Yeast Ataxin-2 (Pbp1) Condensates Regulate TORC1 Activity and Autophagy in Response to Cellular Redox State

Yeast ataxin-2, also known as Pbp1 (Poly(A) binding protein-binding protein 1), is an intrinsically disordered protein that has earlier been implicated in stress granule formation, RNA biology, and neurodegenerative disease. However, the normal endogenous function of Pbp1 and ataxin-2 remains poorly understood. In this dissertation, I identified Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Unlike the autophagy-deficient atg mutants that harbor severe growth defects, pbp1 null mutants exhibited significantly increased cell growth despite lack of autophagy. I discovered that Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region of Pbp1 forms reversible cross-β fibrils that facilitate phase transition of the protein into either liquid-like or gel-like states in vitro and enables self-association of full-length Pbp1 into pelletable assemblies in vivo. Sequence analysis revealed that Pbp1 LC region contains an unusually high frequency of methionine residues (24 methionines in 150 a.a.) compared to the rest of the yeast proteome. I showed that the phase separation of Pbp1 is mediated by these methionine residues, which are sensitive to H2O2-mediated oxidation and mitochondrial toxins in living cells. I also observed that the phase separation of Pbp1 mediated by its C-terminal LC region is responsive to the activity state of mitochondria and required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy in vivo. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling. These observations offer a mechanistic explanation describing how reversible formation of condensates formed from the LC region of Pbp1 has evolved as a sensor of cellular redox state.