Recombinant Mouse Protein FAM134B (Fam134b)

What is FAM134B and what are its primary functions in cellular biology?

FAM134B is a membrane-shaping protein characterized by reticulon homology domains that plays a crucial role in endoplasmic reticulum (ER) remodeling and ER-phagy (selective autophagy of the ER). It functions as an ER-phagy receptor by binding to LC3 proteins through its LC3-interacting region (LIR), mediating the fragmentation and degradation of ER sheets through autophagy . FAM134B is highly conserved from yeast to humans, indicating its evolutionary importance in cellular homeostasis . In mice and humans, FAM134B dysfunction is associated with hereditary sensory and autonomic neuropathy type 2 (HSAN II), characterized by sensory loss and neurodegeneration .

What are the different isoforms of mouse FAM134B and how do they differ structurally?

Mouse FAM134B exists in at least two distinct isoforms:

• FAM134B-1: The full-length isoform that contains a complete reticulon homology domain (RHD) and four transmembrane domains.

• FAM134B-2: An N-terminal-truncated isoform that is a type I membrane protein with only one transmembrane domain (amino acids 55-88). While FAM134B-2 preserves the cytosolic LC3-interacting region, it partially lacks the reticulon homology domain .

The structural differences between these isoforms are illustrated in this schematic model:

These structural differences contribute to distinct functional roles in ER-phagy, with FAM134B-2 specifically involved in starvation-induced selective ER-phagy of secretory proteins .

How is FAM134B expression regulated across different mouse tissues?

FAM134B shows tissue-specific expression patterns that correlate with its specialized functions:

• Brain: High expression of FAM134B-1, with consistent levels across fed, fasted, and re-fed conditions .

• Liver: Predominant expression of FAM134B-2, which is significantly upregulated during fasting conditions and downregulated upon refeeding .

• Dorsal Root Ganglion (DRG): Particularly high expression of FAM134B, consistent with its role in sensory neuron maintenance .

Expression analysis by qRT-PCR demonstrates that FAM134B-2 expression in mouse liver is significantly increased during fasting (approximately 6-fold) compared to fed conditions, while FAM134B-1 shows minimal changes . This differential regulation suggests distinct physiological roles for each isoform.

What post-translational modifications regulate FAM134B activity in ER-phagy?

FAM134B activity is regulated through multiple post-translational modifications that affect its oligomerization, membrane remodeling capacity, and interaction with autophagy machinery:

• Phosphorylation: ER stress triggers CaMKII-mediated phosphorylation of FAM134B at serine residues within the reticulon domain (particularly Ser151), which enhances FAM134B oligomerization and ER-phagy . This modification is crucial for stress-induced activation of ER-phagy.

• Acetylation: FAM134B is acetylated at lysine 160 (K160) by the acetyltransferase CBP. This modification dramatically enhances FAM134B oligomerization and ER membrane fragmentation activity. Mutation studies show that:

  • FAM134B K160R (deacetylation mimic) reduces self-interaction and membrane fragmentation

  • FAM134B K160Q (acetylation mimic) significantly enhances self-association and membrane remodeling

• Ubiquitination: Ubiquitination of FAM134B, particularly of its reticulon homology domain (RHD), significantly enhances its membrane-remodeling activity. In vitro studies demonstrate that ubiquitinated RHD (Ub-RHD-Ub) creates significantly smaller membrane structures compared to non-ubiquitinated RHD .

These modifications create a complex regulatory network that fine-tunes FAM134B activity in response to cellular stress conditions.

How does FAM134B oligomerization influence its function in ER membrane fragmentation?

FAM134B oligomerization is a critical determinant of its membrane-remodeling activity:

• Oligomerization mechanism: The reticulon domain (RTND, amino acids 84-233) is both necessary and sufficient for FAM134B self-association and oligomerization . Native PAGE and size-exclusion chromatography reveal that FAM134B oligomers have a molecular weight of approximately 450-700 kDa .

