Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling

Introduction Localized protein synthesis plays a critical role in creating subcellular structures by allowing protein production at the site of action and in response to local cellular need. Local translation is involved in diverse processes, including developmental patterning, cellular motility, synaptic plasticity, and protein trafficking through the secretory pathway. Despite this broad importance, few gene expression tools are available that faithfully preserve spatial information. We developed a flexible deep sequencing–based methodology (termed proximity-specific ribosome profiling) that enables precise characterization of localized protein synthesis. We applied our method to analyze translation at the endoplasmic reticulum (ER) in yeast and mammalian cells. Proximity-specific ribosome profiling provides spatiotemporal details of translation at the ER. Biotin ligase is localized to the ER as a fusion protein, where it biotinylates Avi-tagged ribosomes at the ER surface. Ribosome profiling is performed on streptavidinpurified ribosomes and compared to whole-cell profiling to resolve which genes are translated at the ER (1) and how much nascent chain was required to target the ribosome to the translocon (2). Rationale The basis of our approach is to biotinylate ribosomes in intact cells in a manner dependent on their subcellular location. This is accomplished through the coexpression of a spatially restricted biotin ligase (BirA) fusion protein together with ribosomes containing an AviTag, which makes them substrates for BirA. Controlled pulses of biotin are then provided to allow for spatiotemporal control of ribosome labeling. This in vivo biotinylation enables the recovery of ribosomes from defined locations, including those that cannot be purified by classical cell fractionation techniques. Combining this purification strategy with ribosome profiling, the deep sequencing of ribosome-protected mRNA fragments, provides subcodon resolution of which messages were translated at the site of interest. Results We identified several principles used by cells to coordinate translation with ER targeting. Cotranslational targeting to the ER is pervasive and is principally determined by the location of the hydrophobic targeting sequence within the protein, rather than the mechanism of targeting or translocation. The position of this hydrophobic domain within the open reading frame determines the duration of time a targeted ribosome nascent-chain complex (RNC) can associate with the ER. Our data suggest a role for polysomes in retaining mRNAs at the ER, allowing for efficient targeting of RNCs for translocation. Position-specific analyses revealed that distinct translocon complexes engage nascent chains at different points during synthesis. Most proteins engage the ER immediately after or even before the signal sequence or signal anchor emerges from the ribosome. These nascent chains typically undergo a conformational rearrangement within the translocon, the proteinaceous tunnel through which nascent proteins cross the ER membrane. This rearrangement results in a “looped” conformation of the nascent chains, with their N termini facing the cytosol. This conformation is required for signal sequence processing. However, we discovered a class of Sec66-dependent proteins that engage only when they are long enough to adopt the looped conformation. Finally, we monitored the fate of ER-associated ribosomes after translation termination using pulsed-labeling experiments. These data demonstrated that ER-associated ribosomes readily exchanged into the cytosol after at most a few rounds of translation at the ER. Conclusion These results, together with those in an accompanying Report on translation at mitochondria, establish proximity-specific ribosome profiling as a robust and general tool. In principle, this method can be applied to any site that can be specified by a biotinligase fusion protein. Thus, our approach provides in vivo access to a broad spectrum of subpopulations of ribosomes defined either by their subcellular locations or through their interactions with specific factors, such as chaperones. The wheres and whys of protein translation Localized protein synthesis is important for a broad range of biological activities, from specifying the animal body plan to coordinating entry into the secretory pathway. Few tools are available that can investigate translation at specific subcellular sites. Jan et al. present a flexible ribosome profiling–based methodology to enable precise characterization of localized protein synthesis (see the Perspective by Shao and Hegde). Proximity-specific ribosome profiling provides a high-precision tool for looking at the mechanism of localized protein targeting and synthesis in living cells. The approach yielded a high-resolution systems-level view of cotranslational translocation at the endoplasmic reticulum. Williams et al. applied the technique to look at localized mRNA translation at the mitochondrial outer membrane. Science, this issue 10.1126/science.1257521, p. 701; see also p. 748 A new method reveals exactly which proteins are synthesized in the neighborhood of the endoplasmic reticulum and mitochondria. [Also see Perspective by Shao and Hegde] Localized protein synthesis is a fundamental mechanism for creating distinct subcellular environments. Here we developed a generalizable proximity-specific ribosome profiling strategy that enables global analysis of translation in defined subcellular locations. We applied this approach to the endoplasmic reticulum (ER) in yeast and mammals. We observed the large majority of secretory proteins to be cotranslationally translocated, including substrates capable of posttranslational insertion in vitro. Distinct translocon complexes engaged nascent chains at different points during synthesis. Whereas most proteins engaged the ER immediately after or even before signal sequence (SS) emergence, a class of Sec66-dependent proteins entered with a looped SS conformation. Finally, we observed rapid ribosome exchange into the cytosol after translation termination. These data provide insights into how distinct translocation mechanisms act in concert to promote efficient cotranslational recruitment.