Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi-ome, remains undefined. Uncovering the peroxi-ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover peroxisomal proteins in the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi-ome by ~40% and paved the way for performing systematic, whole-organellar proteome assays. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

The highly conserved endoplasmic reticulum (ER) protein translocation channel contains one nonessential subunit, Sec61β/Sbh1, whose function is poorly understood so far. Its intrinsically unstructured cytosolic domain makes transient contact with ER-targeting sequences in the cytosolic channel vestibule and contains multiple phosphorylation sites suggesting a potential for regulating ER protein import. In a microscopic screen, we show that 12% of a GFP-tagged secretory protein library depends on Sbh1 for translocation into the ER. Sbh1-dependent proteins had targeting sequences with less pronounced hydrophobicity and often no charge bias or an inverse charge bias which reduces their insertion efficiency into the Sec61 channel. We determined that mutating two N-terminal, proline-flanked phosphorylation sites in the Sbh1 cytosolic domain to alanine phenocopied the temperature-sensitivity of a yeast strain lacking SBH1 and its ortholog SBH2. The phosphorylation site mutations reduced translocation into the ER of a subset of Sbh1-dependent proteins, including enzymes whose concentration in the ER lumen is critical for ER proteostasis. In addition, we found that ER import of these proteins depended on the activity of the phospho-S/T-specific proline isomerase Ess1 (PIN1 in mammals). We conclude that Sbh1 promotes ER translocation of substrates with suboptimal targeting sequences and that its activity can be regulated by a conformational change induced by N-terminal phosphorylation.