Endosomal Rab cycles regulate Parkin-mediated mitophagy

Essential revisions:

1) Most importantly, the authors do not provide any evidence that endogenous Rab7 and/or endogenous Rab5 is recruited to damaged mitochondria. Such experiments would significantly alter the impact of the authors' findings.

We agree that it would be important to observe mitochondrial recruitment of endogenous RAB5 and RAB7. For this purpose, we obtained commercially available anti-RAB5 and anti-RAB7 antibodies. First, we tested whether these antibodies can be used for immunostaining, and found that the signal stained with anti-RAB5 antibody overlapped with the early endosome marker EEA1 (Figure 7B in the revised manuscript), and the signal stained with anti-RAB7 antibody overlapped with the late endosome/lysosome marker LAMP2 (Figure 1—figure supplement 2A in the revised manuscript). We tested the Rab7 antibody by knocking down endogenous RAB7A by siRNA and showing that the immunostain signal using anti-RAB7 antibody was reduced (Figure 1—figure supplement 2B in the revised manuscript). These results assure us that anti-RAB5 and anti-RAB7 antibodies we used in the revised study properly recognize endogenous RAB5 and RAB7A, respectively.

To test whether endogenous RAB5 and RAB7 are recruited to damaged mitochondria, we used WT, FIS1-/-, and TBC1D15/17 DKO HCT116 cells stably expressing mCherry-Parkin. Under normal growing conditions, neither RAB5 nor RAB7A signals were overlapped with mitochondria (Figure 7C for RAB5 and Figure 1—figure supplement 2C for RAB7A). On the other hand, after mitophagy stimulation by 3hrs of valinomycin treatment, both endogenous RAB5 and RAB7A were recruited to damaged mitochondria especially in FIS1-/- and TBC1D15/17 DKO cells (Figure 7C for RAB5 and Figure 1—figure supplement 2C for RAB7A). These results indicate that not only exogenous, but also endogenous RAB5 and RAB7A are recruited to damaged mitochondria during mitophagy, and loss of mitochondrial Rab-GAPs (TBC1D15 and TBC1D17) stabilizes the association.

2) To finally prove the cascade of events proposed, please show that Rabex-5 and Rab5 are still recruited to OMM of damaged mitochondria upon ablation of Rab7 or MON1/CCZ1. This will confirm that Rab7 and its GEFs are downstream Rabex/Rab5.

In the original manuscript, we have shown that RAB5C recruitment to mitochondria was not inhibited by knocking down of MON1/CCZ1 complex (original Figure 7B and 7C). Now, we show that knocking down RAB7A did not affect the RAB5 recruitment to mitochondria as well (Figure 7D and 7E in the revised manuscript). Furthermore, we tested whether RABGEF1 (Rabex5) recruitment was inhibited by downstream Rabs and Rab-related proteins. As shown in Figure 8—figure supplement 2 in the revised manuscript, in both HeLa and HCT116 cells, neither knocking down MON1/CCZ1 complex nor RAB7A abrogated RABGEF1 recruitment. Therefore, in addition to the original data, these results indicate that RABGEF1 recruitment to damaged mitochondria depends on poly-ubiquitin chains on the surface of damaged mitochondria, but not the downstream Rabs or Rab-related factors.

3) Where in the Rab cascade do the authors place TBC1D15 and TBC1D17? Does depletion of Rab5, Rab7, RABEX5 or MON1A/B&CCZ1 effect the mitochondrial localization of these two Rab GAPs?

TBC1D15 and TBC1D17 were identified as FIS1-binding proteins (Onoue et al., JCS 2013 and Yamano et al., eLife 2014). Indeed, although overexpressed TBC1D15 and TBC1D17 localize in the cytosol, overexpressed FIS1 relocates them to mitochondria (Figure 10A in the revised manuscript). Therefore, mitochondrial localization of these two Rab-GAPs seems to depend on FIS1. However, in order to exclude the possibility that endosomal Rab cycles contribute to the localization of TBC1D15, we knocked down the upstream GEFs and Rabs (RABGEF1, MON1, CCZ1, and RAB7A) and observed endogenous TBC1D15 during mitophagy by immunostaining. As shown in the Figure 10B in the revised manuscript, TBC1D15 still localizes on the mitochondria in cells treated with endosomal Rab siRNAs.