Phosphorylation of mitochondrial matrix proteins regulates their selective mitophagic degradation

Abstract 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 two known matrix-localized protein kinases, Pkp1 and Pkp2. By focusing on a specific matrix phosphoprotein reporter, we also demonstrate that phospho-mimetic and non-phosphorylatable 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. Significance statement Mitochondrial dysfunction underlies many age-related human pathologies. In normal cells, defective mitochondria are often degraded by mitophagy, a process in which these mitochondria are engulfed in autophagosomes and sent for degradation in the lysosome/vacuole. Surprisingly, studies on mitophagy in diverse eukaryotic organisms reveal an unexpected dimension of protein-level selectivity, wherein individual protein species exhibit divergent rates of mitophagic degradation. In this manuscript, we show that this surprising intra-mitochondrial selectivity can be generated by differential phosphorylation of individual mitochondrial protein species, and we identify mitochondrial phosphatases and kinases which contribute to this regulation. By identifying a mechanism which regulates the intra-mitochondrial selectivity of mitophagic degradation, our findings open the door to potential manipulation of the quality control process in the future.