The function of ER-phagy receptors is regulated through phosphorylation-dependent ubiquitination pathways

Abstract

Selective autophagy of the ER (ERphagy) is an important regulator of ER remodeling and critical to maintain cellular homeostasis upon environmental changes. ERphagy receptors link the ER with autophagic membrane thus regulating ERphagy flux. We recently showed that members of the FAM134 family play overlapping and distinct roles during stress-induced ERphagy. Yet the mechanisms on how they are activated remain largely unknown. In this study we analyzed mTOR-mediated dynamic phosphorylation of FAM134 as a trigger of FAM134-driven ERphagy. An unbiased screen of kinase inhibitors revealed CK2 to be essential for FAM134B- and FAM134C-driven ERphagy upon mTOR inhibition. Identified dynamic phosphorylation sites on FAM134C in cells were fitting with predicted CK2 targeting sites, indicating a direct regulatory role of CK2 in FAM134-driven ERphagy. Using super-resolution microscopy, we showed that activity of CK2 is essential for the formation of high-density clusters of FAM134B and FAM134C. Consistently, FAM134B and FAM134C proteins carrying point mutations of selected Serin residues, within their reticulon homology domain, are unable to form high-density clusters. In addition, we provide evidence that the ubiquitination machinery is required for ERphagy and that FAM134B and FAM134C clustering is activated by phospho-dependent ubiquitination. Treatment with CK2 inhibitor SGC-CK2-1 prevents Torin1-induced ERphagy flux as well as ubiquitination of FAM134 proteins and consistently, treatment with E1 inhibitor suppresses Torin1-induced ERphagy flux. Therefore, we propose CK2 dependent phosphorylation of ERphagy receptors precedes ubiquitin-dependent ERphagy flux activation.

Increasing speed and brightness in super-resolution microscopy with renewable fluorophores

“…The hybridization kinetics can be used to read out molecule numbers 15 , e.g. in protein nano-clusters 16,17 . The combination of DNA-based exchangeable fluorophore labels and neural networks for high-emitter mapping enabled fast and multi-target DNA-PAINT microscopy in fixed tissue samples 18 .…”

ER-phagy Receptor’s Intrinsically Disordered Modules Drive ER Fragmentation and ER-phagy

“…The ERphagy receptors are all composed of a membrane-tethering module and of a cytoplasmic intrinsically disordered region (IDR) containing a LC3 interacting motif (LIR) (Adriaenssens et al, 2022;Chino & Mizushima, 2023;Conway et al, 2020;Ferro-Novick et al, 2021;Hubner & Dikic, 2020;Johansen & Lamark, 2020;Lamark & Johansen, 2021;Mizushima, 2022;Popelka & Uversky, 2022;Reggiori & Molinari, 2022). Seminal studies performed to understand the mode-of-action of mammalian ER-phagy receptors of the FAM134 family (FAM134A, FAM134B, FAM134C) established that their phosphorylation, ubiquitylation, and homo-/hetero-oligomerization control ER fragmentation and lysosomal clearance (Berkane et al, 2023;Di Lorenzo et al, 2022;Foronda et al, 2023;Gonzalez et al, 2023;Iavarone et al, 2022;Jiang et al, 2020;Khaminets et al, 2015;Reggio et al, 2021). FAM134 proteins are peculiar ER-phagy receptors because they possess a RHD (light blue in Fig.…”

Reticulon dependent ER-phagy mediates adaptation to heat stress inC. elegans

“…Data in yeast, plant and mammals have shown that reticulons and RHD containing proteins have SAR function for reticulophagy during ER stress (Mochida & Nakatogawa, 2022;Chino & Mizushima, 2023;Reggiori & Molinari, 2022). The mammalian FAM134B and RTN3 proteins participate in remodeling the ER network and the elimination of specific domains by reticulophagy (González et al, 2023;Berkane et al, 2023;Khaminets et al, 2015;Grumati et al, 2017). These pioneer works have raised numerous questions on the functions of these SAR concerning the specificity of the isoforms, their interactions with LC3 and GABARAP members, the categories of ER cargoes and the relations with the types of stress.…”