Mitophagy is an important quality-control mechanism in eukaryotic cells, and defects in mitophagy correlate with aging phenomena and neurodegenerative disorders. It is known that different mitochondrial matrix proteins undergo mitophagy with very different rates but, to date, the mechanism underlying this selectivity at the individual protein level has remained obscure. We now present evidence indicating that protein phosphorylation within the mitochondrial matrix plays a mechanistic role in regulating selective mitophagic degradation in yeast via involvement of the Aup1 mitochondrial protein phosphatase, as well as 2 known matrix-localized protein kinases, Pkp1 and Pkp2. By focusing on a specific matrix phosphoprotein reporter, we also demonstrate that phospho-mimetic and nonphosphorylatable point mutations at known phosphosites in the reporter increased or decreased its tendency to undergo mitophagy. Finally, we show that phosphorylation of the reporter protein is dynamically regulated during mitophagy in an Aup1-dependent manner. Our results indicate that structural determinants on a mitochondrial matrix protein can govern its mitophagic fate, and that protein phosphorylation regulates these determinants.

The mitophagic degradation of mitochondrial matrix proteins inSaccharomyces cerevisiaewas previously shown to be selective, reflecting a pre-engulfment sorting step within the mitochondrial network. This selectivity is regulated through phosphorylation of mitochondrial matrix proteins by the matrix kinases Pkp1 and Pkp2, which in turn appear to be regulated by the phosphatase Aup1/Ptc6. However, these same proteins also regulate the phosphorylation status and catalytic activity of the yeast pyruvate dehydrogenase complex, which is critical for mitochondrial metabolism. To understand the relationship between these two functions, we evaluated the role of the pyruvate dehydrogenase complex in mitophagic selectivity. Surprisingly, we identified a novel function of the complex in regulating mitophagic selectivity, which is independent of its enzymatic activity. Our data support a model in which the pyruvate dehydrogenase complex directly regulates the activity of its associated kinases and phosphatases. This regulatory interaction then determines the phosphorylation state of mitochondrial matrix proteins and their mitophagic fates.

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Mitophagy, or the autophagic degradation of mitochondria, is an important housekeeping function of eukaryotic cells that prevents the accumulation of defective mitochondria due to oxidative damage and spontaneous mutations. The culling of defective mitochondria is thought to delay the onset of aging symptoms, and defects in mitophagy have been linked to late onset disorders such as Parkinson's disease and type II diabetes. We previously demonstrated that different mitochondrial matrix proteins undergo mitophagy at different rates. We now find that dynamic mitochondrial matrix protein phosphorylation and dephosphorylation can generate a segregation principle that would couple with mitochondrial fission and fusion to selectively degrade sub‐sets of mitochondrial proteins on the basis of differential covalent modification. Our data support a model wherein differences in protein‐protein interactions between differentially phosphorylated proteins of the same species can drive a microscopic phase separation which, coupled with fusion‐fission dynamics, may account for the observed selectivity.

The mitophagic degradation of mitochondrial matrix proteins is selective, reflecting a pre-engulfment sorting step. This selectivity is regulated through phosphorylation of mitochondrial matrix proteins by the matrix kinases Pkp1 and Pkp2, which in turn appear to be regulated by the phosphatase Aup1/Ptc6. However, these proteins also regulate the phosphorylation and catalytic activity of the yeast pyruvate dehydrogenase complex. To understand the relationship between these two functions, we tested the effect of deleting PDA1 on mitophagic selectivity. We report that pda1Δ cells show a mitophagy selectivity phenotype nearly identical to that of the pkp1Δ pkp2Δ double mutant. However, this is not due to a role for pyruvate dehydrogenase enzymatic activity in regulating mitophagy. Rather, our data suggest a novel mechanism wherein the pyruvate dehydrogenase complex directly regulates its cognate kinases and phosphatases, independent of its catalytic activity, to determine the phosphorylation state of mitochondrial matrix proteins in response to metabolic cues.