Recombinant Human Protein MON2 homolog (MON2), partial

What is MON2 and what are its primary cellular functions?

MON2 (MON2 homolog, regulator of endosome-to-Golgi trafficking) is a protein encoded by the MON2 gene located on human chromosome 12 . This protein plays critical roles in:

• Regulating trafficking between the Golgi apparatus and endosomes

• Acting as a scaffold to recruit Dop1 to the Golgi

• Contributing to autophagy regulation by activating GABARAP/LGG-1 in C. elegans and GABARAPL2 in mammals

• Supporting longevity mechanisms in various paradigms including mitochondrial respiration defects and reduced insulin/IGF-1 signaling

For researchers studying MON2 function, fluorescently-tagged constructs (e.g., MON-2::GFP) are commonly used to visualize localization and dynamics in live cells, alongside colocalization studies with organelle markers to determine its precise distribution within cellular compartments.

How is MON2 localized within cellular compartments and what techniques best visualize its distribution?

MON2 displays specific subcellular localization patterns that provide insights into its function:

In C. elegans:

• MON-2::GFP is expressed in multiple cell types including intestinal cells, neuronal cells, and hypodermal seam cells

• It colocalizes with RFP::RAB-10 (a Golgi marker) but not substantially with markers for late endosomes/lysosomes (mCherry::RAB-7) or early endosomes (mCherry::RAB-5)

In mammalian cells:

• MON2 colocalizes with giantin (a Golgi marker) and substantially with RAB11 (a recycling endosome marker)

• It does not colocalize with autophagosome (LC3B), lysosomal (LAMP1), or early endosome (EEA1) markers

• During starvation, MON2 translocates from the Golgi to recycling endosomes, potentially linking to its role in longevity

Recommended visualization techniques include confocal microscopy with immunofluorescence using antibodies against endogenous MON2 or expression of fluorescently-tagged MON2 constructs, combined with established organelle markers to precisely determine subcellular distribution.

What structural features distinguish MON2 from other trafficking regulators?

MON2 possesses distinctive structural characteristics that set it apart from related proteins:

• It shares extensive homology with non-catalytic portions of both BIG and Golgi brefeldin A resistance factor subfamilies of Arf GEFs

• Unlike typical large Arf GEFs, MON2 lacks the Sec7 domain that catalyzes nucleotide exchange on Arf1

• The protein primarily localizes to the trans-Golgi network

Researchers investigating MON2 structure should consider:

• Protein domain analysis to identify functional regions

• Structure prediction tools to model three-dimensional organization

• Structural biology techniques (X-ray crystallography or cryo-EM) for detailed structural characterization

• Comparative analysis with related proteins to understand functional divergence

How does MON2 contribute to autophagy regulation and longevity pathways?

MON2 plays a significant role in autophagy and longevity through several mechanisms:

In C. elegans:

• MON-2 is required for longevity of mitochondrial respiration mutants (e.g., isp-1)

• It works with DOP1/PAD-1 to traffic macromolecules between the Golgi and endosome

• MON-2 upregulates autophagy by activating the Atg8 ortholog GABARAP/LGG-1

• It contributes to longevity conferred by various interventions, including mitochondrial respiration inhibition, reduced insulin/IGF-1 signaling, and dietary restriction

In mammals:

• Mammalian MON2 activates GABARAPL2 through physical interaction, increasing autophagic flux

• This suggests an evolutionarily conserved role in mediating autophagy-dependent longevity

Methodologically, researchers should employ lifespan assays in model organisms with MON2 mutations, autophagy flux measurements, and protein-protein interaction studies to further elucidate these mechanisms.

What methods are most effective for studying MON2 trafficking dynamics?

To effectively study MON2 trafficking dynamics, researchers should consider these methodological approaches:

Live-cell imaging techniques:

• Expression of fluorescently tagged MON2 (e.g., MON2-GFP) to visualize movement in real-time

• Dual-color imaging with compartment markers to track movement between organelles

• FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility and exchange rates

Biochemical approaches:

• Subcellular fractionation to isolate different organelles and quantify MON2 distribution

• Immunoprecipitation to identify trafficking-related interaction partners

• Proximity labeling approaches (BioID, APEX) to identify proteins in close proximity to MON2

Perturbation strategies:

• Generation of domain-specific mutations to assess impact on trafficking

• Use of trafficking inhibitors to disrupt specific pathways

• RNAi or CRISPR knockout of suspected trafficking partners

The evidence that MON2 translocates from the Golgi to recycling endosomes during starvation provides a valuable experimental paradigm for studying condition-dependent trafficking dynamics.

