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Proteome of Fam134A/B/C knock-out MEFs

ABSTRACT: Selective degradation of endoplasmic reticulum (ER) via autophagy receptors is important to maintain cell homeostasis. In this study we identify FAM134A/RETREG2 and FAM134C/RETREG3 as bona fide ER-phagy receptors containing a classical and functional LIR motif. In contrast to the other family member FAM134B/RETREG1, over-expressed FAM134A and C are in a relatively inactive state, under basal conditions, and require an activation signal to fragment significant amounts of ER. Global proteomes analysis of knockout MEFs suggests distinct and overlapping functions of the three FAM134. Common functions are the maintenance of the ER shape and delivery of mis-folded pro-collagen I to lysosomes. Single knockout of Fam134a or Fam134c lead to swollen ER and massive intracellular accumulation of pro-collagen I. In this context, Fam134a but not Fam134c over-expression is able to recover collagen I levels, in Fam134b knockout MEFs, indicating distinct modes of action. Moreover, we provide evidence that Fam134a has a LIR-independent function in maintaining collagen I homeostasis in a co-receptor complex together with Fam134c. This pathway works in parallel to Fam134b, which in contrast is self-sufficient to drive ER-phagy.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Paolo Grumati

LAB HEAD: Paolo Grumati

PROVIDER: PXD021690 | Pride | 2021-06-23

REPOSITORIES: Pride

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			Action	DRS
	20180601_fb_pg_Famprot_KO_A_1.raw	Raw
	20180601_fb_pg_Famprot_KO_A_2.raw	Raw
	20180601_fb_pg_Famprot_KO_A_3.raw	Raw
	20180601_fb_pg_Famprot_KO_B_1.raw	Raw
	20180601_fb_pg_Famprot_KO_B_2.raw	Raw

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Critical functions of FAM134 family members in endoplasmic reticulum remodeling, ER-phagy and collagen quality control

Project description:Degradation of the endoplasmic reticulum (ER) by selective autophagy (ER-phagy) is vital for cellular homeostasis. Here, we introduce FAM134A/RETREG2 and FAM134C/RETREG3 as new ER-phagy receptors. FAM134A and FAM134C exist in a relatively inactive state under basal conditions and require an activation signal to induce significant ER fragmentation. Molecular modeling and simulations implicate a single compact fold of FAM134A’s reticulon homology domain (RHD). In contrast, the RHDs of FAM134B and FAM134C are able to adopt at least three discrete conformations. These differences result in slower vesicle formation for FAM134A compared to FAM134C and mostly FAM134B. Global proteomic analyses of knockout MEFs indicate both distinct and overlapping roles for the Fam134s, with Fam134a being most distant from Fam134b and Fam134c. Fam134c appears to enhance and facilitate Fam134b’s role in maintaining pro-collagen-I levels and is therefore insufficient to compensate for its loss. By contrast, Fam134a has a particular mode of action in maintaining pro-collagen-I levels and is able to fully compensate loss of Fam134b or Fam134c upon over-expression in its wild-type or LIR mutant form.

2021-06-23 | PXD025626 | Pride

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

Project description:Selective autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is an important regulator of ER remodeling and is critical to maintaining cellular homeostasis during environmental changes. We recently showed that members of the FAM134 family play a role during stress-induced ER-phagy. However, the mechanisms on how they are activated remain largely unknown. In this study, we analyzed mTOR-mediated phosphorylation of FAM134 as a trigger of FAM134-driven ER-phagy. An unbiased screen of kinase inhibitors revealed that CK2 is essential for ER-phagy driven by FAM134B and FAM134C after inhibition of mTOR. Using superresolution microscopy, we showed that CK2 activity is essential for the formation of high-density groups of FAM134B and FAM134C. Continually, the FAM134B and FAM134C proteins that carry point mutations of selected serine residues within their reticulon homology domain are unable to form high-density clusters. Furthermore, we provide evidence that ubiquitination regulates ER-phagy receptors and that dense clustering of FAM134B and FAM134C requires events upstream of ubiquitination. Treatment with the CK2 inhibitor SGC-CK2-1 or mutation of phosphosites prevents Torin1-induced ER-phagy flux as well as ubiquitination of FAM134 proteins, and consistently treatment with E1 inhibitor suppresses Torin1-induced ER-phagy flux. Therefore, we propose that CK2 dependent phosphorylation of ER-phagy receptors precedes ubiquitin-dependent activation of ER-phagy flux.

