TRAPPC2L Human

What is TRAPPC2L and what is its function in human cells?

TRAPPC2L (trafficking protein particle complex 2 like) is a core subunit of the TRAPP (Transport Particle Protein) complexes that function as tethering factors during membrane trafficking. This 140-residue protein interacts with other TRAPP components to mediate the contact between vesicles and target membranes, and is thus involved in vesicle-mediated transport of proteins and lipids . TRAPPC2L is related to the X-linked trafficking protein particle complex 2 and plays an essential role in both TRAPP II and TRAPP III complexes, which regulate distinct intracellular trafficking pathways: secretion and autophagy .

What experimental approaches are commonly used to study TRAPPC2L function?

Common experimental approaches to study TRAPPC2L function include:

• Yeast two-hybrid assays to investigate protein-protein interactions, particularly between TRAPPC2L and other TRAPP complex components

• In vitro binding studies to validate protein interactions identified through other methods

• Size exclusion chromatography to assess TRAPP complex assembly and stability

• Membrane trafficking assays using fibroblasts to examine effects on cellular transport processes into and out of the Golgi apparatus

• Measurement of active RAB11 levels to assess downstream effects of TRAPPC2L function

• Whole genome sequencing to identify disease-causing variants in patient cohorts

What pathogenic variants in TRAPPC2L have been identified and what phenotypes are associated with them?

To date, two pathogenic homozygous missense variants in TRAPPC2L have been identified:

• p.(Asp37Tyr) variant: Reported in two unrelated individuals presenting with:

  • Neurodevelopmental delay

  • Post-infectious encephalopathy-associated developmental arrest

  • Tetraplegia

  • Recurrent rhabdomyolysis episodes

• p.(Ala2Gly) variant: Identified in three affected siblings from an Ashkenazi Jewish family with:

  • Global developmental delay

  • Intellectual disability

  • The variant segregated with the neurodevelopmental phenotype within the family

Both variants lead to membrane trafficking defects but appear to disrupt TRAPP complex function through distinct mechanisms .

How do researchers distinguish between pathogenic and benign variants in TRAPPC2L?

Distinguishing pathogenic from benign variants in TRAPPC2L involves a multi-faceted approach:

• Segregation analysis: Confirming the variant co-segregates with disease phenotype in affected families

• Functional studies:

  • Assessing protein-protein interactions (e.g., TRAPPC2L-TRAPPC6a binding)

  • Evaluating TRAPP complex assembly through size exclusion chromatography

  • Measuring membrane trafficking efficiency in patient-derived fibroblasts

  • Quantifying changes in downstream effectors like active RAB11

• In silico prediction tools: Using computational methods to predict the deleterious effects of variants

• Population frequency data: Pathogenic variants are typically rare or absent in general population databases

• Conservation analysis: Evaluating whether the affected residue is conserved across species, suggesting functional importance

What are the optimal experimental designs to assess TRAPPC2L variant pathogenicity?

An optimal experimental design to assess TRAPPC2L variant pathogenicity should include multiple complementary approaches:

• Protein interaction studies:

  • Yeast two-hybrid assays to screen for disrupted interactions with known binding partners

  • Co-immunoprecipitation to validate interactions in mammalian cell systems

  • In vitro binding assays with purified proteins to quantify binding affinities

• Complex assembly analysis:

  • Size exclusion chromatography to assess complex formation

  • Blue native PAGE to visualize intact complexes

  • Immunofluorescence to examine co-localization of TRAPP components

• Cellular trafficking assays:

  • VSVG-GFP trafficking assay to measure ER-to-Golgi transport

  • CD8 reporter assay for post-Golgi trafficking

  • Comparison between patient-derived and control fibroblasts

• Downstream effector analysis:

  • Active RAB11 pull-down assays

  • RAB GTPase activation assays

• Rescue experiments:

  • Complementation studies with wild-type TRAPPC2L in patient-derived cells

  • Generation of isogenic cell lines using CRISPR/Cas9 to introduce or correct variants

How can researchers effectively model TRAPPC2L-related disorders in experimental systems?

Researchers can effectively model TRAPPC2L-related disorders through several complementary approaches:

• Patient-derived cellular models:

  • Primary fibroblasts from affected individuals

  • Induced pluripotent stem cells (iPSCs) differentiated into relevant cell types (neurons, astrocytes)

• Engineered cellular models:

  • CRISPR/Cas9-generated isogenic cell lines with specific TRAPPC2L variants

  • Inducible knockdown/knockout systems to study dosage effects

• Animal models:

  • Knockin mice harboring patient-specific variants

  • Conditional knockout models to study tissue-specific effects

  • Zebrafish models for high-throughput screening and developmental analyses

• 3D organoid systems:

  • Brain organoids to recapitulate neurodevelopmental aspects

  • Co-culture systems to examine cell-cell interactions

• In silico models:

  • Structural modeling of variant effects on protein interactions

  • Systems biology approaches to predict pathway disruptions

Each model system has distinct advantages and limitations, and combining multiple approaches provides the most comprehensive understanding of disease mechanisms.

