Recombinant Human Protein FAM134B (FAM134B)

What is FAM134B and what are its key structural domains?

FAM134B is a mammalian reticulon-like protein that shapes the ER membrane and functions as an ER-phagy receptor, mediating fragmentation and turnover of ER components. Structurally, FAM134B contains two critical domains: the LC3-interacting region (LIR) and the reticulon-homology domain (RHD) . The LIR domain is responsible for binding to autophagy proteins LC3/GABARAP, while the RHD domain, through its hairpin structure formed by two transmembrane fragments, induces ER membrane remodeling . This unique structural arrangement enables FAM134B to serve as an effective mediator between the ER membrane and the autophagy machinery.

What cellular functions does FAM134B regulate?

FAM134B primarily regulates ER-phagy, a selective autophagy process that maintains ER homeostasis. It accomplishes this by:

• Promoting the formation of autophagosomes through reshuffling and inducing fragmentation of ER membranes

• Facilitating the turnover of damaged and dysfunctional ER components

• Protecting against ER stress by maintaining ER homeostasis

• Forming specialized nanoclusters at ER-phagy initiation sites, which are critical for the efficient engulfment of ER fragments

It's important to note that FAM134B function requires precise regulation, as both deficiency and excessive activity can be detrimental. FAM134B deficiency leads to ER expansion and activation of ER stress pathways, while excessive FAM134B-mediated ER-phagy can impair ER homeostasis, causing ER stress and ultimately cell death .

How does FAM134B expression or mutation affect neuronal health?

FAM134B plays a crucial role in maintaining neuronal health, particularly in sensory and autonomic neurons. Dysfunction of FAM134B causes hereditary sensory and autonomic neuropathy type 2 (HSAN II) . Recent research has also demonstrated that FAM134B-mediated ER-phagy protects cochlear spiral ganglion neurons from cisplatin-induced damage .

Notably, while single knockout of either FAM134B or FAM134C does not lead to severe phenotypes, the combined deletion of both genes (Fam134b/c dKO) in mice results in rapid neuromuscular and somatosensory degeneration, leading to premature death . These double-knockout mice exhibit expanded tubular ER with a transverse ladder-like appearance in their long axons, demonstrating the critical roles of FAM134B and FAM134C in maintaining proper ER structure in axons of both motor and sensory neurons .

What experimental approaches are recommended for studying FAM134B-mediated ER-phagy?

For comprehensive analysis of FAM134B-mediated ER-phagy, researchers should consider a multi-faceted approach:

• In vitro liposome fragmentation assays: These allow assessment of the direct effect of FAM134B or its RHD domain on membrane remodeling. Purified FAM134B protein or its domains can be incubated with liposomes, and the resulting structures can be analyzed by electron microscopy or dynamic light scattering .

• Cellular ER fragmentation assays: Overexpression of wild-type or mutant FAM134B followed by quantification of ER membrane fragmentation using ER markers such as BAP31 or REEP5 .

• Colocalization studies: Examining the colocalization of FAM134B with autophagy markers (LC3B) and ER markers using confocal or super-resolution microscopy .

• Genetic manipulation: Using knockdown or knockout approaches with shRNA, CRISPR-Cas9, or viral vectors like Anc80-Fam134b shRNA to reduce FAM134B expression .

• Rescue experiments: Reintroducing wild-type or mutant FAM134B to knockout cells to assess functional complementation .

How can researchers visualize and quantify FAM134B clustering and oligomerization?

Super-resolution microscopy techniques have proven invaluable for visualizing FAM134B nanoclusters that are smaller than the resolution of conventional light microscopy. Specific approaches include:

• DNA-PAINT super-resolution imaging: This technique has revealed that FAM134B forms nanoscale clusters within the ER network, with some clusters colocalizing with LC3B-II, potentially representing ER-phagy initiation sites .

