Common Principles and Specific Mechanisms of Mitophagy from Yeast to Humans

Abstract

Mitochondria are double membrane-bound organelles in eukaryotic cells essential to a variety of cellular functions including energy conversion and ATP production, iron-sulfur biogenesis, lipid and amino acid metabolism, and regulating apoptosis and stress responses. Mitochondrial dysfunction is mechanistically linked to several neurodegenerative diseases, cancer, and ageing. Excessive and dysfunctional/damaged mitochondria are degraded by selective autophagic pathways known as mitophagy. Both budding yeast and mammals use the well-conserved machinery of core autophagy-related genes (ATGs) to execute and regulate mitophagy. In mammalian cells, the PINK1-PARKIN mitophagy pathway is a well-studied pathway that senses dysfunctional mitochondria and marks them for degradation in the lysosome. PINK1-PARKIN mediated mitophagy relies on ubiquitin-binding mitophagy adaptors that are non-ATG proteins. Loss-of-function mutations in PINK1 and PARKIN are linked to Parkinson´s disease (PD) in humans, and defective mitophagy is proposed to be a main pathomechanism. Despite the common view that yeast cells lack PINK1- and PARKIN-homologs and that mitophagy in yeast is solely regulated by receptor-mediated mitophagy, some studies suggest that a ubiquitination-dependent mitophagy pathway also exists. Here, we will discuss shared mechanisms between mammals and yeast, how mitophagy in the latter is regulated in a ubiquitin-dependent and -independent manner, and why these pathways are essential for yeast cell survival and fitness under various physiological stress conditions.

Keywords: PARKIN; PINK1; autophagy; cancer; mitophagy; quality control; ubiquitin.

Figure 1

Mitochondrial quality control pathways. In response to moderate mitochondrial damage, misfolded or damaged proteins are initially degraded by the mitochondrial protease–chaperone network (1.). The inner mitochondrial membrane-localized AAA⁺ metalloproteases (i-AAA and m-AAA) and the mitochondrial matrix localized ATP-dependent Lon (Pim1 in yeast) protease are main players removing misfolded/damaged proteins. Heat stress-induced aggregation-prone cytosolic proteins are partly imported into mitochondria for degradation in a Hsp104-dependent disaggregation manner. Stalled mitochondrial precursor proteins are degraded via distinct pathways following ubiquitination and proteasomal degradation (2.). These cytosolic ubiquitin-proteasome system (UPS) pathways prevent an accumulation of stalled precursors on the organelle’s surface, safeguarding the translocation channel from being clogged, and thus restore full protein import capacity. Upon mild/local mitochondrial oxidative damage, mitochondrial-derived vesicles (MDVs) are generated that are degraded in the lysosome (3.). In response to severe mitochondrial damage or when other quality control pathways fail, the entire organelle is selectively removed via mitophagy at the cellular level (4.). All levels of mitochondrial quality control presumably occur in parallel, yet the exact interplay and regulation is a matter of current research.

Figure 2

Molecular mechanism of Atg32-mediated mitophagy. (A) Regulation of Atg32 activity. Upon starvation or treatment with rapamycin, Tor1 (target of rapamycin 1), an evolutionarily conserved Ser/Thr kinase, is inactivated, releasing Sin3–Rpd3–Ume6 transcription repressor complex from the ATG32 promoter, and thus allowing a strong transcriptional induction of ATG32. Translated Atg32 is translocated to mitochondria and proteolytically processed by the Yme1 i-AAA protease at its C-terminus. Subsequently, Atg32 is phosphorylated by the Casein kinase 2 (CK2) at two residues, Ser114 and Ser119, that allow interaction with the scaffold protein Atg11 and Atg8 at the PAS. Atg11 may also recruit Dnm1 and the ERMES complex components for mitochondrial fission before mitochondria are sequestered into double-membrane autophagosomes. Atg32 phosphorylation is reversed by a phosphatase Ppg1 and the Far complex. Note that Yme1-dependent processing of Atg32 was observed when mitophagy was initiated by nitrogen starvation. (B) Schematic diagram of functional domains of Atg32 (S. cerevisiae) and Bcl-2-L-13 (mouse). Atg8-interacting motif/LC3-interacting region (AIM/LIR); Ppg1 binding region (Ppg1 BR); pseudo-receiver (PsR) domain; transmembrane domain (TM); S (Ser). The potential phosphorylation sites (S81/83/85) in Atg32 are shown upstream of the AIM motif. The kinase (s) that phosphorylates Bcl-2-L-13 at Ser²⁷² upstream of the functional LIR motif is still unknown. The protein size is shown by the number of amino acid residues.

Figure 3

Interplay of ceramide synthesis, mitochondrial damage, and mitophagy in baker’s yeast. (A). Regulation of Atg32-dependent mitophagy in the wild type (WT) and isc1∆ mutant strains. S. cerevisiae WT cells, when grown over time on non-fermentable carbon sources, maintain a basal level of mitophagy limiting excessive mitophagy. This apparently depends on the recruitment of Isc1 to mitochondria that catalyzes ceramide synthesis from sphingolipids. Mitochondrial ceramides, in turn, may activate mitochondrial localized protein phosphatase Ppg1, thus counteracting CK2-mediated phosphorylation of Atg32 and limiting excessive mitophagy. The isc1Δ cells show mitochondrial damage and fragmentation linked to increased protein expression of Dnm1. The isc1∆ mutant cells show enhanced Atg32-mediated mitophagy promoting cellular viability. (B) Isc1 and Atg32-dependent cellular viability. The mitophagy-deficient atg32Δ strain does not show any defect in the cell growth and viability. The ceramide-deficient isc1Δ strain can still maintain cell viability (∼80% of the wild-type level) by enhancing mitophagy, which acts as a protective mechanism here. However, additional deletion of ATG32 in isc1∆ cells (i.e., isc1Δatg32Δ double mutant) drastically reduces cell survival as Isc1-dependent enhancement of mitophagy cannot operate in the absence of Atg32.

Figure 4

Ubiquitin-dependent mitophagy in mammals and yeast. (A) The PINK1–PARKIN pathway. Upon mitochondrial damage, PINK1 kinase is stabilized on the mitochondrial surface and recruits the E3 ubiquitin ligase PARKIN. PINK1 phosphorylates both ubiquitin (Ub) and PARKIN at the residue Ser65 and generates pSer65-Ub and pSer65-PARKIN, respectively. Mitochondrial localization of PARKIN and its ubiquitin ligase activity is enhanced drastically (∼4400-fold) when pSer65-Ub binds to pSer65-PARKIN. This can create a feedforward loop by providing additional ubiquitin molecules for PINK1 phosphorylation. Some PARKIN substrates (labeled as “Y”) are degraded by the ubiquitin-proteasome system (UPS) during mitophagy. Mitochondria decorated with polyubiquitin/phospho-ubiquitin chains (shown on “X”) are recognized by ubiquitin-binding mitophagy receptors and sequestered by autophagosomes for lysosomal degradation. (B) Ub-dependent mitophagy in yeast. Upon mitophagy induction in respiring cells with treatment with rapamycin, mitochondrial outer membrane (MOM) proteins can be ubiquitinated by unknown E3 ubiquitin ligase(s). The autophagy machinery recognizes ubiquitinated mitochondria for subsequent vacuolar degradation. The cytosolic Ubp3-Bre5 deubiquitinase complex can inhibit mitophagy when it is recruited to mitochondria presumably by removing ubiquitin moieties from MOM proteins.