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Abstract

One of the fundamental requirements for protein function and cell viability is the accurate targeting of proteins to the cellular compartments in which they operate. For approximately one third of all proteins, this begins with their targeting to the endoplasmic reticulum (ER), which is often coupled directly to protein synthesis. In eukaryotes, cotranslational ER targeting largely depends on the universally conserved Signal Recognition Particle (SRP). According to textbook models, SRP recognizes and binds targeting signals on nascent chains when they emerge from the ribosome. This interaction transiently slows translation while SRP targets the ribosome to the ER membrane by binding to its receptor. The ribosome then docks onto the translocon, allowing the nascent chain to be inserted into the ER, as SRP dissociates. However, several aspects of this model are debated or are yet to be explored using approaches capable of capturing the complex sequence of events in living cells and the variability across the proteome. In particular, significant gaps remain in our understanding of the cellular substrate spectrum of SRP, the extent of overlap between alternative targeting pathways, and the molecular cues that mediate SRP’s initial recruitment to ribosomes. Understanding these aspects is also essential to be able to place ER targeting within the broader network of other cotranslational maturation processes, such as chaperone assisted folding. Additionally, previous observations in bacteria have hinted at potential roles for SRP in later stages of ER translocation, which remain to be further explored. To address these questions, I employed ribosome profiling based approaches to map the timing of SRP interactions translatome wide in the eukaryotic model organism Saccharomyces cerevisiae, thus defining SRP’s substrate pool and the molecular features mediating its recruitment. I further investigated the association of ribosomes with membranes at codon resolution and dissected the contribution of individual factors in cells lacking key components, including SRP, the nascent polypeptide associated complex (NAC), and the ribosome associated chaperone Ssb. My findings support the classical signal hypothesis that SRP engages targeting signals after their emergence from the ribosomal exit tunnel, refuting more complex models of early pre loading of SRP. Assessing prevalence of ribosome collisions as a readout of translation slowdown during SRP mediated targeting, the data revealed not only an SRP induced slowdown upon binding but also an SRP independent slowdown that coincided with the ribosomal emergence of targeting signals. Challenging the long held assumption that SRP is the main factor targeting proteins that contain N terminal cleavable signal peptides, I find that most of these proteins are targeted independently of SRP. Instead, transmembrane domain containing proteins rely fully on SRP for targeting and constitute the major SRP substrates. This clear separation between the targeting routes aligns with mutually exclusive downstream maturation pathways suggesting that SRP sorts nascent chains based on the properties of their emerging signals into distinct biosynthetic trajectories. I further explore the postulated role of NAC in enhancing the efficiency and specificity of ER targeting. I find that while a fraction of NAC interactions coincide with SRP binding to its substrates, SRP specificity and targeting efficiency is globally unaffected in cells lacking NAC. Moreover, NAC preferentially binds SRP independent ER proteins, delaying targeting until a possible handover to unknown targeting factors. Finally, I establish that SRP repeatedly interacts with ribosomes that are already membrane associated, revealing a so far uncharacterized role after initial ER membrane targeting. SRP rebinding to multipass membrane proteins becomes necessary due to the unanticipated extent of ribosome dissociation from the translocon prior to the completion of translocation. Supporting the functional relevance of this process, I show that ribosome undocking facilitates access of Ssb to support the folding of the soluble domains of membrane proteins. Overall, this work presents a proteome wide identification of cotranslational targeting and SRP interactions in yeast, revealing the determinants and distinct features of different targeting pathways. The findings uncover a role for SRP in the maturation of its substrates beyond their initial delivery to the ER. Moreover, the data highlight the widespread use of SRP independent targeting routes by the majority of signal peptide containing proteins. Finally, the temporal sequence of nascent chain interactions during cotranslational targeting reveals previously unrecognized roles for cytosolic chaperones and NAC in the maturation of ER destined proteins.