What is known about the differential roles of TRAPPC2L in TRAPP II versus TRAPP III complexes?

Although both TRAPP II and TRAPP III contain TRAPPC2L as a core component, their differential functions remain an area of active investigation:

• Functional specialization:

  • TRAPP II primarily functions in intra-Golgi and endosomal trafficking pathways

  • TRAPP III is involved in autophagy and ER-to-Golgi trafficking

• Complex-specific interactions: TRAPPC2L may interact with different subunits depending on the complex, contributing to complex-specific functions .

• GEF activity regulation: TRAPP complexes function as guanine nucleotide exchange factors (GEFs) for RAB GTPases. TRAPPC2L may contribute to the specificity of this GEF activity, with variants showing effects on RAB11 activation .

• Tissue-specific roles: The observation that mutations in different TRAPP subunits lead to distinct diseases suggests possible tissue-specific functions of different TRAPP complexes .

• Membrane targeting: Different TRAPP complexes interact with distinct vesicle populations (COPII vs. COPI), and TRAPPC2L may contribute to this specificity .

Comparing TRAPPC2L with Other TRAPP Subunits

Investigating potential functional redundancy between TRAPPC2L and other TRAPP subunits requires specialized experimental approaches:

• Comparative loss-of-function studies:

  • Paired knockdown/knockout experiments of TRAPPC2L and related subunits

  • Quantitative assessment of phenotypic severity with single vs. double knockdown

  • Temporal control of gene silencing using inducible systems

• Cross-complementation experiments:

  • Overexpression of one subunit in cells lacking another

  • Structure-guided design of chimeric proteins combining domains from different subunits

  • Rescue experiments in patient-derived cells

• Interaction mapping:

  • Systematic protein-protein interaction studies between TRAPPC2L and other subunits

  • Competition binding assays to identify shared binding partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Evolutionary analyses:

  • Comparative genomics across species to identify co-evolution patterns

  • Identification of lineage-specific adaptations in TRAPP architecture

  • Analysis of paralogs like TRAPPC2 and TRAPPC2L for functional divergence

• Quantitative proteomics:

  • Stoichiometry analysis of TRAPP complexes in different tissues

  • Protein turnover studies using pulse-chase labeling

  • Proximity labeling to identify tissue-specific interactors

What are the key technical challenges in studying TRAPPC2L function and how can they be addressed?

Researchers face several significant technical challenges when studying TRAPPC2L:

• Complex assembly dynamics:

  • Challenge: TRAPP complexes are multi-subunit assemblies with dynamic composition

  • Solution: Employ advanced structural biology techniques like cryo-EM to capture various assembly states and crosslinking mass spectrometry to map interaction networks

• Tissue-specific functions:

  • Challenge: Different tissues may utilize TRAPPC2L-containing complexes in unique ways

  • Solution: Develop tissue-specific conditional knockout models and perform comparative proteomics across tissue types

• Temporal dynamics of trafficking:

  • Challenge: Membrane trafficking occurs rapidly and involves multiple compartments

  • Solution: Implement live-cell imaging with super-resolution microscopy and synchronized trafficking assays

• Distinguishing direct vs. indirect effects:

  • Challenge: Separating primary effects of TRAPPC2L dysfunction from secondary consequences

  • Solution: Develop acute protein degradation systems (e.g., auxin-inducible degron) to observe immediate effects of TRAPPC2L loss

• Functional redundancy:

  • Challenge: Potential compensation by related proteins like TRAPPC2

  • Solution: Generate double knockout models and perform detailed comparative analyses of paralogs

What emerging research directions might advance our understanding of TRAPPC2L in human health and disease?

Several promising research directions could significantly advance our understanding of TRAPPC2L:

• Systems-level analysis of membrane trafficking networks:

  • Integration of proteomics, functional genomics, and computational modeling

  • Network analysis to identify compensatory pathways and critical nodes

  • Synthetic genetic interaction screens to map functional relationships

• Disease-specific therapeutic approaches:

  • Small molecule screening to identify compounds that can rescue trafficking defects

  • Gene therapy approaches for neurodevelopmental disorders caused by TRAPPC2L variants

  • Identification of downstream effectors that could serve as therapeutic targets

• Broader phenotypic spectrum investigation:

  • Systematic screening for TRAPPC2L variants in diverse patient cohorts

  • Exploration of potential roles in additional disorders based on the pattern observed with other TRAPP subunits

  • Investigation of carrier phenotypes in heterozygous individuals

• Developmental roles of TRAPPC2L:

  • Characterization of TRAPPC2L function during embryonic and postnatal development

  • Investigation of potential roles in neuronal migration, synaptogenesis, and circuit formation

  • Temporal-specific conditional knockout models to identify critical developmental windows

• Integration with cellular stress responses:

  • Investigation of TRAPPC2L's potential role in cellular stress responses, given the occurrence of rhabdomyolysis in patients

  • Examination of membrane trafficking adaptations under various stress conditions

  • Analysis of potential roles in quality control pathways like ERAD (ER-associated degradation)