Correction CELL BIOLOGY Correction for “Phosphorylation of mitochondrial matrix proteins regulates their selective mitophagic degradation,” by Panagiota Kolitsida, Jianwen Zhou, Michal Rackiewicz, Vladimir Nolic, Jörn Dengjel, and Hagai Abeliovich, which was first published September 23, 2019; 10.1073/pnas.1901759116 (Proc. Natl. Acad. Sci. U.S.A. 116, 20517–20527). The authors note that, due to a printer’s error, part of the Acknowledgments section appeared incorrectly. “Global Innova- tion Fund Grant I-111-412.7-2014 (to H.A. and J.D.)” should instead have appeared as “German-Israeli Foundation for Scientific Research and Development Grant I-111-412.7-2014 (to H.A. and J.D.)”. The online version has been corrected. Published under the PNAS license. First published November 18, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1918594116 24374 | PNAS | November 26, 2019 | vol. 116 | no. 48 www.pnas.org Downloaded by guest on September 28, 2021 Phosphorylation of mitochondrial matrix proteins regulates their selective mitophagic degradation Panagiota Kolitsidaa, Jianwen Zhoub, Michal Rackiewiczb, Vladimir Nolica, Jörn Dengjelb,c,d, and Hagai Abeliovicha,d,1 aInstitute of Biochemistry, Food Science and Nutrition, Hebrew University of Jerusalem, 7612001 Rehovot, Israel; bDepartment of Biology, University of Fribourg, 1700 Fribourg, Switzerland; cDepartment of Dermatology, Medical Center–University of Freiburg, 79104 Freiburg, Germany; and dFreiburg Institute for Advanced Studies, University of Freiburg, 79104 Freiburg, Germany Edited by Beth Levine, The University of Texas Southwestern Medical Center, Dallas, TX, and approved August 27, 2019 (received for review March 7, 2019) Mitophagy is an important quality-control mechanism in eukary- mitochondrial matrix protein reporters undergo mitophagy at dras- otic cells, and defects in mitophagy correlate with aging phenom- tically different rates, indicating the existence of a preengulfment ena and neurodegenerative disorders. It is known that different sorting mechanism (31). We were also able to show that altering mitochondrial matrix proteins undergo mitophagy with very mitochondrial dynamics through deletion of the DNM1 gene different rates but, to date, the mechanism underlying this selectivity affected the selectivity that is observed, even though mitochondrial at the individual protein level has remained obscure. We now present dynamics is not absolutely essential for mitophagy per se, as also evidence indicating that protein phosphorylation within the mito- confirmed by others (32). These results led us to speculate that chondrial matrix plays a mechanistic role in regulating selective mitochondrial dynamics, through repeated mitochondrial fission mitophagic degradation in yeast via involvement of the Aup1 mito- and fusion cycles, can distill defective components from the network chondrial protein phosphatase, as well as 2 known matrix-localized while sparing other molecular species (31, 33). However, while fis- protein kinases, Pkp1 and Pkp2. By focusing on a specific matrix sion and fusion can “shake up” the network by mixing components, phosphoprotein reporter, we also demonstrate that phospho-mimetic and nonphosphorylatable point mutations at known phosphosites a distillation process would also require some kind of physical in the reporter increased or decreased its tendency to undergo segregation principle. A hint into the nature of the putative segre- mitophagy. Finally, we show that phosphorylation of the re- gation principle could be related to the function of the mitochon- drial phosphatase Aup1 (also known as Ptc6). Aup1 was originally porter protein is dynamically regulated during mitophagy in an CELL BIOLOGY Aup1-dependent manner. Our results indicate that structural deter- identified by virtue of a synthetic genetic interaction with the minants on a mitochondrial matrix protein can govern its mitophagic Atg1 protein kinase (34). Loss of this gene leads to defects in fate, and that protein phosphorylation regulates these determinants. mitophagy when assayed by some methods, but not by other methods (34, 35). The finding that mitophagy could be protein- mitophagy | autophagy | protein phosphorylation | phosphatase | specific at the intramitochondrial level suggests the possibility that Saccharomyces cerevisiae Aup1 could be involved in generating and maintaining mitophagic selectivity, as this would explain the different experimental out- comes obtained by using different reporters and conditions. In the itochondrial autophagy, or mitophagy, is considered an present work, we demonstrate that perturbations of mitochondrial Mimportant quality-control mechanism in eukaryotic cells protein phosphorylation caused by mutating AUP1 and additional (1–3). Defects in mitophagic clearance of malfunctioning mito- chondria have been proposed to play a role in the pathogenesis Significance of neurological disorders such as Parkinson’s, Alzheimer’s and Huntington’s diseases (4–8) and may be associated with addi- tional aging-related pathologies (9). As with other forms of se- Mitochondrial dysfunction underlies many age-related human lective autophagy, mitophagy is induced by the activation of a pathologies. In normal cells, defective mitochondria are often receptor protein or by its recruitment to the organellar surface. degraded by mitophagy, a process in which these mitochondria are engulfed in autophagosomes and sent for degradation in The receptor interacts with the autophagic machinery to mediate the lysosome/vacuole. Surprisingly, studies on mitophagy in the engulfment of specific mitochondria that are designated for diverse eukaryotic organisms reveal an unexpected dimension degradation (10, 11). In many mammalian cell types, loss of of protein-level selectivity, wherein individual protein species mitochondrial membrane potential stabilizes the PINK1 protein exhibit divergent rates of mitophagic degradation. In this re- kinase on the outer mitochondrial membrane (12). This leads port, we show that this surprising intramitochondrial selectiv- to the PINK1-dependent recruitment and phosphorylation of ity can be generated by differential phosphorylation of Parkin, a ubiquitin E3 ligase (13–15), as well as to the local pro- – individual mitochondrial protein species, and we identify mi- duction of phospho-ubiquitin (16 19). Phospho-ubiquitylation of tochondrial phosphatases and kinases that contribute to this mitochondrial outer membrane proteins recruits the soluble regulation. By identifying a mechanism that regulates the mitophagy receptors NDP52 and optineurin, which link the de- intramitochondrial selectivity of mitophagic degradation, our fective mitochondrial compartment with the autophagic machin- findings open the door to potential manipulation of the ery and mediate its degradation (20). Mitophagy has also been quality-control process in the future. implicated in specific developmental transitions in mammals, such – as muscle, neuron, and erythrocyte differentiation (21 25). In Author contributions: P.K., J.D., and H.A. designed research; P.K., J.Z., M.R., and V.N. yeast cells, the Atg32 mitophagy receptor is a type 2 mitochondrial performed research; P.K. contributed new reagents/analytic tools; P.K., J.Z., M.R., J.D., outer membrane protein that is found on all mitochondria (26, 27) and H.A. analyzed data; and P.K., J.D., and H.A. wrote the paper. but undergoes posttranslational modifications that activate it, The authors declare no conflict of interest. presumably on specific mitochondria that are thus marked for This article is a PNAS Direct Submission. degradation (28–30). Published under the PNAS license. An important question is whether the representation of matrix 1To whom correspondence may be addressed. Email: [email protected]. proteins in mitochondria destined for degradation is identical to the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. average representation in the general mitochondrial network. We 1073/pnas.1901759116/-/DCSupplemental. previously determined that, in Saccharomyces cerevisiae,different First published September 23, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1901759116 PNAS | October 8, 2019 | vol. 116 | no. 41 | 20517–20527 genes encoding mitochondrial kinases affect the selectivity of Aco1 (1 of 2 mitochondrial aconitase isoforms in yeast) over the mitophagy. We also show that point mutations at specific phospho- time course of stationary-phase mitophagy (34). To test the in- sites in a known mitochondrial matrix phosphoprotein, Mdh1, affect volvement of Aup1 in mitophagy using more up-to-date meth- the mitophagic efficiency of the protein, and that mitophagic effi- ods, we followed the effects of deleting AUP1 in yeast expressing ciency of Mdh1-GFP is determined downstream of Aup1 function. a chimeric Mdh1-GFP fusion protein. As previously shown for Thus, our data indicate that targeting of individual protein molecules other reporter proteins, delivery of the GFP chimera to the for mitophagy depends on specific structural determinants and can be vacuole results in degradation of the native Mdh1 moiety, while further regulated by protein phosphorylation. the residual GFP portion is resistant to vacuolar proteases, generating the appearance of free GFP in immunoblots (36). Results This can then be used as a semiquantitative readout of the ef- A Role for Mitochondrial Phosphatases and Kinases in Regulating the ficiency of mitophagy (Fig. 1). We find that Aup1 is required for Intramitochondrial