Recombinant Mouse Protein FAM134C (Fam134c)

What is the basic structure of FAM134C and how does it compare to other FAM134 family members?

FAM134C is part of the FAM134 protein family, which represents the first characterized ER-phagy receptor family. All FAM134 proteins (FAM134A, FAM134B, and FAM134C) share strong homology in the LC3-interacting region (LIR) and contain a reticulon homology domain (RHD) . The human version of FAM134C has a canonical length of 466 amino acids with a molecular weight of approximately 51.4 kDa, although two isoforms have been identified .

Despite their similarities, each family member has unique characteristics. For example, while FAM134B overexpression is sufficient to promote ER-phagy, FAM134A and FAM134C appear inactive under steady-state conditions and require activation . The RHDs of these proteins also exhibit different dynamics, which may contribute to their distinct functions in ER homeostasis .

What is the primary function of FAM134C in cellular physiology?

FAM134C functions as an endoplasmic reticulum-anchored autophagy regulator. Upon activation following cellular stress, it induces ER fragmentation and mediates the delivery of ER components into lysosomes through sequestration into autophagosomes via interaction with ATG8 family proteins . The protein promotes ER membrane curvature and tubulation, which are required for subsequent fragmentation and engulfment into autophagosomes .

Additionally, FAM134C plays a significant role in collagen quality control in a LIR motif-dependent manner and mediates NRF1-enhanced neurite outgrowth, indicating its importance in neuronal development . Recent studies have shown that FAM134C can act cooperatively with FAM134B to enhance the degradation of misfolded pro-collagen, demonstrating its importance in protein quality control mechanisms .

How does FAM134C contribute to ER homeostasis?

FAM134C provides critical functions in maintaining ER homeostasis, and its absence results in massive deformation of the ER network and swelling of the ER . Upon ER stress, FAM134C can induce significant fragmentation and degradation of the ER via the lysosomal pathway. This process, known as ER-phagy, helps cells adapt to ER stress by selectively removing damaged or excess ER components .

The protein's ability to promote ER membrane curvature and tubulation is essential for the subsequent fragmentation and engulfment of ER portions into autophagosomes . Through this mechanism, FAM134C plays a crucial role in the quality control of the ER, particularly during cellular stress conditions when protein misfolding may occur.

How is FAM134C activity regulated at the post-translational level?

FAM134C activity is tightly regulated through post-translational modifications (PTMs), particularly phosphorylation. Research has identified that casein kinase 2 (CK2) phosphorylates FAM134C at multiple serine residues . This phosphorylation acts as an inhibitory mechanism that keeps FAM134C inactive under basal conditions.

Importantly, immunoprecipitation-mass spectrometry (IP-MS) studies have demonstrated that FAM134C, but not FAM134A or FAM134B, interacts with CK2 α, α I, and β subunits . Pharmacological inhibition of CK2 with the inhibitor CX4945 enhances FAM134C binding efficiency with endogenous LC3B and promotes its lysosomal localization and degradation, supporting the role of CK2-mediated phosphorylation in regulating FAM134C activity .

What triggers the activation of FAM134C during cellular stress?

FAM134C exists predominantly in an inactive state under basal conditions but becomes activated in response to various cellular stressors, particularly those affecting ER homeostasis . The mTOR inhibitor Torin1 has been shown to be a potent activator of FAM134C, inducing its delivery to lysosomes with approximately 20-fold increased acidification upon 6 hours of treatment .

The activation process involves the relief of inhibitory phosphorylation by CK2. Under stress conditions, phosphatases may counteract CK2 activity, or CK2 itself may be inhibited, allowing FAM134C to interact more efficiently with LC3 proteins and mediate ER-phagy . This stress-responsive activation mechanism ensures that ER-phagy occurs primarily under conditions that require remodeling of the ER or elimination of damaged ER components.

What is the significance of the LC3-interacting region (LIR) in FAM134C function?

