Recombinant Xenopus laevis Ragulator complex protein LAMTOR1 (lamtor1)

What is LAMTOR1 and what role does it play in cellular signaling?

LAMTOR1 (Late Endosomal/Lysosomal Adaptor, MAPK And MTOR Activator 1) functions as the foundational subunit of the pentameric Ragulator complex, which also includes LAMTOR2-5. This complex plays critical roles in multiple cellular pathways:

• It serves as a guanine nucleotide exchange factor (GEF) for Rag GTPases

• It mediates the recruitment of Rag GTPases to lysosomal membranes

• It anchors the mTORC1 complex to lysosomes for activation

• It participates in MAPK signaling

LAMTOR1 specifically wraps around the other subunits (LAMTOR2-5) and stabilizes the complex. Through N-terminal myristoylation and palmitoylation, LAMTOR1 anchors the entire Ragulator complex to the lysosomal membrane . This anchoring is essential for the complex's function, as protein levels of LAMTOR2-5 are reduced in cases of LAMTOR1 deficiency.

How does LAMTOR1 differ between Xenopus laevis and other model organisms?

When comparing LAMTOR1 across species, there are both conserved domains and species-specific variations:

While the core functional domains are conserved, these subtle sequence variations may affect protein-protein interactions, especially in cross-species studies. Researchers should consider these differences when designing experiments that rely on specific molecular interactions or when using recombinant proteins from different species .

What expression systems are optimal for producing recombinant Xenopus laevis LAMTOR1?

Several expression systems can be used to produce recombinant Xenopus laevis LAMTOR1, each with specific advantages:

How can I verify the functional activity of recombinant LAMTOR1 in experimental settings?

Verifying functional activity of recombinant LAMTOR1 requires assessing both its binding capabilities and functional effects:

Binding Assays:

• Co-immunoprecipitation with other Ragulator components (LAMTOR2-5)

• Pull-down assays with RagA/B and RagC/D GTPases

• Interaction studies with BORC complex components, particularly lyspersin

Functional Assays:

• mTORC1 activation assay measuring phosphorylation of S6K and 4E-BP1

• Lysosomal localization assay using confocal microscopy

• GEF activity assay toward Rag GTPases

A critical control experiment is to test the ability of recombinant LAMTOR1 to rescue phenotypes in LAMTOR1-deficient cells, such as:

• Restoration of mTORC1 recruitment to lysosomes

• Recovery of amino acid-dependent mTORC1 activation

• Normalization of lysosome positioning in response to amino acid availability

What techniques can be used to study LAMTOR1's interaction with the lysosomal membrane?

LAMTOR1's interaction with the lysosomal membrane is primarily mediated by its N-terminal lipid modifications. To study these interactions:

In vitro techniques:

• Liposome binding assays with purified recombinant LAMTOR1

• Surface plasmon resonance to measure membrane association kinetics

• Fluorescence recovery after photobleaching (FRAP) to assess membrane dynamics

In vivo techniques:

• Confocal microscopy with fluorescently tagged LAMTOR1

• Subcellular fractionation followed by Western blotting

• Proximity labeling methods (BioID or APEX) to identify nearby proteins

Critical for these studies is the use of LAMTOR1 mutants lacking myristoylation (G2A mutation) or palmitoylation sites, which should show reduced membrane association. Additionally, N-myristoyltransferase (NMT) inhibitors have been shown to block LAMTOR1 lysosomal localization, offering a pharmacological approach to studying this interaction .

How does the LAMTOR1-BORC interaction regulate lysosome positioning in response to nutrient availability?

The interaction between LAMTOR1 (as part of the Ragulator complex) and BORC (BLOC-1-related complex) represents a sophisticated mechanism for controlling lysosome positioning in response to nutrient status:

Mechanism:

• BORC promotes lysosome dispersal by coupling to the small GTPase Arl8b and kinesins (KIF1B and KIF5B)

• The Ragulator complex interacts with BORC through direct binding of LAMTOR2 to the lyspersin subunit of BORC

• This interaction involves the hydrophobic site on LAMTOR2 and the DUF2365 CE1 region of lyspersin

• Amino acid starvation strengthens this interaction, inhibiting BORC's ability to recruit Arl8b to lysosomes

Experimental Evidence:

• In LAMTOR-deficient cells, lysosomes are abnormally dispersed to the cell periphery

• Conversely, BORC silencing leads to juxtanuclear clustering of lysosomes

• The lysosomal positioning becomes unresponsive to amino acid starvation when either complex is disrupted

• Y2H analyses revealed that mutations in LAMTOR2's hydrophobic patch (L7, L11, V19, L24, L32, V86, L89, V112, L115) abolish interaction with lyspersin

For researchers studying this interaction, point mutations in the binding interfaces provide valuable tools to disrupt this specific interaction without affecting other functions of these complexes.

