New Regulatory Mechanisms in SRP Targeting Pathway

Cotranslational protein targeting by the signal recognition particle (SRP) is a fundamental and evolutionarily conserved process that ensures accurate delivery of nascent proteins to the endoplasmic reticulum (ER). This pathway requires precise coordination between cargo recognition, targeting complex assembly, and membrane-associated handover, yet how these steps are temporally and mechanistically integrated during ongoing translation remains incompletely understood. In this thesis, I combine steady-state, pre-steady-state, and single-molecule fluorescence approaches to dissect the dynamic regulation of SRP function across distinct stages of the targeting cycle. In Chapter 1, I investigate how nascent polypeptide elongation modulates SRP activity. Using Förster resonance energy transfer (FRET) measurements, I show that increasing nascent chain length enhances dynamic excursions of the signal sequence from SRP, shifting the complex into suboptimal conformations with impaired interaction kinetics with the SRP receptor (SR). Furthermore, the nascent polypeptide-associated complex (NAC) amplifies these effects, further antagonizing SRP function. These findings reveal that nascent chain length and NAC together impose a limited temporal window for efficient cotranslational targeting. In Chapter 2, I focus on the membrane-associated stages of the SRP pathway and elucidate the mechanism of ribosome–nascent chain complex (RNC) handover to the ER translocation machinery. I demonstrate that SR remodels and partially destabilizes the SRP–RNC complex by inducing signal sequence release and detachment of the SRP GTPase domain from the ribosome, while paradoxically stabilizing the overall complex through multivalent interactions. GTP hydrolysis subsequently disrupts this stabilization to enable cargo transfer. Importantly, the ER membrane selectively stabilizes a pre-handover intermediate with delayed GTP hydrolysis for correct client proteins, thereby providing a time window for continued nascent chain elongation and productive translocation. In Chapter 3, I explore the expression of the Sec61 complex in insect cells and its reconstitution into nanodiscs to recapitulate the effects of the ER membrane on RNC handover and SRP conformation. Using a copolymer-based method, I successfully purified Sec61-containing nanodiscs comprising all three subunits. However, further assays are required to verify the structural and functional integrity of the reconstituted Sec61 translocon, and additional optimization of the purification strategy is needed. Together, this work advances our understanding of the molecular mechanisms underlying SRP-dependent cotranslational targeting. In conjunction with prior studies from our laboratory, it contributes to a high-resolution mechanistic model of this pathway. More broadly, it reveals how multiple regulatory layers are temporally coordinated: how SRP selectively rejects RNCs bearing excessively elongated nascent chains to enhance targeting specificity, and how GTPase activity serves as a molecular timer to ensure cargo handover occurs at the appropriate stage of nascent chain elongation.