Chaperone-like protein p32 regulates ULK1 stability and autophagy

Abstract

Mitophagy mediates clearance of dysfunctional mitochondria, and represents one type of mitochondrial quality control, which is essential for optimal mitochondrial bioenergetics. p32, a chaperone-like protein, is crucial for maintaining mitochondrial membrane potential and oxidative phosphorylation. However, the relationship between p32 and mitochondrial homeostasis has not been addressed. Here, we identified p32 as a key regulator of ULK1 stability by forming complex with ULK1. p32 depletion potentiated K48-linked but impaired K63-linked polyubiquitination of ULK1, leading to proteasome-mediated degradation of ULK1. As a result, silencing p32 profoundly impaired starvation-induced autophagic flux and the clearance of damaged mitochondria caused by mitochondrial uncoupler. Importantly, restoring ULK1 expression in p32-depleted cells rescued autophagy and mitophagy defects. Our findings highlight a cytoprotective role of p32 under starvation conditions by regulating ULK1 stability, and uncover a crucial role of the p32-ULK1-autophagy axis in coordinating stress response, cell survival and mitochondrial homeostasis. Mitophagy is a selective form of autophagy by which mitochondria are degraded in autolysosomes. p32 is a critical regulator of mitochondrial bioenergetics.1 It primarily localizes to the mitochondrial matrix, but has also been reported to be present in other subcellular locations.2-5 Many human tumors exhibit higher p32 expression levels than their nonmalignant counterpart tissues.6-9 Depleting p32 in human cancer cells strongly shifts their metabolism from oxidative phosphorylation to glycolysis.1 Consistently, p32 knockout causes mid-gestation lethality of knockout embryos and defects in oxidative phosphorylation. Mouse embryonic fibroblasts (MEFs) generated from p32 knockout embryos exhibited impaired ATP production and reduced mitochondrial membrane potential, which is in agreement with the observation that p32 silencing leads to increased mitochondrial fragmentation.10,11 Notably, p32 was found to form protein complex with a variety of molecules 7,12,13 and has been suggested that it may act as a multifunctional chaperone protein. [12][13][14] ULK1 has a crucial role in mitophagy induction. 15 Despite the pivotal role of ULK1 in mitochondrial clearance, little is known as how ULK1 itself is regulated. ULK1 is a relatively stable protein and is subject to proteasome-mediated degradation. Post-translational modifications including K63-linked ubiquitylation 16,17 and phosphorylation [18][19][20] have been reported to modulate the rates of ULK1 turnover and kinase activity in different cellular contexts. Hsp90 and Cdc37 have been shown to regulate ULK1 stability and activity by forming complex with ULK1, which subsequently influences Atg13-mediated mitophagy. 21 Here, we found p32 regulates ULK1 stability by forming protein complex with ULK1. The interaction between ULK1 and p32 is crucial for maintaining the steady-state levels and activity of ULK1. We further show that p32 ablation results in a defec...

Mitochondrial nucleic acid binding proteins associated with diseases

“…Silencing p32 profoundly impairs starvation-induced autophagic flux and clearance of damaged mitochondria caused by a mitochondrial uncoupler. These findings support a cytoprotective role for p32 under starvation conditions by regulation of ULK1 stability, and reveal a crucial involvement of the p32-ULK1-autophagy axis in coordinating the stress response, cell survival and mitochondrial homeostasis (69). Mammalian RNase H1 is implicated in mitochondrial DNA replication and RNA processing and is required for embryonic development.…”

Role of Host Cell p32 in Herpes Simplex Virus 1 De-Envelopment during Viral Nuclear Egress

“…The interaction of UL47 with p32 was confirmed in HSV-1-infected cells: p32 coprecipitated with MEF-tagged UL47 in lysates of cells infected with recombinant virus YK536 (MEF-UL47). p32 is a ubiquitous protein in most cell types and a multifunctional protein regulating various cellular functions, including apoptosis, RNA splicing, mitochondrial translation and metabolism, and autophagy (27,(59)(60)(61)(62). We also found that p32 accumulated at the nuclear rim in HSV-1-infected cells and that UL47 was required for the p32 accumulation.…”

A molecular perspective of mammalian autophagosome biogenesis

“…Furthermore, the chaperone protein p32 binds ULK1 and, acting by an unknown E3 ligase, inhibits Lys-48-linked ubiquitination while driving Lys-63-linked ubiquitination. This promotes ULK1 stability and is crucial in both starvation-induced autophagy and mitophagy ( 28 ), the selective removal of mitochondria. In another context, ubiquitination negatively regulates ULK1 signaling ( 29 – 31 ).…”