Most secreted and plasma membrane proteins are synthesized on membrane-bound ribosomes at the endoplasmic reticulum (ER), where they cross the membrane or become embedded in it. Ribosomes synthesizing this subclass of proteins are targeted to the ER membrane by an evolutionarily conserved molecular machine, the signal recognition particle (SRP) and the SRP receptor (SR). SRP binds to signal sequences as they emerge as part of the growing polypeptide chain from the ribosome. It then interacts with the SR, to effect the joining of the ribosome to a membrane-bound protein translocation channel through which the protein is moved into the ER lumen or is integrated into the ER membrane. Both SRP and SR contain essential GTPase domains, which together form the central engine driving the targeting reaction. SRPs from all species also contain an RNA subunit. Here, we propose to investigate how the engine (the SRP and SR GTPases) couples the consumption of energy (GTP hydrolysis) to the work of targeting proteins to the membrane. In addition, we will investigate the mechanism by which SRPRNA controls the interaction of the SRP and the SR. We hypothesize that SRP RNA links signal sequence and/or ribosome occupancy of SRP to the GTPase engine. Specifically, we will: i) Determine how SRP/SR couples substrate delivery to the membrane with GTP hydrolysis to ensure efficient targeting: We will dissect the targeting reaction, making use of well-characterized mutant components that stall SRP-SR interactions at discrete steps. ii) Dissect the structural rearrangements that occur as the targeting reaction proceeds: We will apply structural approaches to get a high-resolution view of intermediates in the targeting reaction and use fluorescence tools to monitor the kinetics of structural rearrangements that mark each step of the targeting cycle. iii) Investigate how the SRP RNA controls the interaction of the SRP and SR and why this control is essential to the targeting reaction: We will identify features of the SRP RNA, the SRP and SR that are important for its function and situate the activity of the SPR RNA in the context of the targeting reaction. In addressing these questions, we will aim at a high resolution definition of core parameters, i.e., parameters that are conserved among all SRP/SR targeting systems, but we will also exploit clues from evolutionary variations on the theme. The core parameters will provide a solid foundation on which to build models that add further structural and regulatory complexities, such as those found in higher eukaryotic systems. Ultimately, we wish to understand SRP-dependent protein targeting in precise mechanistic detail, thus increasing our understanding of how cells expend energy to achieve and maintain their complex organization. A molecular understanding of this highly conserved pathway - that operates in all living cells and is responsible for the proper biogenesis of virtually every signaling protein with which the cell communicates with its environment - is of profound significance to our understanding of cell physiology and pathology at a most fundamental level.
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