• Stress-induced oligomerization: ER stress and starvation enhance FAM134B oligomerization in vivo. These oligomers are partially resistant to denaturing solutions containing SDS and DTT, suggesting strong interactions .

• Functional consequences: Increased oligomerization directly correlates with enhanced ER membrane fragmentation activity, both in vitro using liposome fragmentation assays and in vivo using ER-phagy measurements .

• Experimental assessment: Liposome fragmentation assays provide a quantitative method to assess oligomerization-dependent activity. In this approach, biotinylated liposomes are anchored onto streptavidin-coated glass within a chamber, and spinning-disk confocal microscopy measures the dynamics of liposome fragmentation after addition of recombinant FAM134B proteins .

Mutations or modifications that enhance oligomerization (K160Q, G216R) significantly increase membrane fragmentation activity, while those that reduce oligomerization (K160R) decrease activity .

What phenotypes are observed in FAM134B-deficient mouse models?

FAM134B knockout and mutant mouse models display several characteristic phenotypes:

• Neurological phenotypes:

  • Fam134b knockout mice develop sensory neurodegeneration similar to HSAN II in humans .

  • Fam134b/c double knockout (dKO) mice show reduced body weight at 4 weeks of age compared to single knockouts or wild-type animals .

  • Neuronal hyperexcitability: Fam134b/c dKO mice exhibit dramatically increased neuronal firing rates (13.565 ± 2.118 spikes/s) compared to wild-type (0.40 ± 0.053 spikes/s) .

• Cellular phenotypes:

  • Expanded ER sheets in sensory neurons that degenerate over time .

  • Incomplete budding of ER membranes and severe impairment of ER-phagy flux .

  • Altered ER protein composition, including increased levels of ApoCIII in hepatic microsomes during fasting .

• Molecular phenotypes:

  • Accumulation of misfolded proteins associated with neuronal sensory disorders .

This phenotypic characterization demonstrates the importance of FAM134B in neuronal maintenance, particularly in sensory neurons, and provides insight into the cellular mechanisms underlying HSAN II pathology.

What methodological approaches are recommended for studying FAM134B-mediated ER-phagy in mouse models?

To comprehensively investigate FAM134B-mediated ER-phagy, researchers should consider these methodological approaches:

• In vitro membrane fragmentation assays:

  • Liposome fragmentation assay: Biotinylated liposomes anchored to streptavidin-coated glass are monitored by spinning-disk confocal microscopy to assess membrane remodeling capacity of recombinant FAM134B proteins .

  • This approach allows quantitative comparison of wild-type vs. mutant proteins and assessment of post-translational modification effects.

• Cellular ER-phagy assessments:

  • Inducible expression systems: Generate stable cell lines with inducible expression of EGFP-tagged FAM134B variants in FAM134B knockout backgrounds to ensure controlled expression levels near endogenous amounts .

  • Puncta quantification: Apply Bafilomycin A1 (BafA1) treatment to accumulate EGFP-FAM134B-labeled ER membrane fragments for easier quantification .

  • Co-localization studies: Measure co-localization of FAM134B with autophagosomal markers (LC3) and ER markers (SERCA2, BAP31) .

• In vivo analysis of tissue-specific effects:

  • Tissue fractionation: Isolate microsomes from tissues (particularly liver) and separate ER subfractions using iodixanol density gradient ultracentrifugation (Opti-Prep) .

  • Proteomics analysis: Comparative proteomics of hepatic microsomes from wild-type and FAM134B KO mice under various nutritional states .

  • Leupeptin challenge: Intraperitoneal injection of leupeptin (15 mg/kg body weight) to inhibit lysosomal degradation and visualize accumulated autophagy substrates .

• Neurophysiological assessments:

  • Electrophysiological recordings of sensory neurons to measure firing rates, burst frequency, and duration of excitation in wild-type versus FAM134B-deficient models .

These complementary approaches provide comprehensive insight into FAM134B function at multiple levels, from molecular mechanisms to physiological outcomes.

