Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions

Abstract

Seventy years following the discovery of peroxisomes, their proteome remains undefined. Uncovering the complete peroxisomal proteome, the peroxi-ome, is crucial for understanding peroxisomal activities and cellular metabolism. We used high- content microscopy to uncover the peroxi-ome of the model eukaryote – Saccharomyces cerevisiae. This strategy enabled us to expand the known organellar proteome by ∼40% and paved the way for performing systematic, whole-organellar proteome assays. Coupled with targeted experiments this allowed us to discover new peroxisomal functions. 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.

Determining the targeting specificity of the selective peroxisomal targeting factor Pex9

“…Therefore, we sought to find additional proteins that can be targeted by Pex9. To this end, we performed a microscopic screen on a collection of ~40 yeast strains representing a set of recently identified yeast peroxisomal proteins (Yifrach et al, 2021), which were never tested for targeting by Pex9 (Fig. 1A).…”

“…We imaged the entire collection using a high content screening setup and looked for proteins that co-localize with peroxisomes when only Pex9 is expressed as a PTS1 targeting factor. Following analysis of all strains, we were able to identify two proteins that co-localized with peroxisomes in this condition – Fsh3, a newly-identified peroxisomal lipase (Yifrach et al, 2021), and Afr1, a protein required for the formation of pheromone-induced projection in yeast (Konopka, 1993) (Fig. 1B).…”

“…We then performed Molecular Dynamics (MD) simulations of a cargo factor (Pex5 or Pex9) with peptides consisting of six C’ amino acids of four PTS1 proteins that are Pex9 cargo: Mls1, Mls2, Gto1, and Fsh3. Previously we showed that the binding stability of a peptide to Pex5 could be estimated from the constancy of hydrogen bonds (H-bonds) between peptide backbone atoms and specific Pex5 residues, particularly H-bonds of peptide residues −1 and −3 (Yifrach et al, 2021). The Pex5 residues that form these H-bonds are conserved between Pex5 and Pex9 supporting the importance of the C’ tripeptide for binding.…”

“…Pex3-mCherry (a peroxisomal marker), PEX5 deletion (Δpex5 ), and PEX9 constitutive-expression ( TDH3pr-PEX9 )), a query strain was constructed based on an SGA compatible strain (for further information see Table S1). Using the SGA method (Cohen and Schuldiner, 2011; Tong and Boone, 2006) the query strain was crossed with a collection of strains from the SWAT N’-GFP library (Yofe et al, 2016; Weill et al, 2018) containing ~40 strains of recently-identified peroxisomal proteins (Yifrach et al, 2021) together with controls. To perform the SGA in an arrayed format, we used a RoToR benchtop colony arrayer (Singer Instruments).…”

“…We took four approaches to better understand the targeting specificity of Pex9. First, we looked for additional Pex9 cargos amongst the recently identified yeast peroxisomal proteins (Yifrach et al, 2021) and used one such new protein alongside the known cargo to align the PTS1 and look for similar patterns. Second, we performed site-directed mutagenesis on the PTS1 sequences of known Pex9 and Pex5 cargos and looked at their effect on Pex9 dependent targeting to peroxisomes and physical binding.…”