All roads lead to Rome-SRP independent translocation into the endoplasmic reticulum

Translocation into the endoplasmic reticulum (ER) is an initial and crucial biogenesis step for all secreted and endomembrane proteins in eukaryotes. Even in the simple eukaryotic model organism, Saccharomyces cerevisiae, this is no simple task, as over a fifth of its proteome must translocate into the ER. It's been well established that several ER targeting pathways are present in S. cerevisiae, the best known of which relies on the signal recognition particle (SRP). We set out to shed new light on alternative SRP-independent translocation pathways. To do so, we harnessed unbiased and systematic approaches to further our understanding of the mechanisms by which these pathways function, and what measures are in place if they are dysfunctional. By combining hydropathy-based analysis and high throughput microscopy, we uncovered that over 20% of the yeast secretome translocates without the aid of the SRP. Further investigation of these SRP-independent substrates revealed an additional motif for ER targeting and uncovered a network of cytosolic proteins that facilitate SRP-independent targeting and translocation. Finally, by employing a systematic microscopic screen, we revealed that SRPindependent substrates are subject to pre-translocational monitoring that clears the cytosol of proteins that have failed to translocate in a timely manner. These findings highlight the underappreciated complexity of SRP independent translocation and its central role in enabling the extensive flux of proteins into the ER.