What role does LAMTOR1 play in immune cell function and how can recombinant LAMTOR1 be used to study immunomodulation?

LAMTOR1 has emerged as a critical regulator of immune cell function, with particular importance in macrophage polarization and inflammasome regulation:

Macrophage Polarization:

• LAMTOR1 is essential for M2 macrophage polarization

• Lamtor1 deficiency, amino acid starvation, or inhibition of v-ATPase and mTOR result in defective M2 polarization and enhanced M1 polarization

• The mechanism involves liver X receptor (LXR) as a downstream target of Lamtor1 and mTORC1

• Production of 25-hydroxycholesterol is dependent on Lamtor1 and mTORC1

Inflammasome Regulation:

• LAMTOR1 interacts with both NLRP3 and HDAC6

• Lamtor1 deficiency abrogates NLRP3 inflammasome activation in murine macrophages

• Myeloid-specific Lamtor1-deficient mice show attenuated severity of NLRP3-associated inflammatory diseases

Experimental Approaches:

• Reconstitution experiments using recombinant LAMTOR1 in Lamtor1-deficient immune cells

• Structure-function analysis using truncated or mutated LAMTOR1 constructs

• In vitro inflammasome assembly assays incorporating recombinant LAMTOR1

• Evaluation of cytokine production and immune cell polarization markers

This research area offers potential therapeutic applications for conditions involving dysregulated inflammation.

How can mutagenesis of recombinant LAMTOR1 inform structure-function relationships in the Ragulator complex?

Targeted mutagenesis of recombinant LAMTOR1 provides valuable insights into structure-function relationships:

Key Functional Domains for Mutagenesis:

• N-terminal lipidation sites (G2 for myristoylation, C3 for palmitoylation)

• Regions interacting with LAMTOR2-5 (wrap-around region)

• Surfaces involved in Rag GTPase binding

• BORC interaction interface

Experimental Design Approach:

• Generate point mutations or deletion constructs of recombinant LAMTOR1

• Express in LAMTOR1-deficient cells to assess rescue capability

• Evaluate specific functions (membrane binding, complex assembly, mTORC1 activation)

• Perform co-immunoprecipitation to identify altered interaction partners

Example Mutational Analysis Results:

These structure-function studies can be further enhanced by combining mutagenesis with structural techniques such as cryo-electron microscopy and crystallographic analyses that have been applied to the Ragulator complex .

What are common challenges in working with recombinant LAMTOR1 and how can they be addressed?

Researchers working with recombinant LAMTOR1 frequently encounter several challenges:

Challenge 1: Low solubility and aggregation

• Solution: Express with solubility-enhancing tags (MBP, SUMO)

• Use mild detergents during purification

• Optimize buffer conditions (pH 7.2-7.5, 150-300 mM NaCl)

• Include stabilizing agents like glycerol (10-15%)

Challenge 2: Loss of N-terminal lipid modifications

• Solution: Use eukaryotic expression systems that support lipid modifications

• Co-express with N-myristoyltransferase for bacterial systems

• Consider chemical methods for post-purification lipidation

Challenge 3: Improper folding affecting functional assays

• Solution: Refold protein gradually using step-wise dialysis

• Validate folding with circular dichroism spectroscopy

• Include positive controls (commercially validated proteins) in functional assays

Challenge 4: Batch-to-batch variability

• Solution: Standardize expression and purification protocols

• Implement quality control testing for each batch

• Quantitatively assess activity using standardized assays

• Document and report specific storage conditions and shelf-life

How should researchers interpret seemingly contradictory results between LAMTOR1 studies in different model systems?

When interpreting contradictory results across model systems, consider these methodological factors:

Source of Discrepancies:

• Species-specific variations in LAMTOR1 sequence and function

• Different expression systems affecting post-translational modifications

• Cell type-specific binding partners and regulatory mechanisms

• Variations in experimental conditions (nutrient availability, stress conditions)

Systematic Approach to Resolve Contradictions:

• Directly compare protein sequences and identify variations

• Assess the completeness of the Ragulator complex in each model system

• Evaluate experimental conditions, particularly nutrient status

• Consider cell type-specific expression of interaction partners

Case Study Example:
Studies of LAMTOR1's role in mTORC1 activation may show different magnitudes of effect across species. This could be due to:

• Different affinities for Rag GTPases

• Variations in amino acid sensing mechanisms

• Cell type-specific dependencies on the Ragulator complex

To resolve such contradictions, researchers should conduct parallel experiments with LAMTOR1 from different species under identical conditions, or perform cross-species complementation studies to identify functionally conserved domains .

