Selective autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is an important regulator of ER remodeling and essential to maintain cellular homeostasis during environmental changes. We recently showed that members of the FAM134 family play a critical role during stress-induced ER-phagy. However, the mechanisms on how they are activated remain largely unknown. In this study, we analyze phosphorylation of FAM134 as a trigger of FAM134-driven ER-phagy upon mTOR (mechanistic target of rapamycin) inhibition. An unbiased screen of kinase inhibitors reveals CK2 to be essential for FAM134B- and FAM134C-driven ER-phagy after mTOR inhibition. Furthermore, we provide evidence that ER-phagy receptors are regulated by ubiquitination events and that treatment with E1 inhibitor suppresses Torin1-induced ER-phagy flux. Using super-resolution microscopy, we show that CK2 activity is essential for the formation of high-density FAM134B and FAM134C clusters. In addition, dense clustering of FAM134B and FAM134C requires phosphorylation-dependent ubiquitination of FAM134B and FAM134C. Treatment with the CK2 inhibitor SGC-CK2-1 or mutation of FAM134B and FAM134C phosphosites prevents ubiquitination of FAM134 proteins, formation of high-density clusters, as well as Torin1-induced ER-phagy flux. Therefore, we propose that CK2-dependent phosphorylation of ER-phagy receptors precedes ubiquitin-dependent activation of ER-phagy flux.

Selective macroautophagy/autophagy of the endoplasmic reticulum, known as reticulophagy/ER-phagy, is essential to maintain ER homeostasis. We recently showed that members of the autophagy receptor family RETREG/FAM134 are regulated by phosphorylation-dependent ubiquitination. In an unbiased screen we had identified several kinases downstream of MTOR with profound impact on reticulophagy flux, including ATR and CSNK2/CK2. Inhibition of CSNK2 by SGC-CK2-1 prevented regulatory ubiquitination of RETREG1/FAM134B and RETREG3/FAM134C upon autophagy activation as well as the formation of high-density RETREG1- and RETREG3-clusters. Here we report on additional resource data of global proteomics upon CSNK2 and ATR inhibition, respectively. Our data suggests that the function of CSNK2 is mainly limited to the ER/reticulophagy and Golgi/Golgiphagy, while ATR inhibition by VE-822 affects the vast majority of organelles/selective autophagy pathways.

Calcium (Ca²⁺) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca²⁺ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca²⁺ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca²⁺ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.

Legionella pneumophila (LP) secretes more than 300 effectors into the host cytosol to facilitate intracellular replication. One of these effectors, SidH, 253 kDa in size with no sequence similarity to proteins of known function is toxic when overexpressed in host cells. SidH is regulated by the LP metaeffector LubX which targets SidH for degradation in a temporal manner during LP infection. The mechanism underlying the toxicity of SidH and its role in LP infection are unknown. Here, we determined the cryo-EM structure of SidH at 2.7 Å revealing a unique alpha helical arrangement with no overall similarity to known protein structures. Surprisingly, purified SidH came bound to a E. coli EF-Tu/t-RNA/GTP ternary complex which could be modeled into the cryo-EM density. Mutation of residues disrupting the SidH-tRNA interface and SidH-EF-Tu interface abolish the toxicity of overexpressed SidH in human cells, a phenotype confirmed in infection of Acanthamoeba castellani. We also present the cryo-EM structure of SidH in complex with a U-box domain containing ubiquitin ligase LubX delineating the mechanism of regulation of SidH. Our data provide the basis for the toxicity of SidH and into its regulation by the metaeffector LubX.

The endoplasmic reticulum (ER) constitutes a sophisticated network of membranes that commands diverse molecular processes comprising protein folding, lipid synthesis, and calcium storage. Safeguarding the ER's integrity against insults, achieved through a process known as ER-phagy, is imperative for optimal functionality at both the individual protein and organelle levels. Dysregulation in ER-phagy has been associated with various human diseases, including hereditary sensory and autonomic neuropathy (HSAN). The exploration of ER-phagy gained momentum with the discovery of ER-phagy receptors, exemplified by FAM134B—the first identified mammalian ER-phagy receptor. FAM134B’s interaction with autophagy modifiers forms molecular tethers, connecting the ER with autophagic membranes. The clustering of FAM134B with other reticulon homology domain (RHD) -containing proteins amplifies the capacity, versatility, and variability of ER remodeling, thereby facilitating adaptive responses to diverse cellular requirements. Belonging to the FAM134 protein family, FAM134B shares an LC3-interacting region (LIR) amino acid sequence with FAM134A and FAM134C. However, the limited understanding of the coordinated functions of FAM134 family members necessitates a detailed investigation into the molecular mechanism and regulation of these proteins. In this work, I present findings highlighting the role of each of the individual FAM134 proteins as well as the molecular mechanisms governing their ER-phagy functions. To this end, an interdisciplinary approach was employed to explore the dynamics within FAM134 protein functions and how clustering and post-translational modification regulate FAM134-dependent ER remodeling and the selective degradation of ER components at both macro- and nanoscale resolutions. The activity of both FAM134B and FAM134C can be influenced by inhibiting mTOR, the master kinase of autophagy, using Torin1. Employing a chemical screening approach, several downstream kinases – in particular CK2, emerge as crucial for Torin1-induced ER-phagy driven by FAM134B and FAM134C. The investigation underscores the fundamental role of phosphorylation sites within the RHD of FAM134 proteins in inducing ER-phagy upon Torin1 treatment. Furthermore, we demonstrate that the phosphorylation of FAM134B and FAM134C is essential for their subsequent ubiquitination and the formation of high-density receptor clusters. This suggests that the phosphorylation-dependent ubiquitination of both FAM134 proteins acts as the driving force behind the formation of high-density clusters at active ER-phagy sites. These findings provide valuable insights into the regulatory mechanisms governing FAM134B and FAM134C in the context of ER-phagy, emphasizing the interplay between phosphorylation, ubiquitination, and cluster formation in the complex regulation of ER remodeling. Overall, these discoveries expand our understanding of the molecular principles behind ER- phagy activation and may pave the way for the development of novel strategies to modulate ER-phagy, offering new avenues for therapeutic exploration. Ultimately, the investigation of interconnected cellular processes and the intricate regulatory networks of FAM134 proteins' functions may serve as a cornerstone for future studies, fostering advancements in our understanding of cellular physiology and pathology.