Zala, C. A. | Search bioRxiv

bioRxiv2026

TSPAN3 defines a distinct intracellular trafficking route to secretory multivesicular bodies

Extracellular vesicles (EVs) comprise molecularly diverse populations generated through multiple membrane-trafficking pathways, yet the intracellular basis of this heterogeneity remains poorly understood. Here, we identify the EV-associated tetraspanin TSPAN3 as a marker of a secretory multivesicular body (MVB) population that is molecularly and functionally distinct from canonical CD63-positive compartments. Using endogenous genome editing, live-cell and super-resolution microscopy, electron microscopy, quantitative EV secretion assays, and complementary proteomic approaches, we show that TSPAN3 localizes to fusion-competent MVBs but exhibits limited overlap with CD63 during secretion. Unlike CD63, which extensively traffics through the plasma membrane and depends on YXX{Phi}-mediated endocytic retrieval, TSPAN3 reaches secretory MVBs predominantly through an intracellular trafficking route that relies on a dileucine-containing sorting region. Orthogonal proximity-labeling and affinity-purification proteomics revealed that TSPAN3-positive compartments are associated with a selective LC3/ATG8-related membrane network, including GABARAPL2 and proteins involved in endosomal membrane remodeling and fusion. Perturbation of residues required for this association impaired localization to LC3-positive compartments and reduced secretory MVB fusion. Consistent with these findings, pharmacological disruption of autophagy- and endolysosomal-associated pathways differentially altered TSPAN3-positive EV secretion. Finally, proximity-labeled EV proteomics demonstrated that TSPAN3-associated EVs possess cargo signatures distinct from CD63-associated EVs, with greater representation of endosomal and endolysosomal proteins suggesting that tetraspanin-associated membrane nanodomains retain molecular signatures consistent with their intracellular trafficking history. Together, our findings identify TSPAN3 as a marker of a previously unrecognized secretory MVB population distinguished by its intracellular trafficking, molecular interactions, and EV composition, supporting a model in which distinct tetraspanin-organized membrane nanodomains are associated with different intracellular trafficking routes and molecularly distinct EV populations.

cell biology↗

bioRxiv2026

Stoichiometry-dependent specificity in biotin enrichment: a benchmarking framework for proximity labeling proteomics

Proximity labeling methods (including, BioID, TurboID, ultraID), along with surface proteomics and microdomain mapping, enable proteome-wide identification of spatially proximal proteins via MS-based analysis. These workflows require specific enrichment of biotinylated proteins using affinity purification, yet enrichment specificity can often be compromised by non-specifically bound proteins. As labeling strategies are increasingly applied to complex biological samples with low protein input or low biotin stoichiometry, accurately distinguishing true targets from background becomes a major analytical challenge. Despite its critical impact on data quality and interpretation, the influence of biotinylation level and protein input on enrichment performance remains poorly characterized, limiting the reliability of proximity labeling experiments. To address this, we establish a quantitative benchmarking framework that systematically evaluates biotin enrichment under controlled conditions, including scenarios of low biotin stoichiometry. Using this setup, we show that enrichment specificity strongly depends on biotin stoichiometry: higher levels of biotinylation in samples yield high specificity, whereas low biotinylation increases non-specific background. Reduced protein input further limits recovery of true targets, yet maintains enrichment specificity, highlighting sensitivity constraints of enrichment-based workflows. We apply this framework to biotinylated extracellular vesicle (EV) cargo uptake in recipient cells using ultraID-CD63 labeling. Detection of the most abundant EV cargo proteins under low biotinylation conditions indicates that current workflows approach the lower bounds of biotin enrichment sensitivity. Together, these standards provide a practical reference for evaluating and optimizing biotin enrichment workflows, supporting quantitative and reproducible proximity labeling in proteomics.

molecular biology↗