A current view on molecular mechanisms and machineries driving unconventional pathways of protein secretion.

Exploring the effects of Golgi Reassembly and Stacking Proteins in lipid membranes.

Many proteins can reach the cell surface through a Golgi-independent unconventional protein secretion (UPS) pathway, particularly under cellular stress conditions. However, the molecular mechanisms that mediate UPS remain largely elusive. In this study, VPS26A-containing retromer complex, along with the sorting nexin SNX27, is identified as a regulator of UPS of transmembrane proteins, including the trafficking-deficient ∆F508 mutant CFTR, which causes cystic fibrosis, and the SARS-CoV-2 spike protein, associated with COVID-19. A targeted CRISPR knockout screen identified VPS26A as a key contributor in the UPS of ∆F508-CFTR. Subsequent molecular analyses revealed that SNX27 recruits ∆F508-CFTR to the VPS26A-VPS35-VPS29 retromer complex, facilitating its transport to the cell surface under UPS-inducing conditions. Additionally, VPS26A and SNX27 are necessary for UPS of the spike protein, enabling the formation of intact SARS-CoV-2 virions. These findings suggest that the retromer complex and SNX27, known for their roles in recycling endosomes, mediate previously unrecognized functions in the UPS of transmembrane proteins.

Misfolded proteins lacking signal sequence can be secreted into the extracellular space via an unconventional protein secretion (UcPS) process termed misfolding-associated protein secretion (MAPS), which involves HSP70 and a membrane-associated HSP70 co-chaperone named DNAJC5. Here, we show that DNAJC5 can be palmitoylated by several DHHC palmitoyl acyltransferases in human cells. Among them, DHHC11 has a modest activity toward DNAJC5, but its overexpression enriches DNAJC5 in a Golgi-associated compartment, which correlates with increased secretion. Mutagenesis studies show that a minimum DNAJC5 module (DC95) consisting of the palmitoyl acceptor-enriched cysteine string (CS) domain plus the C-terminal 62 residues and a short upstream segment is sufficient to drive palmitoylation, Golgi translocation and secretion. In contrast, removal of 5 residues from DC95 abolishes its palmitoylation, Golgi association and secretion. These findings suggest that the palmitoylation sites of DNAJC5 act with flanking sequences to control its subcellular localization and UcPS function.

Unconventional protein secretion (UcPS) enables the export of cytosolic proteins through pathways that bypass the canonical endoplasmic reticulum-Golgi secretory route. Although increasingly recognized as essential for intercellular communication, stress responses, and tissue homeostasis, UcPS remains difficult to quantify due to low secretion efficiency, high intracellular background, and the challenge of distinguishing active secretion from passive leakage. Recent methodological advances, including NanoLuc split luciferase-based reporters and the Retention Using Selective Hooks (RUSH) system for synchronized protein transport, have improved sensitivity and temporal control of trafficking. Here, we present complementary protocols integrating these tools to provide a highly sensitive, quantitative workflow centered on a split NanoLuc (HiBiT/LgBiT) complementation assay for monitoring UcPS in mammalian cells. The Basic Protocol describes a robust luminescence-based secretion assay, while the Support Protocols detail the generation of stable HiBiT reporter cell lines, approaches for probing UcPS mechanisms using siRNA-mediated gene knockdown and pharmacological perturbation, and the incorporation of the RUSH system to synchronize cargo release and identify potential trafficking intermediates. Together, these protocols provide a sensitive, scalable, high-throughput toolkit that enables analysis of UcPS mechanisms across diverse cargo proteins, cell types, and perturbations. This methodological framework allows for rigorous dissection of UcPS pathways in both physiological and disease-relevant contexts. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Split luciferase complementation assay for quantifying UcPS in mammalian cells Support Protocol 1: Generation of stable cell lines expressing HiBiT-tagged cargo proteins for the split luciferase assay Support Protocol 2: siRNA-mediated knockdown to assess the role of candidate genes in UcPS Support Protocol 3: Pharmacological perturbation of UcPS Support Protocol 4: Integration of the RUSH system to synchronize UcPS.