How is the MON2-Dop1 interaction regulated and what are its functional implications?

The MON2-Dop1 interaction represents a key aspect of MON2 function with significant implications:

Interaction details:

• MON2 forms a complex with Dop1, a large cytoplasmic protein conserved from humans to protozoa

• MON2 acts as a scaffold to recruit the Golgi-localized pool of Dop1

• This interaction is critical for proper membrane organization and trafficking

Functional consequences:

• Deletion of MON2 results in mislocalization of Dop1 from the Golgi

• This mislocalization leads to defects in cycling between endosomes and the Golgi

• While MON2 is dispensable for yeast viability, Dop1 is essential, suggesting Dop1 has additional functions beyond its interaction with MON2

Research methodologies for studying this interaction:

• Co-immunoprecipitation to confirm physical association

• Fluorescence microscopy to track co-localization

• Genetic epistasis experiments to determine functional relationships

• Domain mapping to identify critical interaction regions

• Functional rescue experiments to test conservation across species

The observation that a conditional allele of Dop1 affects both endosome-to-Golgi transport and endoplasmic reticulum organization suggests this interaction has broad implications for cellular membrane dynamics.

What expression systems are optimal for producing recombinant MON2 protein?

For researchers working with recombinant MON2 protein, selection of appropriate expression systems is crucial:

Expression optimization strategies:

• Vector design considerations include promoter strength, codon optimization, and fusion tags

• Growth conditions should be optimized, with lower temperatures (15-25°C) often improving folding

• For membrane-associated proteins like MON2, detergent screening or amphipol stabilization may be necessary

Purification approaches:

• Affinity tags (His, GST, FLAG) for initial capture

• Size exclusion chromatography to remove aggregates

• Ion exchange chromatography to separate different conformational states

Considering MON2's role in membrane trafficking, co-expression with binding partners like Dop1 might improve stability and solubility during recombinant production.

What genetic models are available for studying MON2 function in vivo?

Several genetic models can be utilized to study MON2 function:

C. elegans models:

• MON-2 mutants have been established and characterized for longevity defects

• Transgenic strains expressing MON-2::GFP provide tools for localization studies

• RNAi knockdown approaches have been employed for functional studies

Yeast models:

• MON2 deletion strains show defects in endosome-Golgi trafficking

• These can be used for complementation studies with human MON2

Mammalian cell models:

• CRISPR/Cas9 knockout or knockdown cell lines

• Fluorescent tagging of endogenous MON2

• Inducible expression systems for controlled studies

Research findings indicate that genetic inhibition of mon-2 does not affect expression of several stress response factors (SKN-1, ATFS-1, HIF-1, or CEP-1) in wild-type or isp-1 mutant backgrounds , providing valuable information for designing genetic interaction studies.

How can researchers analyze MON2's role in organelle communication networks?

To analyze MON2's role in organelle communication, particularly among mitochondria, the Golgi, and autophagosomes, researchers should consider:

Organelle proximity analysis:

• Super-resolution microscopy to visualize potential contact sites

• Electron microscopy for ultrastructural examination

• Split fluorescent protein approaches to detect proximity

Dynamic tracking methods:

• Multi-color live cell imaging to simultaneously track MON2 and organelles

• Photoactivatable fluorescent proteins to track subpopulations

• FRAP or photoactivation to measure exchange rates

Functional assays:

• Cargo tracking to measure transport between organelles

• Organelle-specific sensors to measure signaling

• Lipid transfer assays to assess non-vesicular transport

The observation that "MON2 translocated to the recycling endosome from the Golgi during starvation" provides a valuable experimental paradigm for studying dynamic changes in MON2-mediated organelle communication.

How does MON2 function contribute to cellular response during stress conditions?

MON2 appears to play important roles in cellular stress responses:

Starvation response:

• MON2 translocates from the Golgi to recycling endosomes during starvation

• This translocation correlates with MON2's role in starvation-induced longevity

• MON2 is required for longevity conferred by dietary restriction

Mitochondrial stress:

• Mitochondrial isp-1 mutation increases MON-2 protein levels post-transcriptionally

• MON-2 is required for the longevity of mitochondrial respiration mutants

• This suggests MON2 participates in mitonuclear communication pathways

Methodological approaches to study stress responses:

• Time-course analysis of MON2 localization during various stressors

• Quantitative proteomics to measure MON2 levels and modifications during stress

• Genetic interaction studies with stress response pathways

• Transcriptional profiling in wild-type versus MON2-deficient cells under stress

The evidence that genetic inhibition of daf-2 or cco-1 increases MON-2::GFP levels indicates that MON2 responds differentially to distinct stress pathways, suggesting complex integration into cellular stress response networks.

