Abstract
Peroxisomes perform beta-oxidation of branched and very-long chain fatty acids, which leads to the formation of reactive oxygen species (ROS) within the peroxisomal lumen. Peroxisomes are therefore prone to ROS-mediated damages. Here, using light to specifically and acutely induce ROS formation within the peroxisomal lumen, we find that cells individually remove ROS-stressed peroxisomes through ubiquitin-dependent pexophagy. Heat shock protein 70 s mediates the translocation of the ubiquitin E3 ligase Stub1 (STIP1 Homology and U-Box Containing Protein 1) onto oxidatively-stressed peroxisomes to promote their selective ubiquitination and autophagic degradation. Artificially targeting Stub1 to healthy peroxisomes is sufficient to trigger pexophagy, suggesting a key role Stub1 plays in regulating peroxisome quality. We further determine that Stub1 mutants found in Ataxia patients are defective in pexophagy induction. Dysfunctional peroxisomal quality control may therefore contribute to the development of Ataxia.
Highlights
Peroxisomes perform beta-oxidation of branched and very-long chain fatty acids, which leads to the formation of reactive oxygen species (ROS) within the peroxisomal lumen
We found that Stub[1] mutants discovered in Ataxia patients are deficient in their ability to trigger this form of pexophagy, suggesting an interesting link between defective peroxisomal quality control and Ataxia
We validated that diKillerRed-PTS1 properly localized into peroxisomes in NIH3T3 cells, the model cell line used in this study (Fig. 1d, top panels)
Summary
Peroxisomes perform beta-oxidation of branched and very-long chain fatty acids, which leads to the formation of reactive oxygen species (ROS) within the peroxisomal lumen. With the help of EGFP-tagged reporter constructs, we found that ROS-stressed peroxisomes (illuminated with 559 nm light) accumulated ubiquitination (together with a loss of diKillerRed-PTS1 fluorescence due to photobleaching), a characteristic response frequently observed in organelle quality control pathways (Fig. 1d, bottom panels, white arrows)[10]. We elicited peroxisomal ROS generation, and through timelapse imaging found that an ubiquitin E3 ligase reporter construct EGFP-Stub[1] translocated to the ROS-stressed peroxisomes (Fig. 3a, b; white arrowheads).
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