Perspectives on TMED-regulated unconventional protein secretion

The endoplasmic reticulum (ER) is a central organelle for protein synthesis and folding, lipid metabolism and calcium signaling, etc. To maintain ER homeostasis, cells employ a specific autophagy process termed ER-phagy (reticulophagy), which depredates ER components via three forms: macro-ER-phagy (involving bulk ER sequestration), micro-ER-phagy (lysosome-direct), and ER-to-lysosome-associated degradation (ERLAD). The identification of specific ER-phagy receptors including FAM134A, FAM134B, FAM134C, TEX264, SEC62, RTN3L, CCPG1, ATL3, CALCOCO1 and others has significantly advanced our understanding of ER quality control mechanisms. In this review we summarize the current knowledge on ER-phagy receptors, and emerging evidence linking ER-phagy dysfunction to various disease pathologies including neurological disorders, cancer, metabolic diseases, cardiovascular diseases, infections and immune disorders. Recent evidence shows that ER-phagy receptors can form novel ER-derived structures, such as ER-tubular bodies (ER-TBs) consisted of ATL3 and RTN3L, which mediate Golgi-bypassing unconventional protein secretion under stress conditions, revealing non-degradative functions of these receptors beyond quality control. Targeting ER-phagy receptors may provide insights into potential therapeutic strategies for diseases associated with this fundamental cellular process.

Endothelial cells detect pathogens through pattern recognition receptors, such as Toll-like receptor 4 (TLR4), which triggers the synthesis and secretion of molecules that initiate the innate immune response. Proteins bearing signal peptides are secreted through the classical endoplasmic reticulum (ER)–Golgi–dependent route, whereas select signal-peptide-lacking cytoplasmic proteins are secreted via less well-characterized ER-Golgi-independent mechanisms, collectively termed unconventional cytoplasmic protein secretion (UCPS). To systematically characterize the secretome of human umbilical vein-derived endothelial cells (HUVECs) and delineate the contribution of UCPS, we performed deep quantitative proteomics on HUVEC cell lysates and conditioned medium before and after TLR4 stimulation with lipopolysaccharide (LPS). Of 5205 proteins detected in either fraction, 381 were enriched in the conditioned medium and therefore classified as secreted. Of these, 333 proteins (87.4%) were secreted via the conventional pathway, and 48 (12.6%) were secreted via UCPS, 43 of which were not previously associated with this process. Predicted functions of UCPS-secreted proteins include redox regulation, proteostasis, cytoskeletal remodeling, and innate immune signaling. We confirmed that α-globin (HBA1), which functions as a redox sensor and regulator of nitric oxide in endothelial cells, is secreted constitutively by UCPS and at higher levels following inflammatory activation. Notably, UCPS cargo identity showed poor concordance with current computational predictors, underscoring the need for empirical datasets. Overall, our findings suggest that the HUVEC secretome includes both conventionally and unconventionally secreted proteins that regulate coagulation, angiogenesis, and immune function. Our findings establish a high-quality secretome dataset for HUVECs, providing a novel resource for future efforts to define the molecular determinants governing UCPS cargo selection and trafficking related to endothelial cell function.

Within neurons, the misfolding and aggregation of certain proteins has been identified as a common feature of many late-onset neurodegenerative diseases (NDs). These aggregate-prone proteins include tau (in both primary tauopathies and in Alzheimer's disease) and alpha-synuclein in Parkinson's disease. There is strong experimental evidence that the upregulation of intracellular clearance pathways (autophagy and ubiquitin-proteasome pathways) can clear aggregate-prone proteins in experimental models. When the flux through these pathways is increased, the levels of aggregate-prone proteins are reduced, resulting in improved cell survival in both cell-based and animal models of ND. More recently, a third strategy for clearing proteins from cells has been identified, via the unconventional secretion of proteins out of the cell. However, secretion may also facilitate the spreading and propagation of disease through a prion-like process. This review explains how the autophagy and unconventional secretion pathways interact and how these impact ND.

Unconventional protein secretion (UcPS) exports diverse signal peptide-lacking cargoes, yet its cargo selectivity remains poorly understood. Here, we identify TMED proteins as key regulators of vesicle-dependent UcPS, mediating selective cargo release via translocation into secretory carriers. TMED proteins act as translocators, facilitating cargo passage across lipid bilayers with assistance from HSP90 chaperones and partial cargo unfolding. Selectivity arises during translocation, where TMED cytoplasmic tails bind specific cargoes. The ER-Golgi intermediate compartment (ERGIC) is essential for TMED-mediated translocation and release. TMED homo-oligomerization enhances translocation, while hetero-tetramerization inhibits it. ERGIC localization promotes homo-oligomerization, which is further stabilized by cargo binding, forming a feed-forward mechanism to enhance translocation. These findings establish TMED proteins as central regulators of cargo diversity in UcPS, with their oligomerization and subcellular localization modulating translocation efficiency.

