu-Ou (2016) ATPase and GTPase Tangos Drive Intracellular Protein Transport. Trends Biochem Sci 41:1050-1060
Ariosa, Aileen; Lee, Jae Ho; Wang, Shuai et al. (2015) Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting. Proc Natl Acad Sci U S A 112:E3169-78
von Loeffelholz, Ottilie; Jiang, Qiyang; Ariosa, Aileen et al. (2015) Ribosome-SRP-FtsY cotranslational targeting complex in the closed state. Proc Natl Acad Sci U S A 112:3943-8
Guo, Huan; Xiong, Yi; Witkowski, Piotr et al. (2014) Inefficient translocation of preproinsulin contributes to pancreatic ? cell failure and late-onset diabetes. J Biol Chem 289:16290-302

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