How do FAM134B mutations associated with HSAN II affect protein function at the molecular level?

FAM134B mutations associated with HSAN II have distinct effects on protein function:

• Loss-of-function mutations:

  • Most HSAN II-associated mutations result in loss of FAM134B function, leading to impaired ER-phagy and accumulation of expanded ER sheets in sensory neurons .

  • These mutations ultimately cause sensory neuron degeneration, consistent with the phenotype observed in FAM134B knockout mice .

• The paradoxical G216R mutation:

  • Interestingly, the G216R mutation appears to act as a gain-of-function mutation under experimental conditions.

  • FAM134B G216R demonstrates enhanced ability to induce ER scission and ER-phagy compared to wild-type FAM134B when overexpressed in cultured cells .

  • The puncta structures formed by FAM134B G216R are positive for both BAP31 (ER marker) and LC3 (autophagosome marker), confirming authentic ER-phagy rather than simple aggregation .

  • Rescue experiments with expression at endogenous levels confirm that G216R is a gain-of-function mutant for ER membrane fragmentation and ER-phagy .

• Functional consequences in neurons:

  • Despite the apparent gain-of-function in vitro, evidence suggests that G216R-induced excessive ER-phagy may negatively affect neuronal survival .

  • This suggests that balanced FAM134B activity is crucial for neuronal homeostasis, with both insufficient and excessive ER-phagy being potentially detrimental.

Understanding these nuanced effects is essential for developing therapeutic strategies for HSAN II and other FAM134B-related disorders.

How do FAM134B and ARL6IP1 interact to regulate ER-phagy?

Recent research has uncovered an important functional relationship between FAM134B and ARL6IP1 in regulating ER-phagy:

• Protein-protein interaction:

  • ARL6IP1, another ER-shaping protein containing a reticulon homology domain, directly interacts with FAM134B to form heteromeric multi-protein clusters required for effective ER-phagy .

• Regulatory mechanisms:

  • Ubiquitination of ARL6IP1 promotes the formation of these heteromeric clusters, enhancing ER-phagy efficiency .

  • These membrane-embedded clusters of ubiquitinated ARL6IP1 and FAM134B are critical for effective ER remodeling and subsequent degradation .

• Phenotypic parallels:

  • Similar to FAM134B mutations, disruption of ARL6IP1 is associated with sensory loss in humans .

  • Disruption of Arl6ip1 in mice causes expansion of ER sheets in sensory neurons that degenerate over time, mirroring the phenotype of FAM134B-deficient mice .

  • Primary cells from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux .

• Conceptual model:

  • The clustering of ubiquitinated ER-shaping proteins (including both FAM134B and ARL6IP1) facilitates the dynamic remodeling of the ER during ER-phagy.

  • This cooperative action is particularly important for neuronal maintenance, explaining why defects in either protein can lead to similar neurological phenotypes .

This emerging understanding of cooperative protein networks in ER-phagy provides new avenues for investigating neurodegeneration mechanisms and potential therapeutic targets.

What signaling pathways regulate FAM134B expression and function during nutritional stress?

FAM134B expression and activity are regulated by distinct signaling pathways during nutritional stress:

• Transcriptional regulation of FAM134B-2 in liver:

  • Fasting significantly upregulates FAM134B-2 expression in mouse liver (approximately 6-fold increase) .

  • Promoter analysis using luciferase reporter gene assays identified C/EBPβ as a key transcriptional regulator of FAM134B-2 .

  • C/EBPβ expression and activity are increased in mouse liver during fasting conditions .

  • Liver-specific C/EBPβ transgenic (L-C/EBPβ tg) mice show increased FAM134B-2 expression even under fed conditions .

  • Conversely, liver-specific C/EBPβ knockout (L-C/EBPβ KO) mice show decreased FAM134B-2 expression during fasting .

• Post-translational modification signaling:

  • ER stress activates calcium/calmodulin-dependent protein kinase II (CAMKII), which phosphorylates FAM134B at serine residues in the reticulon domain, enhancing its oligomerization and activity .