What are the implications of MON2 dysfunction for understanding human disease mechanisms?

While direct links to human disease were not explicitly mentioned in the search results, MON2's cellular functions suggest several potential pathological connections:

Neurodegenerative diseases:

• MON2's role in autophagy regulation suggests potential involvement in protein aggregation disorders

• Defects in endosome-Golgi trafficking could affect neuronal function

Aging-related disorders:

• Given MON2's contribution to longevity pathways , dysfunction might accelerate aging processes

• This could contribute to age-related pathologies across multiple systems

Metabolic disorders:

• The relationship between MON2 and mitochondrial function suggests roles in metabolic regulation

• MON2 dysfunction might affect cellular energy homeostasis

Research approaches should include analysis of MON2 mutations or expression changes in disease cohorts and creation of disease-relevant models with MON2 alterations.

What computational approaches can help predict MON2 structure-function relationships?

Computational methods offer powerful tools for understanding MON2 function:

Structural bioinformatics:

• Homology modeling based on related proteins like BIG and Golgi brefeldin A resistance factors

• Molecular docking to identify potential interaction interfaces with partners like Dop1

• Molecular dynamics simulations to understand conformational dynamics

Network analysis:

• Protein-protein interaction network construction

• Integration of multi-omics data to place MON2 in biological pathways

• Genetic interaction network analysis

Evolutionary analysis:

• Comparative genomics across species (MON2 is conserved from humans to protozoa)

• Analysis of selection pressures on different domains

• Identification of co-evolving protein families

Machine learning applications:

• Prediction of post-translational modification sites

• Identification of trafficking motifs or localization signals

• Integration of diverse data types to predict condition-specific functions

The extensive homology of MON2 "with the noncatalytic parts of both the BIG and Golgi brefeldin A resistance factor subfamilies of Arf GEFs" provides a solid foundation for computational approaches, particularly structural modeling and evolutionary analyses.

What emerging technologies will advance understanding of MON2 functions?

Cutting-edge technologies that could drive MON2 research forward include:

Advanced imaging approaches:

• Lattice light-sheet microscopy for high-speed 3D imaging of trafficking events

• Super-resolution microscopy (STORM, PALM, STED) to resolve suborganelle localization

• Correlative light and electron microscopy (CLEM) to connect dynamics with ultrastructure

Proximity labeling technologies:

• TurboID or miniTurbo for rapid biotin labeling of proteins near MON2

• Split-TurboID systems to detect specific interaction events

• APEX2 proximity labeling for electron microscopy visualization

Optogenetic and chemical-genetic tools:

• Optogenetic recruitment or inactivation of MON2 at specific organelles

• Rapidly degradable MON2 variants using systems like dTAG

• Chemically induced dimerization to manipulate MON2 interactions

Single-cell approaches:

• Single-cell proteomics to analyze MON2 levels across cell populations

• Spatial transcriptomics to map MON2 expression in tissues

• Single-cell metabolomics to correlate MON2 function with metabolic states

These technologies would be particularly valuable for understanding the dynamic translocation of MON2 during starvation and its relationship to autophagy and longevity pathways.

How can researchers best study the interplay between MON2 and mitochondrial function?

The connection between MON2 and mitochondrial function represents an important area for future research:

Evidence for MON2-mitochondria connections:

• MON-2 is required for longevity of mitochondrial respiration mutants

• Mitochondrial isp-1 mutation increases MON-2 protein levels post-transcriptionally

• MON2 contributes to organismal longevity through communication among mitochondria, the Golgi, and autophagosomes

Methodological approaches:

• Mitochondrial morphology and function analysis in MON2-deficient models

• Tracking of mitochondria-derived vesicles and their interaction with the Golgi

• Investigation of mitophagy processes in relation to MON2 function

• Analysis of retrograde signaling from mitochondria to the nucleus in MON2 mutants

The observation that MON2 is required for the longevity conferred by inhibition of mitochondrial respiration suggests a potential role in mitonuclear communication that warrants further investigation.