UC Berkeley

Autophagy is a fundamental process of cellular quality control and maintenance that is used to protect cells during natural, healthy aging. Defects in autophagy are implicated in a host of pathologies of aging, notably neurodegenerative diseases. The Rubicon Homology (RH) proteins are a class of autophagy-regulating proteins defined by their RAB7A binding RH domains, consisting of the proteins Rubicon, Pacer, and PLEKHM1. These proteins bidirectionally regulate autophagy, as determined by the phosphorylation state of RAB7A.

Here, I demonstrate a mitochondrial autophagy (mitophagy) specific form of regulation for RH family proteins. Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding 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.

The stability of the Rubicon protein has recently been established as a natural regulator of autophagy flux in a variety of tissues (adipose, hepatic, cardiac, and neuronal), where cells will stabilize or degrade Rubicon as a means of inhibiting or promoting autophagy flux respectively. To develop a novel pharmacological inducer of autophagy, I sought to mimic this form of regulation by creating a small molecule heterobifunctional degrader of Rubicon that colocalizes Rubicon with the E3 ligase Cereblon, inducing ubiquitinylation and subsequent degradation.