• DBSCAN clustering algorithm: This computational approach can be used to quantify the diameter of FAM134B clusters. Research has shown that ubiquitination-deficient FAM134B (17KR mutant) forms significantly smaller clusters (79 nm) compared to wild-type FAM134B (101 nm) .

• Kinetic analysis of single-molecule data: This approach can determine the number of molecules in nanoscale clusters, revealing that ubiquitination increases the oligomeric state of FAM134B in nanoclusters by approximately fivefold .

• Immunofluorescence with appropriate markers: Co-labeling FAM134B with ER markers (such as REEP5) helps visualize its distribution within the ER network .

What methodologies are effective for analyzing FAM134B post-translational modifications?

Ubiquitination significantly regulates FAM134B function, making the analysis of this modification crucial for understanding the protein's activity. Effective methodologies include:

• In vitro ubiquitination assays: These can be performed using recombinant proteins, including:

  • Purified substrate (full-length GST-FAM134B or His-RHD domains)

  • Ubiquitin (10 μM)

  • ATP (10 mM) and MgCl₂ (10 mM)

  • E3-ligase AMFR (0.8 μM)

  • E1 UBA1 (100 nM)

  • E2 UBE2G2 (0.8 μM)

  The reaction mixture can be analyzed by SDS-PAGE, western blotting, or mass spectrometry .

• Mass spectrometry analysis: For detailed characterization of ubiquitination sites, samples can be processed with SDC buffer (1% sodium deoxycholate, 0.5 mM TCEP, 2 mM chloroacetamide, and 50 mM Tris-HCl pH 8.5), digested with trypsin, and analyzed by LC-MS .

• Comparison of wild-type vs. ubiquitination-deficient mutants: Creating mutants where lysine residues are replaced with arginine (such as the 17KR mutant) enables functional studies of ubiquitination's role in FAM134B activity .

How does FAM134B interact with the broader autophagy machinery?

FAM134B serves as a bridge between the ER and autophagy machinery through several key interactions:

• Direct interaction with LC3/GABARAP: FAM134B binds directly to LC3 and GABARAP family proteins through its LIR domain, facilitating the recruitment of autophagy machinery to ER fragments .

• Protein complex formation: Mass spectrometry analysis has identified MAP1LC3B and GABARAP among the most enriched proteins in the FAM134B interactome . Importantly, ubiquitination status significantly influences these interactions, with ubiquitination-deficient FAM134B exhibiting reduced interactions with autophagy proteins .

• RHD protein interactions: FAM134B forms homodimers and also interacts with several other RHD-containing proteins, creating a network of ER-shaping proteins that collectively regulate ER structure and dynamics .

• Lack of interaction with ubiquitin-binding autophagy receptors: Notably, autophagy receptors containing ubiquitin-binding domains (such as p62, OPTN, and TAX1BP1) were not detected in the FAM134B interactome, suggesting that FAM134B ubiquitination does not serve as a recruitment signal for these proteins . This is supported by the absence of significant colocalization between FAM134B clusters and p62 in cells .

What is the relationship between FAM134B and cellular stress responses?

FAM134B-mediated ER-phagy plays a critical role in cellular stress responses, particularly:

• Protection against oxidative stress: In cochlear spiral ganglion neurons, FAM134B-mediated ER-phagy protects against cisplatin-induced damage, which involves reactive oxygen species (ROS) generation. This protection can be measured using MitoSOX Red and DCFH-DA probes to evaluate ROS levels .

• ER stress modulation: FAM134B deficiency leads to ER expansion and activation of ER stress pathways. Conversely, FAM134B overexpression can promote ER turnover, potentially alleviating ER stress under certain conditions .

• Fine-tuned regulation: The relationship between FAM134B and stress responses requires precise regulation, as both insufficient and excessive FAM134B activity can be detrimental. Excessive ER-phagy can impair ER homeostasis, causing ER stress and leading to cell death .

How should researchers design experiments to study FAM134B's role in neurodegeneration?

When studying FAM134B's role in neurodegeneration, researchers should consider:

What are the functional differences between FAM134B and related proteins in neuronal health?

