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bioRxiv · 10.1101/2023.08.28.555228

A RAB7A Phosphoswitch Coordinates Rubicon Homology Protein Regulation of PINK1/Parkin-Dependent Mitophagy

Tudorica, D. A.·Basak, B. L. F.·Cordova, A. P.·Khuu, G.·Rose, K.·Lazarou, M.·Holzbaur, E.·Hurley, J. H.
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Abstract

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a cytoprotective mitophagy response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding protein that acts as a negative regulator of autophagy. The structure of the Rubicon-RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon-RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

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Tudorica, D. A., Basak, B. L. F., Cordova, A. P., Khuu, G., Rose, K., Lazarou, M., Holzbaur, E., Hurley, J. H.. 2023-08-29. A RAB7A Phosphoswitch Coordinates Rubicon Homology Protein Regulation of PINK1/Parkin-Dependent Mitophagy. https://doi.org/10.1101/2023.08.28.555228

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