The efficacy of anticancer treatments, including radiotherapy, depends on the activation of type I IFN signaling. However, its regulatory networks and mechanisms remain to be elucidated. Here, we report that tumor cell–intrinsic type I IFN signaling can be transferred to macrophages via secretory autophagy, inducing CXCL9hi macrophages and enhancing CD8+ T cell–mediated antitumor immunity. Mechanistically, K63-linked ubiquitination at the K167 site of phosphorylated STAT2 (p-STAT2) facilitates its binding to LC3B, promoting the loading of p-STAT1 and p-STAT2 into extracellular vesicles and intercellular transference from tumor cells to macrophages, which, however, is suppressed by USP5-mediated STAT2 deubiquitination. Genetic depletion or pharmacological inhibition of USP5 promotes autophagy-dependent unconventional protein secretion of p-STAT1 and p-STAT2, leading to the induction of CXCL9+ macrophages. This process promotes the expression of T cell chemokines and upregulates the antigen presentation machinery, thereby enhancing radiation-induced CD8+ T cell antitumor immunity and radiotherapy efficacy. Our findings reveal a critical role of USP5 in type I IFN–induced antitumor immunity, providing potential targets for improving the efficacy of radiotherapy.

In addition to the classical secretory pathway, which involves the transport of endoplasmic reticulum (ER)-derived cargo through the cisternal stacks of the Golgi apparatus, several Golgi-independent transport routes contribute to the delivery of membrane-bound and soluble proteins to the plasma membrane (PM) or the extracellular space. Some of these transport events bypassing the Golgi stacks-collectively referred to as unconventional protein secretion (UcPS)-employ vesicular intermediates. However, the nature of these intermediates and the intracellular pathways utilized by various unconventional cargo are only partly understood. Notably, beyond its role in conventional secretory trafficking at the ER-Golgi interface, the intermediate compartment (IC) has been identified at the crossroads of two distinct vesicular UcPS pathways, participating in the sorting and Golgi-independent trafficking of both ER-synthesized (ER translocation signal-containing) and leaderless cytoplasmic cargo. Additionally, findings indicating that the IC directly interacts with the endocytic recycling system may help clarify how various unconventional cargo proteins reach the cell surface. Given that the central IC elements and recycling endosomes (REs)-defined by the GTPases Rab1 and Rab11, respectively-are closely associated with, and may even link the Golgi stacks, this chapter explores the connection between the classical and unconventional secretory routes and their potential divergence at the level of the Golgi ribbon.

TMED10 is involved in unconventional protein secretion and ER-Golgi trafficking. TMED10 may exert protumorigenic or oncosuppressive functions in different tumor types, but its role in human cutaneous melanoma has never been explored. Here, TMED10 expression has been investigated in human benign melanocytic tumors and cutaneous malignant melanoma (cMM). This study utilized in silico analysis on various publicly available web platforms to investigate TMED10 gene expression in normal skin and human melanoma at different Clark levels, and immunohistochemistry and digital morphometric analysis of TMED10 protein levels in 60 human samples of benign melanocytic tumors and cMMs at different Breslow T category. In silico analysis revealed that TMED10 is upregulated in cMM compared to normal skin. TMED10 expression in cMM positively correlated with Clark level at diagnosis, and higher expression was associated with the BRAFV600E mutation and poorer patient survival. TMED10 co-expression with TMED2, TMED8, HSP90AA1 and HSP90B1, along with Gene Ontology enrichment analysis, supported its role in ER-Golgi trafficking and unconventional protein secretion in human melanoma. Digital morphometric analysis of immunohistochemical investigation of 60 human specimens confirmed a significant increase in TMED10 protein levels with Breslow thickness-based T category in tumor cells and tumor-associated blood vessels. At variance, TMED10 immunoreactivity in infiltrating lymphocytes was reduced in T3 and T4 cMM. These findings point to a pro-tumorigenic function of TMED10 in human cMM and pave the way to further studies aimed at identifying its contribution to tumor progression, neovascularization, and immune escape, and its potential as a therapeutic target in cMM.

The La nucleoprotein/Sjögren syndrome type B antigen (La/SSB) is an RNA-binding factor in the nucleus that is associated with autoimmune diseases. In this study, we show that La/SSB is a biomarker for liver cancer. We detected full-length La/SSB in several liver cancer cell lines, but not in colorectal cancer cell lines. Importantly, serological analysis using a hepatocellular carcinoma (HCC) mouse model and human samples demonstrated that extracellular detection of La/SSB is specific for HCC. Furthermore, we found that the addition of exogenous La/SSB extract induces extracellular signal-regulated kinase (ERK) activation, which leads to accelerated cell growth. This study suggests that the La/SSB is a novel biomarker for HCC, and in the microenvironment, it may act as a growth factor in the progression of liver cancer.