What controls are essential when studying LAMTOR1's role in protein complex assembly?

When investigating LAMTOR1's role in complex assembly, these controls are critical:

Positive Controls:

• Wild-type LAMTOR1 expression to establish baseline complex formation

• Known LAMTOR1-interacting proteins (LAMTOR2-5, Rag GTPases) as verification of binding capability

• Amino acid stimulation to trigger complex assembly and mTORC1 recruitment

Negative Controls:

• LAMTOR1-deficient cells to confirm phenotype and absence of compensatory mechanisms

• Mutant LAMTOR1 lacking key interaction domains

• Non-interacting proteins to verify binding specificity

• Amino acid starvation conditions to demonstrate nutrient-responsive dynamics

Technical Controls:

• Expression level normalization across different LAMTOR1 variants

• Subcellular fractionation quality controls

• Antibody specificity validation

• Co-immunoprecipitation with reverse pull-down

Physiological Readouts:

• mTORC1 activation status (phosphorylation of S6K, 4E-BP1)

• Lysosomal positioning (peripheral vs. juxtanuclear)

• MAPK pathway activation

These controls help distinguish direct LAMTOR1-mediated effects from indirect consequences and establish causality in complex biological systems .

How is LAMTOR1 involved in cancer biology and are there therapeutic implications?

Recent research has uncovered LAMTOR1's involvement in cancer biology:

Cancer-Related Functions:

• cGAS degradation in response to chemotherapy

• LAMTOR1 ablation impedes cGAS degradation, potentially enhancing anti-tumor immune responses

• N-myristoyltransferase-1-mediated myristoylation of LAMTOR1 promotes bladder cancer progression

• LAMTOR1 serves as a scaffold connecting multiple cancer-related signaling pathways

Therapeutic Considerations:

• Inhibition of LAMTOR1 myristoylation may serve as a potential therapeutic strategy

• LAMTOR1-mediated lysosomal positioning affects cancer cell migration and invasion

• Targeting the LAMTOR1-BORC interaction could disrupt cancer cell adaptations to nutrient stress

• Modulation of LAMTOR1 in tumor-associated macrophages could shift polarization from tumor-promoting M2 to tumor-suppressing M1 phenotypes

For researchers studying LAMTOR1 in cancer contexts, recombinant protein tools provide valuable controls for validating antibody specificity and for reconstitution experiments in knockout systems .

What analytical techniques are being developed to study the dynamics of LAMTOR1-mediated complex assembly?

Advanced analytical techniques for studying LAMTOR1-mediated complex assembly include:

Real-time Imaging Approaches:

• Single-molecule tracking of fluorescently tagged LAMTOR1 components

• FRET-based sensors for monitoring protein-protein interactions

• Photoactivatable or photoswitchable LAMTOR1 for pulse-chase experiments

• Super-resolution microscopy to visualize nanoscale organization

Biochemical and Biophysical Methods:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes

• Surface plasmon resonance for kinetic analysis of complex assembly

• Native mass spectrometry to determine complex stoichiometry

• Cryo-electron microscopy for structural analysis of assembled complexes

Computational Approaches:

• Molecular dynamics simulations of LAMTOR1-mediated interactions

• Network analysis of LAMTOR1 interactome in different cellular contexts

• Machine learning approaches to predict complex assembly from multi-omics data

These emerging techniques are providing unprecedented insights into the dynamics and regulation of LAMTOR1-containing complexes .

How might comparative analysis of LAMTOR1 across species inform evolutionary understanding of lysosomal signaling?

Comparative analysis of LAMTOR1 across species offers insights into the evolution of lysosomal signaling:

Evolutionary Conservation Analysis:

• Core functions of LAMTOR1 (mTORC1 activation, lysosomal anchoring) are conserved across vertebrates

• Xenopus LAMTOR1 shares key structural features with mammalian counterparts

• Variations in sequence may reflect species-specific regulatory mechanisms

Research Approach:

• Phylogenetic analysis of LAMTOR1 sequences across diverse species

• Cross-species complementation studies to identify functionally conserved domains

• Comparative analysis of interaction networks in different model organisms

• Assessment of tissue-specific expression patterns across evolutionary lineages

This evolutionary perspective can reveal which aspects of LAMTOR1 function represent ancient cellular mechanisms versus more recently evolved specializations. For instance, the core role in mTORC1 activation appears widely conserved, while some immune functions may represent more recent adaptations in vertebrates .