Recombinant Pongo abelii Ragulator complex protein LAMTOR3 (LAMTOR3)

What is LAMTOR3 and what is its role in cellular signaling?

LAMTOR3 (Late Endosomal/Lysosomal Adaptor and MAPK and MTOR Activator 3), also known as MP1 or MAPKSP1, is a critical component of the Ragulator complex involved in amino acid sensing and activation of mTORC1, a signaling complex promoting cell growth in response to growth factors, energy levels, and amino acids . The protein functions as an adapter that enhances the efficiency of the MAP kinase cascade, facilitating the activation of MAPK2 . LAMTOR3 specifically interacts with MAP kinase kinase MAP2K1/MEK1, MAP kinase MAPK3/ERK1, and MAP kinase MAPK1/ERK2 .

As part of the pentameric Ragulator complex, which contains Lamtor1/p18, Lamtor2/p14–Lamtor3/MP1, and Lamtor4/p10–Lamtor5/HBXIP, LAMTOR3 helps tether the mTORC1 complex to the lysosomal surface, serving as an essential activation platform for metabolic signaling . The complexity of its interactions makes it a valuable subject for comparative studies between humans and non-human primates.

What are the known physical properties of recombinant Pongo abelii LAMTOR3 protein?

The recombinant Pongo abelii LAMTOR3 protein, when expressed with a His-tag, has an expected calculated molecular weight of approximately 15.8 kDa, with an observed molecular weight of approximately 15 kDa on SDS-PAGE . Though specific data for the orangutan version is limited, human LAMTOR3 is reported to have a molecular weight of 13.6 kDa .

For optimal results in experimental work, the recombinant protein is typically stored at temperatures below -20°C and shipped in frozen conditions with appropriate stabilizing buffers, commonly containing components such as Tris-HCl, NaCl, DTT, and glycerol . When working with this protein, researchers should minimize freeze-thaw cycles to maintain functionality and structural integrity.

What are the recommended approaches for studying LAMTOR3 protein interactions within the Ragulator complex?

To study LAMTOR3 interactions within the Ragulator complex, a multi-faceted approach is recommended:

• Co-immunoprecipitation (Co-IP): This technique allows detection of protein-protein interactions between LAMTOR3 and other components of the Ragulator complex or interacting proteins like MEK1 and ERK1/2. Use specific antibodies against LAMTOR3 or its binding partners .

• Proximity Ligation Assays (PLA): This method offers visualization of protein interactions in situ with high sensitivity and specificity. It's particularly useful for detecting transient interactions between LAMTOR3 and components of the MAP kinase pathway .

• Structural Biology Approaches: The Ragulator complex has been extensively evaluated using cryo-electron microscopy and crystallographic analyses, revealing its multifunctional roles in response to cellular conditions through interactions with effector proteins .

• FRET (Förster Resonance Energy Transfer): For studying dynamic interactions between LAMTOR3 and its binding partners in living cells, FRET can provide temporal and spatial resolution of protein-protein interactions.

• Mutational Analysis: Introducing specific mutations in LAMTOR3 can help identify key residues involved in protein-protein interactions and functional domains critical for its role in the Ragulator complex.

These methodologies provide complementary information and should be used in combination for a comprehensive understanding of LAMTOR3's interaction network.

How can researchers effectively express and purify recombinant Pongo abelii LAMTOR3 for functional studies?

For optimal expression and purification of recombinant Pongo abelii LAMTOR3, follow this methodological workflow:

• Expression System Selection: E. coli is commonly used for LAMTOR3 expression due to its simplicity and high yield . The protein sequence should cover the full length (Met 1-Val124) for complete functionality.

• Construct Design:

  • Include an N-terminal His-tag for efficient purification

  • Optimize codon usage for the expression host

  • Consider adding a cleavable tag if tag-free protein is needed for specific assays

• Expression Conditions:

  • Induce protein expression at lower temperatures (16-18°C) to enhance proper folding

  • Use rich media (e.g., TB or 2xYT) supplemented with appropriate antibiotics

  • Optimize induction time and IPTG concentration

• Purification Protocol:

  • Lyse cells in buffer containing 20mM Tris-HCl, 100mM NaCl, 5mM DTT, pH 8.0

  • Perform initial purification using Ni-NTA affinity chromatography

  • Follow with size exclusion chromatography for higher purity

  • Ensure endotoxin levels are <1.0 EU per μg of protein as determined by the LAL method

• Quality Control:

  • Verify purity (>95%) using SDS-PAGE

  • Confirm identity using mass spectrometry

  • Assess functional activity through appropriate binding assays

• Storage:

  • Store in buffer containing 20mM Tris-HCl, 100mM NaCl, 5mM DTT, 30% Glycerol, pH 8.0

  • Keep at temperatures below -20°C

  • Aliquot to avoid repeated freeze-thaw cycles

This systematic approach ensures production of high-quality recombinant LAMTOR3 suitable for downstream functional and structural studies.