  • CBP-mediated acetylation at K160 dramatically enhances FAM134B oligomerization to induce ER fragmentation and ER-phagy .

  • SIRT7 deacetylates FAM134B to negatively regulate ER-phagy, establishing a regulatory circuit that fine-tunes ER turnover .

• Functional consequences in selective ER-phagy:

  • FAM134B-2 upregulation during fasting specifically mediates selective ER-phagy of secretory proteins such as ApoCIII .

  • Co-immunoprecipitation experiments demonstrate direct interaction between FAM134B-2 and secretory cargo proteins like ApoCIII .

  • This selective degradation helps maintain ER homeostasis during nutritional stress by removing excess secretory proteins when secretion is reduced.

This regulatory network illustrates how cells precisely control ER-phagy through both transcriptional and post-translational mechanisms in response to changing nutritional status.

What techniques are recommended for expressing and purifying recombinant mouse FAM134B for in vitro studies?

For optimal expression and purification of recombinant mouse FAM134B:

• Expression system selection:

  • HEK293T cells have been successfully used for expressing FLAG-tagged FAM134B constructs with good yield and proper folding .

  • For large-scale protein production, baculovirus-insect cell expression systems may provide better yields for membrane proteins while maintaining mammalian-like post-translational modifications.

• Construct design considerations:

  • Include appropriate tags (FLAG, His, etc.) for purification and detection.

  • For domain-specific studies, the reticulon domain (amino acids 84-233) can be expressed separately .

  • When studying ubiquitination effects, gene fusions of Ub to FAM134B RHD (e.g., Ub-RHD-Ub) can be created .

• Purification strategies:

  • For native purification, use gentle detergents like digitonin or DDM to solubilize membrane proteins while preserving structure.

  • Size-exclusion chromatography is particularly important for FAM134B studies as it allows separation of monomeric and oligomeric forms (450-700 kDa) .

• Quality control assessments:

  • Native PAGE to assess oligomerization state .

  • Western blotting with specific antibodies to confirm identity and integrity.

  • Liposome binding assays to confirm membrane interaction capability .

• Storage considerations:

  • For FAM134B antibodies: "Do not aliquot the antibody" to maintain stability .

  • For purified proteins: flash-freeze in small aliquots with glycerol or sucrose as cryoprotectants.

These methodological approaches ensure production of high-quality recombinant FAM134B suitable for downstream functional studies.

How can researchers differentiate between FAM134B-1 and FAM134B-2 isoforms in experimental systems?

Differentiating between FAM134B isoforms requires specialized techniques:

• mRNA detection strategies:

  • isoform-specific RT-PCR: Design primers targeting FAM134B-1 or FAM134B-2 specific sequences (as shown in gel electrophoresis of RT-PCR products from mouse brain and fasted liver) .

  • qRT-PCR with isoform-specific primers can quantify relative expression levels of each isoform under different conditions .

  • 5' RACE (Rapid Amplification of cDNA Ends) can identify distinct transcriptional start sites for different isoforms .

• Protein detection methods:

  • Western blotting: FAM134B-1 and FAM134B-2 can be distinguished by their molecular weight differences (FAM134B-1 appears at a higher molecular weight than FAM134B-2) .

  • Comparison with recombinant standards: Include recombinant FLAG-FAM134B-1 and FLAG-FAM134B-2 generated in HEK293T cells as molecular weight references .

  • Isoform-specific antibodies: When available, use antibodies targeting unique N-terminal regions.

• Experimental model selection:

  • Tissue choice: Brain predominantly expresses FAM134B-1, while fasted liver predominantly expresses FAM134B-2 .

  • Nutritional status: Fasting significantly upregulates FAM134B-2 in liver, facilitating detection and study .

• Functional differentiation:

  • Membrane topology analysis using prediction tools like Protter (http://wlab.ethz.ch/protter/start/) can identify structural differences .

  • Co-immunoprecipitation studies to identify isoform-specific protein interactions .