RESUMO
Selective macro-autophagy is an intracellular process by which large cytoplasmic materials are selectively sequestered and degraded in the lysosomes. Substrate selection is mediated by ubiquitylation and recruitment of ubiquitin-binding autophagic receptors such as p62, NBR1, NDP52 and Optineurin. Although it has been shown that these receptors act cooperatively to target some types of substrates to nascent autophagosomes, their precise roles are not well understood. We examined selective autophagic degradation of peroxisomes (pexophagy), and found that NBR1 is necessary and sufficient for pexophagy. Mutagenesis studies of NBR1 showed that the amphipathic α-helical J domain, the ubiquitin-associated (UBA) domain, the LC3-interacting region and the coiled-coil domain are necessary to mediate pexophagy. Strikingly, substrate selectivity is partly achieved by NBR1 itself by coincident binding of the J and UBA domains to peroxisomes. Although p62 is not required when NBR1 is in excess, its binding to NBR1 increases the efficiency of NBR1-mediated pexophagy. Together, these results suggest that NBR1 is the specific autophagy receptor for pexophagy.
Assuntos
Autofagia/fisiologia , Peroxissomos/metabolismo , Proteínas/metabolismo , Western Blotting , Linhagem Celular , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia Confocal , Microscopia Eletrônica , Peroxissomos/ultraestruturaRESUMO
Peroxisomes are metabolic organelles necessary for anabolic and catabolic lipid reactions whose numbers are highly dynamic based on the metabolic need of the cells. One mechanism to regulate peroxisome numbers is through an autophagic process called pexophagy. In mammalian cells, ubiquitination of peroxisomal membrane proteins signals pexophagy; however, the E3 ligase responsible for mediating ubiquitination is not known. Here, we report that the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy. Expression of PEX2 leads to gross ubiquitination of peroxisomes and degradation of peroxisomes in an NBR1-dependent autophagic process. We identify PEX5 and PMP70 as substrates of PEX2 that are ubiquitinated during amino acid starvation. We also find that PEX2 expression is up-regulated during both amino acid starvation and rapamycin treatment, suggesting that the mTORC1 pathway regulates pexophagy by regulating PEX2 expression levels. Finally, we validate our findings in vivo using an animal model.