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A Mitochondria-Driven Quality Control Pathway Eliminates Mislocalized Peroxisomal Membrane Proteins

ABSTRACT: Protein quality control (PQC) systems are essential for maintaining a healthy proteome by removing aberrant proteins, including mislocalized proteins. However, the mechanisms for eliminating mislocalized Peroxisomal Membrane Proteins (PMPs) have remained elusive. Here, using quantitative proteomics in peroxisome biogenesis–deficient cells, we observed widespread downregulation of PMPs without corresponding changes in mRNA levels, indicating selective post-transcriptional turnover. An unbiased CRISPR screen uncovered an inter-organelle PQC axis in which mitochondria act as a surveillance hub to clear mislocalized PMPs, thereby safeguarding cellular proteostasis when peroxisome biogenesis fails. We show that when PMPs fail to reach their native peroxisomal destination, they are rerouted to mitochondria, where the mitochondrial outer membrane resident E3 ligases MUL1 and MARCH5 act redundantly to ubiquitinate them and promote their degradation via the proteasome. Our findings reveal an additional regulatory layer of organelle cross-talk, highlighting a dual role for mitochondrial E3 ligases, previously implicated in de novo peroxisome biogenesis, in actively participating in peroxisomal protein quality control. Notably, simultaneous loss of peroxisomes and mitochondrial E3 ligases markedly impairs cell survival, highlighting the essential role of this pathway. These findings provide critical insights into the pathology of organelle dysfunction, including peroxisome biogenesis disorders, and reveal broader mechanisms of inter-organelle protein quality control.

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ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Keren Bendalak

LAB HEAD: Itay Koren

PROVIDER: PXD069489 | Pride | 2026-05-14

REPOSITORIES: Pride

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Project description:Several proteomics studies have been performed in the past in order to define the mammalian peroxisomal proteome. During the last decade, it has as well become evident, that a significant number of proteins is shared between several subcellular compartments and that specialized proteins subsets are constituents of membrane contact zones, which bridge two adjacent organelle to allow metabolite exchange. In this respect, a number of proteins, which have been considered as mere contaminants in previous studies, might be indeed proteins, which are to a minor extent indeed truly associated with peroxisomes. Ongoing technical improvements in mass spectrometry (MS) allow to identify and to quantify the enrichment of such less abundant proteins in individual fractions. Accordingly, this study reassessed the proteome of mouse liver peroxisomes by parallel isolation of peroxisomes from a mitochondria- and a microsome-enriched pre-fraction combining density gradient centrifugation with a SWATH-MS quantitative proteomics approach to unveil novel peroxisomal or peroxisome-associated proteins. In total, 1071 proteins from the MS-samples were identified in amounts, which allowed their quantification in order to assess their enrichment in either high density peroxisomal or low-density gradient fractions, containing the bulk of organelle material. Combining the data from isolations from the mitochondria- and microsome-enriched prefractions allowed to identify specific protein clusters characteristic for mitochondria, the ER and peroxisomes. Among the proteins, which were significantly enriched in the peroxisomal cluster, were several proteins, which were not previously associated with the organelles implying that they are novel peroxisomal candidates. In order to validate the organelle proteomics strategy, five of the candidates were selected for further colocalization studies. The peroxismal import of two candidates, HTATIP2 and PAFAH2, which contain potential peroxisome targeting sequences 1, could be confirmed by overexpression in HepG2 cells. Two other candidates, SAR1B and PDCD6, which are known from ER exit sites, did not directly colocalize with peroxisomes but were found to reside at ER sites which often surrounded peroxisomes. Hence, both proteins might concentrate at peroxisome-ER membrane contact sites, which might be co-purified with peroxisomes. Intriguingly, the last candidate, OCIA domain-containing protein 1, was previously described to decrease mitochondrial branching and network formation. In this work, we not only validate its secondary peroxisomal localization but also observed a reduction in peroxisome numbers in response OCIAD1 expression. Hence, OCIAD1 appears to be a novel protein, which has an impact on both mitochondrial and peroxisomal maintenance.

Project description:Peroxisome biogenesis diseases (PBDs) are characterized by global defects in peroxisomal function and can result in severe brain, liver, kidney, and bone malfunctions. PBDs are due to mutations in peroxisome biogenesis factors (PEX genes) that are responsible for peroxisome assembly and function. Increasing evidence suggests that peroxisome import functions decline during aging. However, the transcriptome profiling of peroxisome import defects and how they affect disease development are still lacking. PEX5 encodes the cytoplasmic receptors for peroxisome-targeting signal types 1. We generate knock-in human HEK293 cells mutant using CRISPR to transiently express PEX5 cysteine 11 to alanine mutant (PEX5C11A), which blocks PEX5 recycling and exerts dominant negative effect on PEX5 mediated peroxisome import. To identify conserved responses, we perform transcriptomic analysis on Drosophila oenocyte-specific Pex1, Pex12 and Pex5 knockdowns and on human cells with impaired peroxisome import (through expressing PEX5C11A and applying PEX5 siRNA respectively). PEX5C11A induction triggers vast transcriptomic changes, including decreased oxidative phosphorylation, increased MAPK signaling and HIPPO signaling. PEX5 siRNA specifically decreases spliceosome activity and increases cholesterol metabolism. Using gene set enrichment analysis (GSEA), we identify protein processing in endoplasmic reticulum pathway, specifically ER-associated protein degradation (ERAD) pathway is induced in all PEX knockdowns in Drosophila. Peroxisome dysfunction elevates eIF2α phosphorylation in both Drosophila and human cell culture independent of XBP1 activation, suggesting increased integrative stress response (ISR). Moreover, peroxisome stress decreases ribosome biogenesis genes and impairs ribosome biogenesis in flies and human cells. Specifically, peroxisome stress impairs the 5'-ETS cleavage activity during the ribosome biogenesis and dampens 40S small ribosomal export in both flies and human. Our results suggest that reduced ribosome biogenesis and elevated ISR could be conserved cellular response to peroxisome import stress.

			Action	DRS
	92502-13.zip	Other
	Seq92502_DIA_480Ex2.raw	Raw
	Seq92503_DIA_480Ex2.raw	Raw
	Seq92504_DIA_480Ex2.raw	Raw
	Seq92505_DIA_480Ex2.raw	Raw

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A Mitochondria-Driven Quality Control Pathway Eliminates Mislocalized Peroxisomal Membrane Proteins

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