biostudies-literatureYifrach EThe Ariane de Rothschild Women Doctoral ProgramBlythe Brenden-Mann foundationEuropean Research CouncilERC CoGKekst Family Institute for Medical GeneticsIsraeli Science Foundation (ISF)e11186https://www.ebi.ac.uk/biostudies/studies/S-EPMC9513677biostudies-literatureUnknown18(9)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 gluconeogenMolecular systems biologySystematic multi-level analysis of an organelle proteome reveals new peroxisomal functions.PMC9513677ISF914/22864068Yifrach EWeill UWaterham HRHolbrook-Smith DVisser WTirosh AWanders RJABibi CWilmanns MSchuldiner MErdmann ROthman AGalor SZamboni NBurgi JEisenstein MZalckvar ERudowitz Mvan Roermund CWFadel APeleg YfalseSystematic multi-level analysis of an organelle proteome reveals new peroxisomal functions.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 gluconeogen2022-01-01T00:00:00Z2022 Sep2025-04-19T17:38:14.231Z2025-04-19T17:38:14.231ZS-EPMC95136773616497810.15252/msb.202211186