How does Pongo abelii LAMTOR3 compare structurally and functionally to its human ortholog?

While specific comparative data between human and Pongo abelii LAMTOR3 is limited in the current literature, analysis of these proteins can be approached through several methodological frameworks:

• Sequence Alignment Analysis: Comparative sequence analysis typically reveals high conservation between human and orangutan LAMTOR3, reflecting the evolutionary importance of this protein in fundamental cellular processes. Key functional domains and binding motifs are likely preserved across these species.

• Structural Comparison: Though no direct structural comparison is available in the search results, researchers can use homology modeling techniques to predict the tertiary structure of Pongo abelii LAMTOR3 based on the human structure, followed by molecular dynamics simulations to identify any species-specific structural features.

• Functional Conservation Assessment: Experimental comparison of biochemical properties such as binding affinities to other Ragulator components, GEF activity, and interactions with the MAP kinase pathway would provide insights into functional conservation or divergence.

• Cellular Localization Patterns: Immunofluorescence studies comparing the subcellular localization of LAMTOR3 in human and orangutan cells could reveal any differences in distribution or dynamics.

Given the importance of the Ragulator complex in fundamental cellular processes like amino acid sensing and mTORC1 activation, substantial functional conservation is expected between human and orangutan LAMTOR3 proteins, with potentially subtle species-specific adaptations that warrant further research.

What evolutionary insights can be gained from studying LAMTOR3 across primate species including Pongo abelii?

Studying LAMTOR3 across primate species offers valuable evolutionary insights through several methodological approaches:

• Phylogenetic Analysis: Constructing phylogenetic trees based on LAMTOR3 sequences from multiple primate species, including Pongo abelii, can illuminate the evolutionary history of this gene and identify lineage-specific selection pressures.

• Selection Pressure Analysis: Calculating dN/dS ratios (non-synonymous to synonymous substitution rates) across different regions of the protein can identify functionally important domains under purifying selection versus regions experiencing adaptive evolution.

• Structural Evolution Study: Mapping sequence variations onto three-dimensional structures can reveal how structural constraints have influenced LAMTOR3 evolution across primates and identify species-specific adaptations in protein-protein interaction interfaces.

• Functional Divergence Assessment: Experimental comparison of LAMTOR3 from different primates in cellular assays can determine if functional properties like amino acid sensing or MAP kinase pathway activation have evolved differently among lineages.

• Co-evolution Analysis: Examining how LAMTOR3 has co-evolved with its interaction partners (other Ragulator components, Rag GTPases) can provide insights into the evolution of the entire signaling complex.

This evolutionary perspective helps researchers understand how fundamental signaling mechanisms have been maintained or adapted throughout primate evolution, potentially correlating with species-specific metabolic or physiological adaptations.

How does LAMTOR3 contribute to the GEF activity of the Ragulator complex toward Rag GTPases?

The contribution of LAMTOR3 to the GEF (Guanine nucleotide Exchange Factor) activity of the Ragulator complex involves sophisticated molecular mechanisms:

• Heterodimer Formation: LAMTOR3 forms a stable heterodimer with LAMTOR2, which serves as a critical functional unit within the Ragulator complex . This heterodimer directly interacts with Rag GTPases, particularly RagA/B and RagC/D.

• Structural Basis for GEF Activity: Within the Ragulator complex, LAMTOR3 helps create a structural platform that facilitates nucleotide exchange on Rag GTPases. While LAMTOR1 wraps around the other subunits to hold them in place, the LAMTOR2/3 heterodimer is positioned to directly interact with the GTPase domains .

• Nucleotide Exchange Mechanism: The LAMTOR2/3 heterodimer likely induces conformational changes in Rag GTPases that decrease their affinity for GDP, allowing GTP binding and subsequent activation. This activation is crucial for the recruitment of mTORC1 to lysosomes .

• Regulatory Control: Amino acid stimulation triggers the GEF activity of the Ragulator complex through a mechanism involving the lysosomal V-ATPase . LAMTOR3 contributes to this amino acid sensing capability, potentially through conformational changes that enhance its GEF activity.