Age-related macular degeneration (AMD) is the most common disease of the elderly that leads to the loss of sight. So far, no satisfactory therapy exists for this complex eye disease. The appearance of extracellular deposits, called drusen, on the outside of the retinal pigment epithelium (RPE) is considered to be the main clinical hallmark of AMD. Whilst the mechanisms of drusen formation are not well known, secreted material from the RPE, during its degeneration, is thought to contribute to the development of AMD. Various unconventional protein secretion (UPS) pathways are considered to be routes for the delivery of material which form the drusen. The two main forms of UPS are secretory autophagy, which is responsible for the cleansing of cellular debris from the RPE cells and endosomal secretion which carries material outside of the cell via exosomes. These pathways are unconventional in the sense that they comprise the delivery of material to the exterior of cells by bypassing the Golgi apparatus. Although secretory autophagy and exosome release are regarded as different routes by which cells exude material, they share similarities, such as common molecular participants and that their routes converge. Therefore, manipulation of these two processes might be useful in a therapy against AMD by diminishing the destructive drusen progression in the vicinity of the RPE.

The unfolded protein response (UPR) initiated under endoplasmic reticulum (ER) stress can not only maintain the ER homeostasis but also modulate the secretion of proteins and lipids that transmit ER stress signals among cells. Exosomes are multivesicular body (MVB)-derived extracellular vesicles, constituting the unconventional protein secretion pathway. Whether and how the secretion of exosomes is regulated by the UPR remains largely unknown. Here, we reported that ER stress induces exosome secretion in an UPR-dependent way. Activation of PERK and IRE1α, two of the UPR branches, represses the acidification and catabolic activity of lysosomes. This blocked MVB-lysosome fusion, redirecting MVBs from lysosomal degradation to plasma membrane fusion, resulting in exosome release. Calcium-mediated activation of PERK, in the absence of ER stress, is sufficient to suppress lysosomal degradation and augment exosome secretion, partly through its downstream factor ATF4. Our study revealed a function of PERK and IRE1α in modulating lysosome activity and dictating the fate of MVBs, facilitating cell-to-cell communication via exosomes.

IL-33 is a key driver of type 2 inflammation and implicated in pathology of chronic obstructive pulmonary disease (COPD) and asthma. However, the mechanism for IL-33 secretion and regulation in the context of chronic airway disease is poorly understood. We previously reported an airway disease–associated isoform IL-33Δ34 that escapes nuclear sequestration and is tonically secreted from epithelial cells. Here, we describe how this IL-33Δ34 isoform interacts with HSP70 within cells and is targeted to secretory organelles through coordinated binding to phosphatidylserine (PS) and delivered to compartments for unconventional protein secretion (CUPS). Once secreted, extracellular HSP70 (eHSP70) in complex with IL-33Δ34 stabilizes the cytokine by inhibiting oxidation and degradation, which results in enhanced IL-33Δ34-receptor binding and activity. We further find evidence that IL-33 along with mediators of the proteostasis network HSP70, HSP90, and the Chaperonin Containing TCP1 (CCT) complex are dysregulated in human chronic airway disease. This phenomenon is reflected in the differential extracellular vesicle (EV) proteome in bronchial wash from COPD and asthma samples, which could mark disease activity and potentiate IL-33 function. This study confirms proteostasis intermediates, chiefly HSP70, as chaperones for noncanonical IL-33 secretion and activity that may be amenable for therapeutic targeting in the chronic airway diseases COPD and asthma.

Tubular ER structures shaped by ER-phagy receptors engage in stress-induced Golgi bypass.

Endocytic tethers modulate unconventional GAPDH secretion.

ABSTRACT The secretion of proteins that do not follow the well-characterized endoplasmic reticulum (ER)-Golgi apparatus pathway, known as unconventional protein secretion (UCPS), is gradually revealing its complexities. Our study has identified an ER-based tubulovesicular network, termed ER tubular body (ER-TB), as a central compartment in this process. We demonstrate that ER-TBs are formed by two reticulophagy receptors, ATL3 and RTN3L, under conditions of cellular stress. In addition to their role in stress-induced secretion, the activation of UCPS via ER-TBs facilitates cell surface trafficking of trafficking-deficient transmembrane proteins such as ΔF508-CFTR. Furthermore, their involvement in ER remodeling and vesicle trafficking suggests a potential role in viral replication, particularly in the formation of membrane compartments utilized by positive-strand RNA viruses. By uncovering ER-TBs as key cellular structures in stress-induced UCPS and demonstrating their regulation by autophagy-related factors, our findings offer valuable insights into protein homeostasis, viral pathogenesis, and potential therapeutic strategies for diseases linked to trafficking defects.