This manuscript addresses an important question concerning the molecular mechanisms of cargo sorting in the TGN and the delivery of cargo to the plasma membrane and the endo-lysosomal system. The study extends previous work from the von Blume laboratory on the role of Cab45 in regulating sorting of secretory cargo. Using a combination of secretome/MS analysis, biochemical analyses and a RUSH assay to determine the kinetics of cargo exit from the Golgi, the authors propose that Cab45 plays a calcium independent role in regulating packaging and Golgi export of lysosomal hydrolases.

The results are of potential interest however there are significant gaps in the study and inconsistencies in the findings relating to the conclusions and consequence of Cab45 deficiency on trafficking of lysosomal hydrolases. Overall, as it stands the data does not provide a clear and convincing working model on the impact of Cab45 deficiency on the post-Golgi trafficking of lysosomal enzymes. The conclusion that in the absence of Cab45, PSAP and PRGN are not efficiently captured by their receptors and are misrouted to the cell surface (shown in model), and inferred in the abstract, is misleading. There is no evidence that PSAP is misrouted directly to the cell surface from the TGN. The levels of hypersecretion are in fact very modest. From immunoblotting, PSAP is increased by ~30% and there is no significant difference for PGRN (Fig. 2). The use of long secretion times were used to align with the secretome/ analysis. Shorter secretion times are required and the difference between the secretome data and IB data needs to be reconciled. The location of intracellular PSAP and PGRN in Cab45 deficient cells should also be examined to determine if the intracellular pool is correctly localised to late endosomes/lysosomes. The RUSH system to measure kinetics of exit is very nice (however, additional information is needed, see below) and could be used to track the post-Golgi destination and itinerary using a combination of organelle markers; in addition, TIRF can be used to detect direct transport to the cell surface. Given the cargo used in the RUSH analysis is PSAP-mCherry, a time course study could be easily performed to determine the levels of exported PSAP-mCherry in the medium and calculate the % of the total cargo that is exported under the different conditions. Indeed, I was surprised that this was not carried out given the establishment of the RUSH system for these cargoes and the questions addressed in the paper.

The basis for the alteration of lysosome distribution in Cab45 depleted cells is unclear and, I note that the authors do not suggest a possible mechanism. One possibility is that the homeostasis of the endosomal-lysosome system has been perturbated (not just lysosomal positioning) as a consequence of altered membrane flux between the endosomal compartments and the TGN arising from Cab45 deficiency (and altered calcium levels). TGN modifications and altered trafficking flux has been reported to influence the organisation of endosomes/lysosomes in other systems. The morphology and distribution of TGN, and endosomes (recycling endosomes, early endosomes and late endosomes) should be analysed. In addition, the distribution of the M6P-R should also be determined to assess whether M6P receptor recycling between the TGN and endosomes has been perturbed. Given these collective concerns, overall, I think the current study is very premature for publication.

Additional comments/questions

1. Do the exported lysosomal enzymes have a Man-6-P signal? This would also be useful information to indicate the pathway for export, eg. direct from the TGN or indirect from acidic endosomes/lysosomes. Can you blot for the presence of Man-6-P this as there are reports of antibodies (single chain) to detect Man-6-P?

2. In Fig 2A why are there size differences of PSAP and PGRN between the control and Cab45 KD samples from the supernatants?

3. The secretion assay has used starvation conditions for 24 h. The impact of cell lysis following this extended starvation period has not been incorporated into the analysis. This represents an important control.

4. In Fig, 3E. more data points are required for PGRN (and also ideally PSAP to increase significance). Currently there are only the minimum three data points. PGRN cannot claimed to be significant, yet the text says that “PGRN and CatD proteins was only mildly enhanced” (page 10)

5. In Figure 4 using the RUSH system, and discussed above, the destination of PSAP should be established

6. Is regulated secretion affected by Cab45KD in the system under study? Calcium is known to be required for oligomerisation and packaging of cargo into regulated secretory vesicles. Perhaps this is not relevant for HeLa cells.