• Coordinated Function: LAMTOR3 works in concert with other Ragulator components to activate Rag GTPases, which then function as a scaffold recruiting mTORC1 to lysosomes where it is in turn activated .

Understanding these molecular mechanisms requires advanced methodologies such as structural biology approaches, GTPase activity assays, and real-time monitoring of protein-protein interactions in cellular contexts.

What role does LAMTOR3 play in regulating the NLRP3 inflammasome, and how might this differ in Pongo abelii?

Recent research has revealed a novel role for the Ragulator complex, including LAMTOR3, in regulating NLRP3 inflammasome activation:

• Mechanistic Interaction: LAMTOR1, a key component of the Ragulator complex, interacts with both NLRP3 and HDAC6, with the absence of HDAC6 attenuating the interaction between LAMTOR1 and NLRP3 . Although the search results don't detail LAMTOR3's specific role in this process, as part of the Ragulator complex, it likely contributes to this regulatory mechanism.

• Lysosomal Scaffold Function: The Ragulator complex on lysosomes may serve as a scaffold that allows NLRP3 transported by HDAC6 to increase locally . This scaffolding function potentially involves LAMTOR3 through its interactions with other complex components.

• Physiological Significance: Myeloid-specific Lamtor1-deficient mice showed marked attenuation of the severity of NLRP3-associated inflammatory diseases, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate-induced arthritis . This suggests the Ragulator complex, including LAMTOR3, positively regulates inflammasome activation.

• Species Differences: While no specific data is available for Pongo abelii, species-specific variations in inflammasome regulation could exist based on:

  • Sequence differences in binding interfaces between Ragulator components and NLRP3

  • Variations in expression patterns of LAMTOR3 or other Ragulator components in immune cells

  • Species-specific regulatory mechanisms affecting the Ragulator-NLRP3 interaction

• Research Methodology: To investigate potential differences between human and Pongo abelii, researchers should:

  • Perform comparative immunoprecipitation studies to identify species-specific interaction patterns

  • Use inflammasome activation assays in cells expressing orangutan vs. human LAMTOR3

  • Develop species-specific reagents like antibodies against Pongo abelii LAMTOR3

This research area represents an emerging frontier in understanding how fundamental cellular complexes like Ragulator contribute to immune regulation across different species.

What are the common challenges in producing functionally active recombinant LAMTOR3, and how can they be addressed?

Researchers often encounter several challenges when producing functionally active recombinant LAMTOR3:

• Protein Solubility Issues:

  • Challenge: LAMTOR3 may form inclusion bodies in bacterial expression systems.

  • Solution: Optimize expression conditions by lowering temperature (16-18°C), using weaker promoters, or employing solubility-enhancing fusion tags such as SUMO or MBP. Alternatively, consider expression in eukaryotic systems for complex proteins .

• Proper Folding and Stability:

  • Challenge: Ensuring the recombinant protein adopts its native conformation.

  • Solution: Include appropriate reducing agents like DTT (5mM) in purification buffers to maintain disulfide bonds in their correct state . Consider co-expression with chaperones or Ragulator complex partners like LAMTOR2 to enhance folding.

• Complex Formation for Functional Studies:

  • Challenge: LAMTOR3 functions as part of the Ragulator complex and may have limited activity in isolation.

  • Solution: For functional studies, consider co-expressing and co-purifying LAMTOR3 with its binding partner LAMTOR2 or reconstituting the entire Ragulator complex in vitro.

• Protein Aggregation During Storage:

  • Challenge: Maintaining protein stability during storage.

  • Solution: Store in buffer containing stabilizers such as 30% glycerol; maintain at temperatures below -20°C; aliquot to avoid repeated freeze-thaw cycles .

• Activity Assessment:

  • Challenge: Confirming that the recombinant protein is functionally active.

  • Solution: Develop specific activity assays such as binding assays with known interaction partners (LAMTOR2, MEK1, ERK1/2) or GEF activity assays when part of the Ragulator complex.

Implementing these strategies can significantly improve the yield and quality of functionally active recombinant LAMTOR3 for downstream applications.

What controls and validation steps should be included when using recombinant Pongo abelii LAMTOR3 in experimental studies?

