The peroxisome counteracts oxidative stresses by suppressing catalase import via Pex14 phosphorylation
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Abstract
Most of peroxisomal matrix proteins including a hydrogen peroxide (H 2 O 2 )-decomposing enzyme, catalase, are imported in a peroxisome-targeting signal type-1 (PTS1)-dependent manner. However, little is known about regulation of the membrane-bound protein import machinery. Here, we report that Pex14, a central component of the protein translocation complex in peroxisomal membrane, is phosphorylated in response to oxidative stresses such as H 2 O 2 in mammalian cells. The H 2 O 2 -induced phosphorylation of Pex14 at Ser232 suppresses peroxisomal import of catalase in vivo and selectively impairs in vitro the interaction of catalase with the Pex14-Pex5 complex. A phosphomimetic mutant Pex14-S232D elevates the level of cytosolic catalase, but not canonical PTS1-proteins, conferring higher cell resistance to H 2 O 2 . We thus suggest that the H 2 O 2 -induced phosphorylation of Pex14 spatiotemporally regulates peroxisomal import of catalase, functioning in counteracting action against oxidative stress by the increase of cytosolic catalase.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on April 18, 2020, follows.
Summary
This study investigates whether peroxisomal import is regulated by phosphorylation. The authors initially identify peroxide-triggered phosphorylation on Pex14 using PhosTag gels, then identify the sites, and use mutations to probe their importance. The key discovery is that S232 on Pex14 is phosphorylated (among other sites) in response to H2O2, that a phosphomimetic mutation of this site impairs catalase import, and that the reduced import of catalase during H2O2 treatment is important to maintaining viability during the stress. In vitro interaction studies suggest that the phosphomimetic Pex14 mutant can bind Pex5L, but does not form a stable ternary complex with Pex5L and catalase. The other …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on April 18, 2020, follows.
Summary
This study investigates whether peroxisomal import is regulated by phosphorylation. The authors initially identify peroxide-triggered phosphorylation on Pex14 using PhosTag gels, then identify the sites, and use mutations to probe their importance. The key discovery is that S232 on Pex14 is phosphorylated (among other sites) in response to H2O2, that a phosphomimetic mutation of this site impairs catalase import, and that the reduced import of catalase during H2O2 treatment is important to maintaining viability during the stress. In vitro interaction studies suggest that the phosphomimetic Pex14 mutant can bind Pex5L, but does not form a stable ternary complex with Pex5L and catalase. The other phosphorylation sites seem to have less of an effect, but may affect other PTS1 import substrates. The primary conceptual advance here is that peroxisome import machinery can be regulated by phosphorylation to affect import of some, but not other substrates. The referees agreed that the conceptual advance of identifying a new regulatory aspect of peroxisomal import is appropriate for publication in eLife, but that the data are currently insufficiently complete to fully support the manuscript's claims.
Essential Revisions
The mechanism proposed by the authors for regulation of catalase import involves Pex14 phosphorylation. Yet it is Pex5 that recognizes catalase in the cytosol and is required for chaperoning catalase across the peroxisome membrane. Thus, to understand the mechanism of regulation, their crucial in vitro experiments examining substrate-Pex5-Pex14 interactions need to use the appropriate substrate-Pex5 complexes. Mammals have two Pex5's, a short Pex5 responsible for PTS1 import, and Pex5L, which binds to Pex7 and helps guide PTSII-containing proteins into the peroxisome. The authors do not provide any justification in the manuscript for why Pex5L was used in the in vitro binding experiments, and they do not provide any comparative experiments using the short Pex5. The authors must address this concern in order to justify the extrapolation of the in vitro experiments to the situation in cells.
The phospho-serine rich site identified by the authors is predicted to be a PEST sequence by bioinformatic searches using the sequence for rat Pex14. PEST sequences are typically found on short-lived proteins and act as a signal for turnover by the proteasome or calcium-dependent calpain proteases. In several instances, Fig 1B, Fig 2A, Fig 2D, etc. it appears that oxidative stress results in a reduction of Pex14, consistent with a hypothesis that this proline and serine rich site is functioning like a PEST sequence. In Fig 4F, phosphorylated Pex14 is detected in the cytosolic fraction, which the authors claim is non-specific. An alternative explanation is that Pex14 is being extracted from the peroxisome and turned over upon H2O2 treatment. The dynamics of Pex14 turnover and its contribution to peroxisome import dynamics is not explored by the authors, but has important implications for their hypothesis. The authors should carefully consider the possibility that phosphorylation regulates Pex14 turnover, which impacts import dynamics. If the authors have data on the turnover of Pex14 and its mutants under different conditions, this would be important to include. At the very least, this alternative explanation for regulation should be discussed in a revised manuscript.
The microscopy experiments present in Figures 3 and 4 are not very convincing and are incomplete. It is difficult to see catalase in the cytosol in the S-to-D mutants. The control images stained for SKL are not shown, confounding the analysis. Further, the localization of the Pex14 mutants, while appearing punctate in the images, was not confirmed by colocalization with another PMP. Finally, equal expression of the different mutants relative to wild type was not verified (e.g., by SDS-PAGE analysis of parallel transfections). To make the experiment more complete, control SKL images need to be presented, the subcellular localization of the Pex14 mutants verified by colocalization with another PMP, and equal expression of the mutants verified by either quantification of the microscopy or SDS-PAGE.
Loading controls for the experiment in Figure 4D are needed to make this fully interpretable. Quantification of EGFP-PTS1 and HA-catalase in Figure 5C would be helpful to a reader.
Figure 4F is not convincing because the differences claimed are not very easy to appreciate and the degree of reproducibility of the small effects is not clear. To be convincing, this experiment needs to be quantified from multiple replicates and should be accompanied by Total samples to show the levels of the proteins in each sample before fractionation.
The anti-His blot in Fig. 2D is of poor quality and cannot be interpreted with confidence. The Pex14 blot is clear, but is complicated by co-expression and partial co-migration of endogenous and exogenous Pex14 species. This experiment would be improved by either improving the quality of the anti-His blot, or perhaps if the authors preformed a His-pulldown followed by blotting to selectively visualize the exogenous proteins. The other option is to perform the experiment in cells lacking endogenous Pex14. Regardless of the approach taken, the authors should improve the quality of this important figure.
The claimed role of Pex13 is not clear from the results in Figure 5A. This experiment can be improved if the authors perform IP with phospho-specific antibody to substantiate the claim that Phosphorylation of Pex14 alters it complex formation with Pex13. Alternatively an IP via Pex13 could also be performed and show that the pS232 is coming down with Pex13.
The conclusion that phosphorylation is ERK mediated has been shown with a single inhibitor and should be extended to show this more directly by checking Pex14 in ERK KO or siRNA cells.
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