Unconventional protein secretion (UcPS) is an emerging topic in cell biology. Unlike conventional secretion, which requires ER-Golgi trafficking, UcPS bypasses the Golgi and uses unconventional mechanisms to transport cargo out of the cell. Due to the lower efficiency of UcPS compared to conventional secretion, sensitive assays are necessary to detect low amounts of cargo release within a quantifiable range. This work develops a Hibit assay to measure UcPS cargo secretion. When combined with the RUSH system, this assay can also monitor the kinetics of cargo release. The authors further extend this system with a screening approach, identifying several factors that may regulate Tau secretion. Finally, they investigate the effect of Aβ on Tau release. However, the authors overlook that the Hibit system for screening UcPS has already been employed in a separate study to investigate drugs affecting coronavirus-induced UcPS (PMID: 39379362). While they have upgraded this system with the RUSH approach, which should prove helpful in the field by reducing reliance on labor-intensive Western blotting, there are several points that need to be addressed to improve the work.
1. Limitations of the Hibit system: One limitation of the Hibit system is the difficulty in determining whether the secreted signal corresponds to full-length proteins or fragments that contain the Hibit sequence for complementation. This issue is complicated by the authors’ observation that some SOD1 and Tau mutants increased UcPS, which seems counterintuitive, as these mutants are more prone to aggregation, typically inhibiting both type I and type III UcPS unless the cell dies or they are released via MVBs, secretory autophagy, or the MAPS pathway. Alternatively, the increased signal could be due to protease cleavage of Tau or SOD1 mutants within the cell. Therefore, the authors need to clarify: 1) whether the increased release of Tau and SOD1 mutants involves full-length proteins, ideally confirmed by Western blot, 2) whether the secreted Tau or SOD1 mutants are contained within extracellular vesicles (EVs) or are released as free proteins. Additionally, it would be helpful to include GFP secretion data for the SOD1 and Tau mutants as controls. 3) The authors should also consider testing the MAPS pathway or reviewing recent studies on synuclein secretion (PMID: 27295555; PMID: 36626307).
2. EV-related cargo in Figure 3: To strengthen the analysis, it would be useful to assess another EV-related cargo, such as galectin 3, in Figure 3.
3. SCFD1’s role in UcPS: In Figure 2, the authors suggest that SCFD1 is not required for UcPS, which seems unlikely. Many membrane proteins rely on SCFD1 for trafficking to the cell surface, and these proteins are essential for UcPS. For instance, the Na/K ATPase is necessary for FGF2 secretion, and MVB-mediated release of cargo like galectin 3 also depends on SNARE proteins. Therefore, it is crucial for the authors to confirm the efficiency of SCFD1 deletion. They should also assess SNARE function by depleting NSF. Alternatively, the data could be reconsidered or removed to avoid potential confusion.
4. CUPS as a secretory compartment: On lines 98-99, the authors mention CUPS as a type of secretory compartment. However, CUPS is likely a form of the ERGIC that is specific to UcPS. The authors should provide further clarification or explanation regarding this point.
5. EV-mediated versus naked protein release: Lines 185-187 emphasize that some cargoes are released through EVs, but only a small fraction (less than 20%) of these cargoes are actually associated with EVs. This is an important point because it challenges the traditional view that Galectin 3, for example, is primarily released via EVs. The authors should highlight that the majority of the cargoes are still released as free, naked proteins.

The author have answered my questions.