2023-11-16 | PXD043003 | Pride

An mTORC1-CK2 axis controls starvation induced ER-phagy via FAM134C

Project description:The degradation of endoplasmic reticulum (ER) via selective autophagy is driven by the ER-phagy receptors that facilitate the incorporation of ER-fragments into nascent autophagosomes. How these receptors are regulated, in response to ER-phagy-inducing stimuluses, is largely unknown. Here we propose that starvation, as well as mTOR inhibition, triggers ER-phagy primarily through the activation of the ER-phagy receptor FAM134C. In physiological, nutrient reach, conditions FAM134C is phosphorylated by the Casein kinase 2 (CK2) protein at specific residues negatively affecting FAM134C interaction with the LC3 proteins, thereby preventing ER-phagy. Pharmacological or starvation-induced mTORC1 inhibition limits phosphorylation of FAM134C by CK2, hence promoting FAM134C activation and ER-phagy. Moreover, inhibition of CK2 or the expression of a phospho-mutant FAM134C protein is sufficient to stimulate ER-phagy. Conversely, starvation induced ER-phagy is inhibited in cells and mice that lack FAM134C or expressing a phospho-mimetic FAM134C protein. Overall, these data describe a new mechanism regulating ER-phagy and provides an example of cargo selectivity mechanism during starvation induced autophagy.

2022-10-14 | PXD030451 | Pride

Fam134c and Fam134b shape axonal endoplasmic reticulum architecture in vivo.

Project description:Endoplasmic reticulum (ER) remodeling is vital for cellular organization. ER-phagy, a selective autophagy targeting ER, plays an important role in maintaining ER morphology and function. The FAM134 protein family, including FAM134A, FAM134B, and FAM134C, mediates ER-phagy. While FAM134B mutations are linked to hereditary sensory and autonomic neuropathy in humans, the physiological role of the other FAM134 proteins remains unknown. To address this, we investigated the roles of FAM134 proteins using single and combined knockouts (KOs) in mice. Single KO in young mice showed no major phenotypes, however, combined Fam134b and Fam134c deletion (Fam134b/cdKO), but not the combination including Fam134a deletion, led to rapid neuromuscular and somatosensory degeneration, resulting in premature death. Fam134b/cdKO mice show rapid loss of motor and sensory axons in the peripheral nervous system. Long axons from Fam134b/cdKO mice exhibited expanded tubular ER with a transverse ladder-like appearance, whereas no obvious abnormalities were observed in cortical ER. Our study unveils critical roles of FAM134C and FAM134B in the formation of tubular ER network in axons of both motor and sensory neurons.

2024-06-24 | PXD050983 | Pride

MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B

Project description:Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of ER homeostasis and function1. The selective incorporation of ER fragments into nascent autophagosomes is facilitated by ER resident proteins, ER-phagy receptors, that bind the autophagosomal LC3 protein via the cytosolic LC3 interacting domain (LIR) (REF). However, the molecular mechanisms that regulate ER-phagy in response to cellular needs are still largely unknown. We found that the MiT/TFE transcription factors - master regulators of lysosomal biogenesis and autophagy2- control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. This pathway is robustly activated in chondrocytes by FGF signaling, a critical regulator of chondrocyte differentiation3. FGF triggers TFEB/TFE3-mediated ER-phagy through JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS-1) protein and inhibition of the insulin signaling. FAM134B knock-down impairs cartilage growth and mineralization in medaka fish, suggesting a physiological role for this process during skeletal growth. Notably, we showed that the TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. Thus, this study identifies MiT/TFE-factors as key transcriptional activators of ER-phagy in response to both metabolic and developmental cues.

