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Global analysis of cotranslational protein targeting using proximity-specific ribosome profiling

  • Author(s): Costa, Elizabeth Annette Boydston
  • Advisor(s): Weissman, Jonathan S
  • et al.
Abstract

Eukaryotic cells are composed of diverse subcompartments, or organelles, that each have unique functions and distinct protein compositions. Many proteins that transit through or populate secretory pathway organelles are delivered during synthesis to the endoplasmic reticulum (ER), which serves as the entry organelle to the secretory pathway. Despite decades of studies characterizing in close molecular detail the protein targeting pathways involved, including that of the universally conserved signal recognition particle (SRP), it is unclear at a global level which proteins utilize each pathway under physiological conditions.

In this dissertation, I describe the use of proximity-specific ribosome profiling to globally measure cotranslational protein targeting to two separate organelles, the ER and mitochondrion. We combined this method with rapid auxin-inducible degradation of SRP to define the in vivo function of SRP in Saccharomyces cerevisiae. Despite the classic view that SRP recognizes N-terminal signal sequences, we show SRP was generally essential for targeting transmembrane domains regardless of their position relative to the N-terminus. By contrast, many proteins containing cleavable N-terminal signal peptides were efficiently cotranslationally targeted to the ER the absence of SRP, arguing for the presence of an additional, currently undescribed cotranslational targeting pathway. Lastly, we reveal an unanticipated consequence of loss of SRP: transcripts normally targeted to the ER were mistargeted to mitochondria, leading to rapid mitochondrial morphological defects. These results elucidate the essential roles for SRP in maintaining both efficiency and specificity of protein targeting.

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