The LC3-interacting region (LIR) is crucial for FAM134C's function as an ER-phagy receptor. This domain mediates binding to ATG8 family proteins (including LC3B), which is essential for the targeting of ER fragments to autophagosomes . Research has shown that mutations in the LIR motif significantly reduce FAM134C's interaction with LC3B, as demonstrated by co-immunoprecipitation experiments .

The regulation of this interaction appears to be modulated by phosphorylation states. For example, phosphomimetic mutations (3D-FAM134C) show reduced interaction with endogenous LC3B, while phospho-null mutations (3A-FAM134C) demonstrate enhanced binding . This indicates that the LIR-mediated interaction with LC3 proteins is a key regulatory point in FAM134C-driven ER-phagy, directly influenced by the protein's phosphorylation status.

What are the most effective methods for studying FAM134C-mediated ER-phagy in vitro?

Several robust methods have been developed to study FAM134C-mediated ER-phagy in vitro:

• Fluorescence-based assays: Human embryonic kidney (HEK) 293 cells stably expressing FAM134C tagged with RFP-EGFP (red fluorescent protein–enhanced green fluorescent protein) can be used to measure FAM134C delivery to lysosomes. The acidic environment inside lysosomes quenches EGFP but not RFP fluorescence, allowing quantification by fluorescence-activated cell sorting (FACS) . This dual-fluorescence approach enables time-resolved monitoring of ER-phagy flux.

• Immunofluorescence colocalization: Analysis of FAM134C colocalization with lysosomal markers (e.g., LAMP1) or autophagosome markers (e.g., LC3) using confocal microscopy provides visual evidence of FAM134C-mediated ER-phagy . This approach can be used with wild-type and mutant FAM134C variants to assess the impact of specific modifications on subcellular localization.

• Pharmacological modulation: Using Torin1 (mTOR inhibitor) to induce ER-phagy and Bafilomycin A1 (BafA1) to block lysosomal degradation allows researchers to track the accumulation of FAM134C in autophagosomes/lysosomes . This approach is particularly useful for studying the kinetics of FAM134C-mediated ER-phagy.

• Time-resolved screening: High-throughput screening using kinase inhibitors combined with time-point measurements of RFP/GFP fluorescence ratios allows identification of regulatory pathways controlling FAM134C activity .

How can researchers effectively express and purify recombinant FAM134C protein?

For recombinant FAM134C protein expression and purification:

• Expression system: Recombinant FAM134C or its functional domains (e.g., GFP-FAM134C-LIR) can be effectively overexpressed in E. coli strain BL21(DE3) . This bacterial expression system is particularly useful for producing protein domains for in vitro studies.

• Protein tagging: Adding tags such as GFP, RFP, or Myc facilitates detection, purification, and functional studies of FAM134C . For instance, Myc-tagged FAM134C has been used to study its subcellular localization, while RFP-EGFP tagging enables monitoring of lysosomal delivery.

• Mutation strategies: Generation of phosphomimetic (3D-FAM134C) and phospho-null (3A-FAM134C) mutants provides valuable tools for studying the functional significance of CK2-mediated phosphorylation . These mutants can be created using site-directed mutagenesis.

• Truncated constructs: Creating fragments such as FAM134C 250-466 (encompassing the C-terminal cytosolic region) is useful for studying specific domains and their post-translational modifications . These truncated constructs are particularly valuable for in vitro kinase assays.

What are the appropriate assays for measuring FAM134C-LC3 interaction strength?

Several assays have been validated for measuring FAM134C-LC3 interaction:

• Co-immunoprecipitation (co-IP): This technique has been successfully used to demonstrate the interaction between FAM134C and endogenous LC3B, as well as to assess how mutations or pharmacological treatments affect this interaction . For example, co-IP experiments have shown reduced interaction of 3D-FAM134C compared to wild-type FAM134C with endogenous LC3B.

• In vitro binding assays: Using purified recombinant proteins (e.g., GFP-FAM134C-LIR and LC3 proteins), researchers can quantitatively measure binding affinities and identify critical residues involved in the interaction .

• Fluorescence colocalization: Microscopy-based colocalization analysis of FAM134C with LC3 provides visual evidence of their interaction in cellular contexts . This approach is complementary to biochemical techniques and offers spatial information.

