Dataset Information

Proteomic analysis of peroxisome-associated PEX5 mutants in Xenopus egg extract.

ABSTRACT: This project sought to identify the features in PEX5 required for interaction with the peroxisomal PEX2-PEX10-PEX12 ubiquitin ligase complex. Peroxisomes equilibrated with different PEX5 mutants (i.e., C11A, AH1, AH2) were purified from Xenopus egg extract, solubilized in digitonin, and PEX5-associated material was immunoprecipitated via a C-terminal FLAG tag. PEX5 without a FLAG tag was used as a negative control. PEX5-interacting peroxins were identified by LC-MS/MS at the Taplin Mass Spectrometry Facility at Harvard Medical School.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Xenopus Laevis (african Clawed Frog)

TISSUE(S): Egg

SUBMITTER: Michael Leszek Skowyra

LAB HEAD: Tom Abraham Rapoport

PROVIDER: PXD035105 | Pride | 2022-08-16

REPOSITORIES: pride

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Publications

PEX5 translocation into and out of peroxisomes drives matrix protein import.

Skowyra Michael L ML Rapoport Tom A TA

Molecular cell 20220804 17

Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal enzymes are imported from the cytosol by the receptor PEX5, which interacts with a docking complex in the peroxisomal membrane and then returns to the cytosol after monoubiquitination by a membrane-embedded ubiquitin ligase. The mechanism by which PEX5 shuttles between cytosol and peroxisomes and releases cargo inside the lumen is unclear. Here, we use Xenopus egg extract to demonstrate that PEX ...[more]

PMID: 35931083

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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.

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