p62/SQSTM1 promotes rapid ubiquitin conjugation to target proteins after endosome rupture during xenophagy

Abstract

Autophagy is a bulk degradation pathway, and selective autophagy to remove foreign entities is called xenophagy. The conjugation of ubiquitin to target pathogens is an important process in xenophagy but when and where this ubiquitination occurs remains unclear. Here, we analyzed the temporal sequence and subcellular location of ubiquitination during xenophagy using time‐lapse observations, with polystyrene beads mimicking invading pathogens. Results revealed accumulation of a ubiquitination marker around the beads within 3 min after endosome rupture. Recruitment of ubiquitin to the beads was significantly delayed in p62‐knockout murine embryonic fibroblast cells, and this delay was rescued by ectopic p62 expression. Ectopic expression of a phosphorylation‐mimicking p62 mutated at serine residue 405 (equivalent to human serine residue 403) rescued this delay, but its unphosphorylated form did not. These results indicate that ubiquitination mainly occurs after endosome rupture and suggest that p62, specifically the phosphorylated form, promotes ubiquitin conjugation to target proteins in xenophagy.

The Role of Autophagy in Acute Myocardial Infarction

“…It is recognized as the only autophagy process that allows selective degradation of soluble cytosolic proteins in lysosomes (Alfaro et al., 2018). In addition to those three kinds of classic autophagy, some special forms of autophagy (selective autophagy) have been described, including mitophagy, pexophagy, ribophagy, xenophagy, and secretory autophagy (Ponpuak et al., 2015; Mao and Klionsky, 2017; An and Harper, 2018; Broda et al., 2018; Tsuchiya et al., 2018). Since macroautophagy is the most extensively studied form of autophagy, here in this review, the role of macroautophagy in acute myocardial infarction will be discussed (hereafter referred to as “autophagy”).…”

Anaplasmataceae: Dichotomous Autophagic Interplay for Infection

“…Xenophagy is a selective autophagy whereby intracellular pathogens are tag ge d by ubiq ui tin a n d subse quen t targe tin g to autophagosomes for degradation in autolysosomes. Autophagy receptors such as p62/SQSTM1, nuclear domain 10 protein 52 (NDP52) and neighbor of BRCA1 gene 1 (NBR1) have been shown to bind ubiquitinated intracellular pathogens for autolysosome destruction and clearance (31). Autophagy plays an important role in both innate and adaptive immunity to various intracellular pathogens including Mycobacterium tuberculosis, Streptococcus pyogenes, Listeria monocytogenes, and Salmonella enterica (38)(39)(40)(41)(42).…”

“…This leads to the conjugation of LC3-I to phosphatidylethanolamine (PE) to form LC3-II. The p62/SQSTM1 (sequestosome 1) protein acts as a cargo receptor for ubiquitinated targets which are transported to the autophagosome for degradation (30,31). Following phagophore expansion, the phagophore is completely sealed, forming the double membrane autophagosome containing all targeted components.…”

Microtubule inhibitors identified through non-biased screening enhance DNA transfection efficiency by delaying p62-dependent ubiquitin recruitment

“…The pHrodo-conjugated beads were incorporated into MEF cells expressing a GFPfused Ub protein (GFP-Ub MEF cells) 12 , and the assembly of GFP-Ub around the beads was observed in a living cell using time-lapse fluorescence microscopy (Fig. 4B).…”

“…It has been reported that the phosphorylated form of p62 at the amino acid residue S405 (p62 S405) is required for Ub recruitment in the process of selective autophagy 12 .…”