Research on knockout mouse models has revealed important functional relationships between FAM134 family members:

This table demonstrates that FAM134B and FAM134C have redundant functions in maintaining neuronal health, particularly in axonal ER structure. Their combined deletion leads to expanded tubular ER with abnormal transverse ladder-like appearance in axons, without obvious abnormalities in cortical ER . This suggests that these proteins play specialized roles in different neuronal compartments.

How can gain-of-function and loss-of-function approaches be optimized for FAM134B research?

Effective experimental design for FAM134B functional studies should consider:

Loss-of-function approaches:

• RNA interference: shRNA delivered via viral vectors (such as Anc80-Fam134b shRNA) has been successfully used to knockdown FAM134B expression in neuronal models .

• Genetic knockout: Creation of single and combined knockout models, particularly in mice, has revealed important functional redundancies between FAM134 family members .

• Expression level controls: When using knockdown approaches, it's crucial to verify the extent of protein reduction using western blot or immunofluorescence .

Gain-of-function approaches:

What controls and validation steps are essential when studying FAM134B oligomerization?

When investigating FAM134B oligomerization, researchers should incorporate several key controls and validation steps:

• Puncta characterization: Verify that FAM134B puncta represent functional structures rather than aggregates or membrane blebs by confirming their colocalization with relevant markers such as BAP31 and LC3 .

• Expression level normalization: When comparing different FAM134B constructs, ensure equivalent expression levels to avoid artifacts from differential protein abundance .

• Multiple analytical approaches: Combine biochemical methods (such as co-immunoprecipitation) with imaging techniques (confocal and super-resolution microscopy) to provide complementary evidence for oligomerization .

• Protein-protein interaction validation: When identifying novel interaction partners, use techniques such as proximity ligation assays or FRET-based approaches to validate direct interactions in a cellular context .

• Functional assessment: Determine whether changes in oligomerization status correlate with functional outcomes, such as altered ER morphology or autophagy flux, to establish biological relevance .

What are common pitfalls in FAM134B research and how can they be addressed?

Researchers working with FAM134B should be aware of several common challenges:

• Distinguishing between physiological and pathological ER-phagy: Both insufficient and excessive FAM134B-mediated ER-phagy can be detrimental . Researchers should carefully titrate expression levels and include appropriate controls to distinguish between physiological and pathological effects.

• Neuronal compartment specificity: FAM134B and related proteins may have differential effects in cell bodies versus axons . Experimental designs should account for this compartmentalization by specifically examining different neuronal regions.

• Redundancy among family members: Functional redundancy between FAM134B and FAM134C means that single knockout approaches may fail to reveal phenotypes . Consider combined knockdown or knockout strategies when studying these proteins.

• Tissue and cell-type specificity: FAM134B functions may vary across different tissues and cell types. Research designs should account for this by including appropriate tissue-specific controls and validation in multiple cell types when possible .

• Context-dependent ubiquitination: The ubiquitination state of FAM134B significantly impacts its function . Researchers should consider how experimental conditions might alter this post-translational modification.

How can researchers interpret contradictory results regarding FAM134B function?

When faced with seemingly contradictory results about FAM134B function, researchers should consider:

• Expression level differences: Results may vary based on whether FAM134B is expressed at physiological or supra-physiological levels. Overexpression can lead to gain-of-function effects that may not represent normal physiology .

• Cell type and context specificity: FAM134B function may be influenced by the cellular milieu, including the presence of interacting partners or regulatory machinery that may vary across cell types .

• Redundancy mechanisms: Compensatory mechanisms, particularly involving other FAM134 family members, may mask phenotypes in certain experimental settings .

• Methodological differences: Variations in experimental approaches, from protein purification methods to imaging techniques, can lead to apparently contradictory results. Careful standardization and reporting of methodological details are essential .

• Temporal dynamics: The timing of observations may be critical, as FAM134B function may change during development or in response to various stressors .