Subcellular Fractionation: Defining HRD1-Dependent TLR3 Trafficking across the ER–Golgi–Endosomal Axis

(Invent Biotechnologies Inc.)

Precise intracellular trafficking is crucial for Toll-like receptor 3 (TLR3)– mediated innate immune signaling. In their 2025 study, Zhao et al. demonstrate that the E3 ubiquitin ligase HRD1 is indispensable for TLR3 transport from the endoplasmic reticulum (ER) to endosomal compartments, where ligand recognition and signal transduction occur. Through a combination of ubiquitination assays, genetic perturbation, and organelle-resolved biochemical analysis, the authors establish HRD1 as a critical positive regulator of TLR3 signaling rather than a mediator of receptor degradation. Central to these conclusions is the use of targeted subcellular fractionation approaches, including endosome, ER, and Golgi isolation, which allowed direct mapping of TLR3 localization across the secretory and endolysosomal pathways.

HRD1-Mediated Ubiquitination as a Licensing Step for TLR3 Signaling

TLR3 is synthesized in the ER and requires coordinated trafficking through the Golgi apparatus to reach endosomes, where it engages double-stranded RNA and initiates TRIF-dependent signaling cascades leading to IRF3 and NF-κB activation. Zhao et al. identify HRD1 as a key factor required for this process. Loss of HRD1 markedly impaired TLR3-driven type I interferon and inflammatory cytokine production, despite minimal effects on total TLR3 protein abundance.

Mechanistically, HRD1 catalyzes ubiquitination of TLR3 in a manner that promotes receptor trafficking rather than proteasomal degradation. This finding expands the functional scope of ER-associated ubiquitination, positioning HRD1 as a regulator of innate immune receptor maturation and intracellular routing. Importantly, distinguishing trafficking defects from altered expression required direct biochemical assessment of receptor localization, achieved through organelle-specific fractionation.

Endosome Isolation Reveals Failure of TLR3 Signal Compartmentalization

A defining feature of TLR3 signaling is its strict dependence on endosomal localization. Using Invent Biotechnologies’ Endosome Isolation Kit (ED-028), Zhao et al. enriched endosomal fractions to directly assess the signaling-competent pool of TLR3. In HRD1-deficient cells, TLR3 was markedly reduced in endosome-enriched fractions, providing direct biochemical evidence that HRD1 is required for delivery of TLR3 to its functional signaling compartment.

This endosome-specific deficit correlated with impaired recruitment of downstream signaling components and reduced activation of antiviral transcriptional programs. The use of ED-028 was therefore essential in linking HRD1-mediated ubiquitination to functional innate immune signaling, rather than relying solely on microscopy or whole-cell lysate analyses.

ER Retention of TLR3 Demonstrated by ER Enrichment

To determine where trafficking was disrupted, Zhao et al. employed Invent Biotechnologies’ ER Enrichment Kit (ER-036) to isolate ER membranes with high purity. These experiments revealed abnormal accumulation of TLR3 within ER fractions in the absence of HRD1, indicating a failure of ER exit rather than accelerated degradation or misfolding-induced loss.

The ER-resolved data provided by ER-036 were critical in establishing HRD1’s role at an early stage of the TLR3 life cycle. Rather than functioning solely in ER-associated degradation, HRD1 facilitates forward trafficking of a correctly folded immune receptor, underscoring a quality-control–linked checkpoint that licenses TLR3 for progression through the secretory pathway.

Golgi Transit as an Intermediate Trafficking Step

In addition to ER and endosomal analyses, Zhao et al. examined Golgi-associated TLR3 using Invent Biotechnologies’ Golgi Isolation Kit (GO-037). Golgi-enriched fractions showed a pronounced reduction of TLR3 in HRD1-deficient cells, reinforcing the conclusion that HRD1-dependent ubiquitination is required for sequential trafficking through the ER–Golgi axis.

By capturing this intermediate compartment, the authors ruled out alternative models in which TLR3 might bypass conventional Golgi transport or be directly shunted to degradation pathways. Instead, the data support a stepwise trafficking model in which HRD1-mediated ubiquitination enables orderly progression of TLR3 toward signaling-competent endosomes.

Implications for Innate Immune Signal Transduction

This study illustrates a broader principle in innate immunity: signal transduction is inherently compartmentalized, and biochemical access to specific organelles is often required to uncover regulatory mechanisms. The combined use of ED-028, ER-036, and GO-037 allowed Zhao et al. to reconstruct the intracellular itinerary of TLR3 and directly link ubiquitination events to spatial control of signaling.

The findings position HRD1 as a trafficking checkpoint that integrates ER quality control with immune receptor activation. More broadly, the work highlights the value of standardized, reproducible subcellular fractionation kits in dissecting immune signaling pathways that cannot be resolved using bulk lysates alone.

Reference

Zhao, L., He, Z., Sun, Y., Sun, M., Liu, Z., Zhou, Z., et al. (2025). Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling. Nature Communications.