Export-deficient monoubiquitinated PEX5 triggers peroxisome removal in SV40 large T antigen-transformed mouse embryonic fibroblasts

Abstract

Peroxisomes are ubiquitous cell organelles essential for human health. To maintain a healthy cellular environment, dysfunctional and superfluous peroxisomes need to be selectively removed. Although emerging evidence suggests that peroxisomes are mainly degraded by pexophagy, little is known about the triggers and molecular mechanisms underlying this process in mammalian cells. In this study, we show that PEX5 proteins fused to a bulky C-terminal tag trigger peroxisome degradation in SV40 large T antigen-transformed mouse embryonic fibroblasts. In addition, we provide evidence that this process is autophagy-dependent and requires monoubiquitination of the N-terminal cysteine residue that marks PEX5 for recycling. As our findings also demonstrate that the addition of a bulky tag to the C terminus of PEX5 does not interfere with PEX5 monoubiquitination but strongly inhibits its export from the peroxisomal membrane, we hypothesize that such a tag mimics a cargo protein that cannot be released from PEX5, thus keeping monoubiquitinated PEX5 at the membrane for a sufficiently long time to be recognized by the autophagic machinery. This in turn suggests that monoubiquitination of the N-terminal cysteine of peroxisome-associated PEX5 not only functions to recycle the peroxin back to the cytosol, but also serves as a quality control mechanism to eliminate peroxisomes with a defective protein import machinery.

Pexophagy and peroxisomal protein turnover in plants

“…PEX5 is ubiquitinated in yeast and mammals as it is recycled back into the cytosol for further import rounds [9], making ubiquitinated PEX5 an attractive candidate pexophagy signal. Indeed, preventing efficient retrotranslocation of PEX5 by addition of a bulky C-terminal tag can trigger peroxisome degradation via autophagy in a mammalian cell line [79]. In addition, knockdown of the docking protein PEX14 reduces pexophagy in mammals [73], which might indicate that mammalian PEX14 is directly required to bind a pexophagy receptor or that PEX14 is indirectly required because it is needed to import the actual pexophagy signal, be it ubiquitinated PEX5, a PEX5-dependent matrix protein, or a PEX5-dependent process (e.g., oxidative damage).…”

A mammalian pexophagy target

“…Thus the identification of a physiological link between mammalian ubiquitination and pexophagy would constitute a significant advance in the field. Two related studies, by Zhang et al 6 (published in this issue) and Nordgren et al 7 now identify PEX5 as the physiological ubiquitination target that promotes pexophagy.…”

“…This naturally raises the question as to why ubiquitination at these residues in PEX5 does not also target it for pexophagy. Indeed this can happen, as shown by the demonstration that the N-terminal mono-ubiquitination of PEX5 at Cys11 induces pexophagy in SV40 T antigen-transformed mouse embryonic fibroblasts 7 . These authors found that PEX5 fused to a bulky C-terminal tag was delivered to peroxisomes and mono-ubiquitinated at Cys11, but its failure to be exported from peroxisomes triggered pexophagy (Fig.…”

“…One possibility is that ATM kinase is only activated, through ROS-mediated dimerization, on peroxisomes, whereas another may be that the E3 ubiquitin ligases (PEX2, PEX10 and PEX12), which are required for PEX5 ubiquitination are themselves located on peroxisomes. However, one should be cautious about the conclusion that the peroxisome-associated RING E3 ubiquitin ligases are responsible for the mono-ubiquitination at K209, because these E3 ligases are also involved in other ubiquitination reactions on PEX5 7 . The loss of monoubiquitination observed here, presumably at K209, following the downregulation of these E3 ligases, could be a consequence of the fact that PEX5 is not in the right stage, or location, in the matrix protein import cycle, for this modification to occur.…”

“…2). Additionally, the cells used in the two studies are different and there is evidence of cell type-specific, as well as cell immortalization-dependent, variations in pexophagy 7 . An unanswered question from the two studies 6, 7 is whether the mechanisms they describe are true for pexophagy activated by different signaling mechanisms or specific to certain cell types.…”