2020-07-15 | PXD015130 | Pride

Transcriptional regulation of FAM134B by TFEB/TFE3 controls ER-phagy in response to starvation and chondrocyte differentiation

Project description:Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function1. The recent identification of ER-phagy receptors has shed light on the molecular mechanism underlining this process; however, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3 - master regulators of lysosomal biogenesis and autophagy2- control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, we discovered that this pathway is activated in chondrocytes by FGF signaling, a critical regulator of cell differentiation 3. FGF signaling induces a JNK-dependent proteasomal degradation of the insulin receptor substrate 1, which inhibits the insulin-PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and FAM134B induction. Consistent with a role of the TFEB/TFE3-FAM134B axis in chondrocytes, FAM134B knock-down impairs cartilage growth and mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.

2020-07-15 | PXD015326 | Pride

Function of ERphagy receptors is regulated via phosphorylation-dependent ubiquitination pathways

Project description: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.

2022-08-17 | PXD036103 | Pride

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Project description:ER degradation by selective autophagy (ER-phagy) is crucial for ER homeostasis. However, it remains unclear how ER scission is regulated for subsequent autophagosomal sequestration and lysosomal degradation. Here, we show that oligomer formation of FAM134B (also referred to as reticulophagy regulator 1 or RETREG1) through its Reticulon domain was required for membrane fragmentation in vitro and ER-phagy in vivo. Under ER stress conditions, activated CAMK2B phosphorylated the Reticulon domain of FAM134B, which enhanced FAM134B oligomerization and activity in membrane fragmentation to accommodate high demand of ER-phagy. Unexpectedly, FAM134BG216R, a variant derived from a type II HSAN patient, exhibited gain-of-function defects, such as hyperactive self-association and membrane scission, which resulted in excessive ER-phagy and sensory neuron death. Therefore, this study revealed a mechanism of ER membrane fragmentation in ER-phagy, along with a signaling pathway in regulating ER turnover, and suggested a potential implication of excessive selective autophagy in human diseases.

2020-01-09 | PXD016661 | Pride

Transcriptional regulation of ER-phagy by MiT/TFE factors controls chondrocyte differentiation and bone formation

Project description:Lysosomal-autophagic degradation of Endoplasmic Reticulum via autophagy (ER-phagy) is emerging as critical regulator of ER homeostasis and function. However, the molecular mechanisms governing ER-phagy are still unknown. Working in chondrocytes, we found that ER-phagy and lysosome biogenesis are co-activated by FGF signaling during hypertrophic differentiation, a mandatory step for bone formation. FGF induced ER-phagy trough IRS1-dependent inhibition of the insulin signaling and activation of MiT/TFE transcription factors, master regulators of lysosome biogenesis. MiT/TFE promoted ER-phagy through the induction of the ER-phagy receptor FAM134B. Notably, the activation of ER-phagy promotes chondrocytes differentiation and secretion of factors required for cartilage replacement by bone. Consistently, medaka fish knock-down for FAM134B have impaired ossification of cranial bones. Thus, ER-phagy is a transcriptionally regulated process that participates to cell differentiation during development.

2020-07-15 | PXD015331 | Pride

Proteomic Landscape of Endoplasmic Reticulum Remodeling During Neurogenesis via Combinatorial Selective ER-phagy

Project description:The endoplasmic reticulum (ER) functions in protein and lipid synthesis, calcium ion flux, and inter-organelle communication, all of which are driven by the ER proteome landscape. ER is remodeled in part through autophagy-dependent protein turnover involving membrane-embedded ER-phagy receptors1,2. A refined tubular ER network is formed in neurons within highly polarized dendrites and axons3,4. Autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic ER boutons, associated with hyper-excitability,5 and the ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy6,7. However, mechanisms and receptor selectivity underlying ER remodeling by autophagy in neurons is limited. Here, we employ genetic, proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy during conversion of stem cells to induced neurons in vitro. Through analysis of single and combinatorial ER-phagy receptor mutants coupled with an allelic series computational framework, we delineate the extent to which each of five receptors contributes to both the magnitude of ER turnover by autophagy and the selectivity of clearance for individual ER proteins. We define specific subsets of reticulon-domain containing ER-tubule shaping proteins or luminal proteins as preferred clients for autophagic turnover via distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, which correlates with aberrant accumulation of ER in axonal structures in ER-phagy receptor or autophagy-deficient cells. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping this critical organelle during transitions in cell states.

2024-01-08 | PXD041069 | Pride

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