• Mutation analysis: Comparing the LC3-binding efficiency of wild-type FAM134C with LIR mutants or phosphorylation-site mutants provides insights into the regulatory mechanisms of this interaction . This approach has revealed that phosphorylation at CK2 target sites inhibits FAM134C-LC3 interaction.

How does FAM134C response to mTOR inhibition differ from other FAM134 family members?

FAM134C exhibits a distinctly stronger response to mTOR inhibition compared to other FAM134 family members. When treated with the mTOR inhibitor Torin1, FAM134C shows approximately 20-fold increased acidification (indicating lysosomal delivery) upon 6 hours of treatment in HEK293 cells . This response is significantly higher than that observed for FAM134B, which shows enhanced lysosomal delivery in HEK293 cells but at much lower levels compared to FAM134C .

This differential response highlights the unique regulatory mechanisms governing each FAM134 family member. While FAM134B is constitutively active, FAM134C predominantly exists in an inactive state under basal conditions and becomes strongly activated upon mTOR inhibition . This suggests that FAM134C may play a particularly important role in stress-induced ER-phagy, especially in response to nutrient deprivation or other stressors that inhibit mTOR signaling.

What kinases regulate FAM134C-mediated ER-phagy under different stress conditions?

A time-resolved screening of kinase inhibitors revealed that several kinases play crucial roles in regulating FAM134C-mediated ER-phagy:

• Casein Kinase 2 (CK2): This kinase has been definitively identified as a negative regulator of FAM134C activity. CK2 phosphorylates FAM134C at multiple serine residues, inhibiting its interaction with LC3B and preventing its lysosomal localization . Pharmacological inhibition of CK2 with CX4945 enhances FAM134C-driven ER-phagy.

• Additional kinases: High-throughput screening showed that inhibition of several kinases prevented Torin1-induced, FAM134-driven ER-phagy . While the specific identities of all these kinases are not detailed in the search results, this suggests a complex regulatory network controlling FAM134C activity under different stress conditions.

The regulation of FAM134C by these kinases likely provides a mechanism for integrating various cellular signals to modulate ER-phagy in response to different types of stress. Further research is needed to fully map this kinase network and understand how different stress conditions may alter FAM134C phosphorylation patterns.

How does FAM134C cooperate with FAM134B in stress-induced ER remodeling?

FAM134C and FAM134B have been shown to cooperate in stress-induced ER remodeling, particularly in the context of misfolded protein degradation. Studies have demonstrated that FAM134C can act in concert with FAM134B and enhances its activity in degrading misfolded pro-collagen . This cooperative action suggests a synergistic relationship between these two ER-phagy receptors during cellular stress.

This cooperation may be particularly important in tissues with high secretory loads, such as those producing large amounts of collagen, where efficient quality control mechanisms are essential for cellular homeostasis.

How can phosphosite-specific antibodies be used to monitor FAM134C activation status?

Phosphosite-specific antibodies represent a powerful tool for monitoring FAM134C activation status:

• Detection of phosphorylation state: A commercially available phospho-CK2 substrate antibody has been successfully used to detect phosphorylated FAM134C, with this detection being blunted by preincubating cells with the CK2 inhibitor CX4945 . Similar approaches could be used to develop antibodies specific to individual phosphorylation sites on FAM134C.

• Tracking activation dynamics: Such antibodies would allow researchers to track the activation status of FAM134C in real-time following various cellular stressors, providing insights into the kinetics of FAM134C dephosphorylation and activation .

• Tissue-specific activation patterns: Phosphosite-specific antibodies could be used in immunohistochemistry to examine tissue-specific patterns of FAM134C activation in various physiological and pathological contexts .

• Validation of phosphosite mutations: These antibodies could validate the effectiveness of phospho-null or phosphomimetic mutations in cellular and in vivo models, ensuring that these mutations accurately recapitulate the relevant phosphorylation states .

What is the potential role of FAM134C in neurodegenerative disorders?