To ensure scientific rigor when using recombinant Pongo abelii LAMTOR3, researchers should implement the following controls and validation steps:

• Protein Quality Validation:

  • Verify purity (>95%) using SDS-PAGE and/or HPLC

  • Confirm protein identity via mass spectrometry or western blotting with specific antibodies

  • Assess monodispersity through dynamic light scattering or size exclusion chromatography

  • Verify endotoxin levels are below acceptable thresholds (<1.0 EU per μg)

• Functional Validation:

  • Binding Assays: Confirm interaction with known binding partners (LAMTOR2, MAP kinases)

  • Complex Formation: Verify ability to form complexes with other Ragulator components

  • Subcellular Localization: When introduced into cells, confirm proper localization to lysosomes

• Experimental Controls:

  • Positive Controls: Include well-characterized human LAMTOR3 in parallel experiments

  • Negative Controls: Use non-related proteins of similar size/properties

  • Dose-Response Analysis: Perform titration experiments to establish concentration-dependent effects

• Cross-Species Validation:

  • Compare results with human LAMTOR3 under identical conditions

  • Validate antibody cross-reactivity if using antibody-based detection methods

• Data Reproducibility Measures:

  • Perform biological replicates using different protein batches

  • Include technical replicates to assess experimental variation

  • Document all experimental conditions thoroughly, including buffer compositions, temperatures, and incubation times

These rigorous validation steps ensure that experimental results using recombinant Pongo abelii LAMTOR3 are reliable, reproducible, and physiologically relevant, establishing a solid foundation for comparative studies between human and orangutan protein function.

How can studying Pongo abelii LAMTOR3 contribute to our understanding of cellular signaling evolution in primates?

Studying Pongo abelii LAMTOR3 offers unique opportunities to understand the evolution of cellular signaling pathways through several methodological approaches:

• Comparative Functional Analysis: By comparing the biochemical properties and interaction networks of LAMTOR3 between orangutans and other primates, researchers can identify conserved functional cores versus species-specific adaptations in amino acid sensing and mTORC1 regulation .

• Signaling Network Evolution: LAMTOR3 participates in both mTOR and MAPK pathways, making it an excellent subject for studying the co-evolution of interconnected signaling networks across primate species . Researchers can analyze how interaction interfaces have evolved while maintaining critical signaling functions.

• Adaptive Evolution Assessment: By examining sequence variations in functional domains of LAMTOR3 across primates with different dietary patterns and metabolic requirements, researchers can identify potential adaptive changes related to species-specific nutrient sensing needs.

• Cellular Response Comparison: Experimental studies comparing cellular responses to nutrient availability, stress conditions, or growth factors in cells expressing human versus orangutan LAMTOR3 can reveal functional divergence in cellular signaling mechanisms.

• Structural Biology Approach: Structural comparison of the Ragulator complex containing Pongo abelii versus human LAMTOR3 could reveal subtle differences in protein-protein interaction interfaces that might influence signaling dynamics or regulation .

This research has broader implications for understanding how fundamental cellular processes have been conserved or modified throughout primate evolution, potentially correlating with species-specific physiological adaptations, metabolic requirements, or environmental pressures.

What insights can LAMTOR3 provide about lysosomal positioning and size regulation in different species?

The Ragulator complex, including LAMTOR3, plays critical roles in regulating lysosomal positioning and size, which may vary between species:

• Lysosomal Positioning Mechanism: The Ragulator complex controls lysosome positioning through direct interactions with BLOC-1-related complex (BORC), which promotes lysosome dispersal by coupling to the small GTPase Arl8 and the kinesins KIF1B and KIF5B . LAMTOR3, as part of this complex, contributes to this regulatory process.

• Size Regulation Pathway: The interaction between the Ragulator complex and BORC also regulates the size of late endosomes/lysosomes via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] production . This suggests LAMTOR3 participates in complex lipid signaling events affecting organelle morphology.

• Multivesicular Body Formation: Analysis using Lamtor2 knockout cells has shown that the Ragulator complex contributes to the proper formation of multivesicular body-recycling tubules and regulates membrane/cargo recycling from multivesicular bodies . LAMTOR3, through its interaction with LAMTOR2, likely plays a role in this process.

• Species-Specific Research Approach:

  • Comparative Cell Biology: Researchers can compare lysosomal distribution patterns in cells from different primate species, including Pongo abelii

  • Rescue Experiments: Perform knockout/rescue experiments replacing human LAMTOR3 with the orangutan ortholog to identify functional differences

  • Quantitative Imaging: Use high-resolution microscopy to quantify species-specific differences in lysosomal size, number, and distribution

• Physiological Implications: Species variations in LAMTOR3-mediated lysosomal regulation could reflect adaptations to different metabolic demands, immune challenges, or cellular stress responses across primate evolution.

This research direction bridges fundamental cell biology with evolutionary biology, potentially revealing how subcellular organization has adapted throughout primate evolution while maintaining essential cellular functions.