Sorting of secretory proteins at the trans-Golgi network by TGN46

Joint Public Review:

Lujan et al make a significant contribution to the field by elucidating the essential role of TGN46 in cargo sorting and soluble protein secretion. TGN46 is a prominent TGN protein that cycles to the plasma membrane and it has been used as a TGN marker for many years, but its function has been a fundamental mystery.

In parallel, it remains unclear how most secreted proteins are targeted from the Golgi to the cell surface. These molecules do not contain conserved sequence motifs or post-translation modifications such as lysosomal hydrolases. Cargo receptors for these secreted proteins have remained elusive.

Therefore, these investigations are likely to have a significant influence on the field.

To gain an insight into the molecular role of TGN46 in sorting, they systematically test the impact of the luminal, transmembrane, and cytosolic domains. Importantly and against the current thinking, they demonstrate that the luminal domain of TGN facilitates sorting. Interestingly, neither the cytosolic nor the length of the transmembrane domain of TGN46 plays a role in cargo export. The effects of TGN46 depletion are specific as membrane-associated VSVG remains unaffected.

Interestingly, TGN46 luminal domain also plays an important role in the intracellular and intra-Golgi localization of TGN46, and it contains a positive signal for Golgi export in CARTS. Rigorous, well-performed data support the experimental evidence.

A speculative part of the manuscript, with some accompanying experimental data, proposes that the luminal domain of TGN46 forms biomolecular condensates that help to capture cargo proteins for export.

One important point to discuss is that the effects of TGN46 KO are partial, suggesting that TGN46 stimulates the Golgi export of PAUF but is not essential for this process. The incomplete block is apparent in Fig 1 and in Fig 5D.

Author Response

We thank the reviewers and the editorial team for their assessment and valuable feedback on our manuscript. Their supporting comments reinforce the significance of our findings.

Regarding the specific point raised about the partial effects observed in the TGN46 KO cell line, we acknowledge the importance of addressing this issue in more detail in the revised version of our manuscript. The partial effects observed when using the TGN46 KO cell line are likely caused by several factors:

1. It is important to consider the phenomenon of cell adaptation/compensation, which is documented to occur in gene knockout cell lines. Cells often respond to genetic perturbations by adapting to compensate the loss of a specific gene. These compensatory effects could potentially mitigate the full impact of TGN46 depletion and might explain the partial effects observed.

2. Our data indicate that the absence of TGN46 reduces PAUF secretion, but does not completely block its export. These results align with our proposed role TGN46 in cargo sorting. In its absence, the secretory proteins likely exit the TGN via alternative routes/mechanisms, such as "bulk flow" or by entering other transport carriers in an uncontrolled manner. The partial redistribution of the TGN46-∆lum mutant into VSVG carriers (Figure 4D) supports this likelihood. Importantly, similar situations are observed when unrelated sorting factors are depleted from the Golgi membranes. For example, when the cofilin/SPCA1/Cab45 sorting pathway is genetically disrupted, the secretion of this pathway's clients is inhibited but not completely halted (e.g., von Blume et al. Dev. Cell 2011; J. Cell Biol. 2012).

3. As suggested by the reviewers, it remains possible that TGN46 is not the sole player for cargo sorting. The existence of redundant or alternative mechanisms cannot be ruled out.

In our revised manuscript, we will provide a more in-depth discussion of these factors and their potential contributions to the observed partial effects in TGN46 KO cells. We believe that a comprehensive exploration of these possibilities will improve our understanding of the role(s) of TGN46 in cargo sorting and TGN export.