A Commander-independent function of COMMD3 in endosomal trafficking

Reviewer #1 (Public review):

G. Squiers et al. analyzed a previously reported CRISPR genetic screening dataset of engineered GLUT4 cell-surface presentation and identified the Commander complex subunit COMMD3 as being required for endosomal recycling of specific cargo proteins, such as transferrin receptor (TfR), to the cell surface. Through comparison of COMMD3-KO and other Commander subunit-KO cells, they demonstrated that the role of COMMD3 in mediating TfR recycling is independent of the Commander complex. Structural analysis and co-immunoprecipitation followed by mass spectrometry revealed that TfR recycling by COMMD3 relies on ARF1. COMMD3 interacts with ARF1 through its N-terminal domain (NTD) to stabilize ARF1. A mutation in the NTD of COMMD3, which disrupts the NTD-ARF1 interaction, failed to rescue cell surface TfR in COMMD3-KO cells. In conclusion, the authors assert that COMMD3 stabilizes ARF1 in a Commander complex-independent manner, which is essential for recycling specific cargo proteins from endosomes to the plasma membrane.

The conclusions of this paper are generally supported by data, but some validation experiments and control conditions should be included to strengthen the study.

(1) Commander-Independent Role of COMMD3:
While the authors provided evidence to support the Commander-independent role of COMMD3-such as the absence of other Commander subunits in the CRISPR screen and not decreased COMMD3 levels in other subunit-KO cells-direct evidence is lacking. The mutation that specifically disrupts the COMMD3-ARF1 interaction could serve as a valuable tool to directly address this question.

(2) Role of ARF1 in Cargo Selection:
The Commander-independent function of COMMD3 appears cargo-dependent and relies on ARF1's role in cargo selection. The authors should investigate whether KO/KD of ARF1 reduces cell surface levels of ITGA6 and TfR.

(3) Impact on TfR Stability:
Figure 7D suggests that TfR protein levels are reduced in COMMD3-KO cells, potentially due to degradation caused by disrupted recycling. This raises the question of whether the observed reduction in cell surface TfR is due to impaired endosomal recycling or decreased total protein levels. The authors should quantify the ratio of cell surface protein to total protein for TfR, GLUT-SPR, and ITGA6 in COMMD3-KO cells.

Reviewer #2 (Public review):

Summary:

The Commander complex is a key player in endosomal recycling which recruits cargo proteins and facilitates the formation of tubulo-vesicular carriers. Squiers et al found COMMD3, a subunit of the Commander complex, could interact directly with ARF1 and regulate endosomal recycling.

Strengths:

Overall, this is a nice study that provides some interesting knowledge on the function of the Commander complex.

Weaknesses:

Several issues should be addressed.

(1) All existing data suggest that COMMD3 is a subunit of the Commander complex. Is there any evidence that COMMD3 can exist as a monomer?

(2) In Figure 9, the author emphasizes COMMD3-dependent cargo and Commander-dependent cargo. Can the authors speculate what distinguishes these two types of cargo? Do they contain sequence-specific motifs?

(3) What could be the possible mechanism underlying the observation that the knockout of COMMD3 results in larger early endosomes? How is the disruption of cargo retrieval related to the increase in endosome size?

Reviewer #3 (Public review):

Summary:

The manuscript by Squiers and colleagues uncovers a Commander-independent function for COMMD3 in endosomal recycling. The authors identified COMMD3 as a regulator of endosomal recycling for GLUT4-SPR through unbiased genetic screens. Subsequently, the authors performed COMMD3 knockout experiments to assess endosomal morphology and trafficking, demonstrating that COMMD3 regulates endosomal trafficking in a Commander-independent manner. Furthermore, the authors identified and confirmed that the N-terminal domain (NTD) of COMMD3 interacts with the GTPase Arf1. Using structure-guided mutations, they demonstrated that the COMMD3-Arf1 interaction is critical for the Commander-independent function of COMMD3.

Overall, the manuscript presents compelling evidence for a Commander-independent role of COMMD3, and I agree with the author's interpretations. The manuscript uses a combination of genetic screening, microscopy, and structural and biochemical approaches to examine and support the conclusions. This is an excellent and intriguing study and I have only a few comments and suggestions to improve the manuscript further.