FAM134C has several characteristics suggesting it could play important roles in neurodegenerative disorders:

• Neuronal development function: FAM134C mediates NRF1-enhanced neurite outgrowth, indicating its importance in neuronal development and potentially in neuronal maintenance .

• ER homeostasis maintenance: The absence of FAM134 proteins results in massive deformation of the ER network and swelling of the ER . Since ER stress is a common feature in many neurodegenerative diseases, dysregulation of FAM134C could contribute to pathogenesis.

• Comparison with FAM134B: Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons . Given the functional similarities between FAM134B and FAM134C, the latter might also be involved in neuronal maintenance and susceptibility to degeneration.

• Protein quality control: FAM134C's role in collagen quality control suggests it may participate in broader protein quality control mechanisms , which are often compromised in neurodegenerative disorders characterized by protein aggregation.

Research into FAM134C's potential role in neurodegenerative disorders is still emerging, but these connections suggest it could be a valuable target for further investigation in conditions characterized by ER stress and protein aggregation.

How does ubiquitination interact with phosphorylation in regulating FAM134C activity?

While the search results focus primarily on phosphorylation of FAM134C, there are indications that ubiquitination may also play an important role in regulating ER-phagy:

The Nature article mentions that "membrane-embedded clusters of ubiquitinated ARL6IP1 and FAM134B are required for effective ER remodelling and ER-phagy" and that "ubiquitination of ARL6IP1 promotes this process" . Since ARL6IP1 interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy, it's possible that similar mechanisms may apply to FAM134C.

The interplay between phosphorylation and ubiquitination could work in several ways:

• Sequential modification: Phosphorylation status might influence subsequent ubiquitination patterns, or vice versa.

• Competitive regulation: Phosphorylation and ubiquitination might compete for the same or nearby residues, creating mutually exclusive regulatory states.

• Cooperative function: Both modifications might be required in specific combinations to achieve full activation or inactivation of FAM134C.

Further research is needed to elucidate the specific roles of ubiquitination in FAM134C regulation and how it interacts with the phosphorylation mechanisms that have been more thoroughly characterized.

What are the key functional differences between FAM134A, FAM134B, and FAM134C?

Despite their structural similarities, the three FAM134 proteins exhibit several key functional differences:

• Basal activity: FAM134B is constitutively active, while FAM134A and FAM134C predominantly exist in an inactive state under basal conditions . This suggests different roles in homeostatic versus stress-induced ER-phagy.

• Response to mTOR inhibition: FAM134C shows the strongest response to Torin1 administration, with approximately 20-fold increased acidification upon 6 hours of treatment in HEK293 cells, significantly higher than FAM134B . This indicates a more pronounced role for FAM134C in stress-induced ER-phagy.

• Regulation mechanism: FAM134C, but not FAM134A or FAM134B, interacts with CK2 α, α I, and β subunits, indicating a unique regulatory mechanism involving CK2-mediated phosphorylation .

• Cooperative functions: FAM134C can act in concert with FAM134B and enhances its activity in degrading misfolded pro-collagen, suggesting complementary roles in protein quality control .

These functional differences likely enable the FAM134 family to orchestrate a nuanced regulation of ER-phagy under different cellular conditions and in different tissues.

How do mutation phenotypes differ between FAM134C and other family members in animal models?

The search results provide limited information on FAM134C mutation phenotypes in animal models, but offer insights on FAM134B:

Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons . This neurodegeneration is also observed in FAM134B-deficient mice . Furthermore, disruption of Arl6ip1 (which interacts with FAM134B) in mice causes an expansion of ER sheets in sensory neurons that degenerate over time .

For comparative analysis, we would need additional research specifically addressing FAM134C knockout or mutation phenotypes in animal models. The functional similarities between FAM134 family members suggest that FAM134C deficiency might also lead to ER abnormalities, potentially with tissue-specific manifestations depending on the relative importance of FAM134C in different cell types.

The cooperative action of FAM134C with FAM134B in degrading misfolded pro-collagen suggests that FAM134C mutations might have particularly pronounced effects in tissues with high collagen production, such as connective tissues, though this requires experimental verification.