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What is mitophagy ?

  • selective recruitment of mitochondria to the pre-autophagosomal site (PAS) or vice-versa
  • vacuolar engulfment
  • internalization into vacuole
  • digestion

Why is it happening ?

  • mitochondrial quality control
  • ensuring mitochondrial health/efficiency status

Why should it be studied ?

  • mitochondrial dysfunction is a hallmark of cancer cells
  • optical atrophy
  • etc.
  • mitochondria are the site of ROS generation
  • at least partially responsible aging

Two signals induce mitophagy

Nitrogen starvation or culturing cells to the post-log phase in a nonfermentable carbon source medium are reliable methods to efficiently induce mitophagy in a wild-type yeast strain. Additionally, mitophagy in yeast has been observed upon mitochondrial dysfunction and under rapamycin treatment, the latter acting via simulation of nitrogen starvation by inhibition of the nitrogen sensing Tor pathway. Presuming, that mitophagy in the post-log phase represents an increased mitochondrial quality control, this data suggests, that mitophagy is subject to two pathways, controlled by different upstream signals.

Why nitrogen starvation ?

alpha-Ketoglutarate may play a role in nitrogen starvation induced mitophagy

While degradation of dysfunctional mitochondria is essential under any nutritional condition (except during strong demands for oxidative phosphorylation for means of ATP production, where even damaged mitochondria might become indispensable), the degradation of mitochondria as a reaction to the absence of nitrogen is not easy to explain. Mechanisticly, the Tor pathway is responsible for nitrogen sensing, therefore TORC1 downstream effectors or TORC1 itself must confer nitrogen starvation induced mitophagy. The fact, that nitrogen starvation induced mitophagy among other general stress responses is not observed in cells deleted for Whi2, connects nitrogen starvation induced mitophagy to induction of STREs.

One possible explanation for this behaviour could be the cell's need for more nitrogen sources, like mitochondrial protein. If this is the reason, we would expect also other mechanisms of protein degradation to be up-regulated. However, this hypothesis is in contradiction with the fact, that protein catabolism involves the TCA, which requires mitochondria and should therefore rather inhibit mitophagy, than induce it.

A different explanation could be an increased need for alpha-Ketoglutarate, which, up to literature, is required for nitrogen assimilation from ammonia. alpha-Ketoglutarate also is a component of the TCA, therefore increased cellular levels of alpha-Ketoglutarate would be degraded by the mitochondria in the TCA, thereby shooting down cellular efforts to increase ammonia assimilation.

The question remains, why mitophagy is required to inhibit the TCA, while intuitively TCA inhibition would be sufficient.

If the second hypothesis is the case, we would also expect the Tor kinase pathway to be related to other alpha-Ketoglutarate associated pathways.

Four proteins are made responsible for assimilation of ammonia using alpha-Ketoglutarate: Gdh1, Gdh2, Gdh3 and Glt1.

  • Deletion of Gdh1 and overexpression of glycerol kinase Gut1, leads to increased growth and ethanol production.
  • Gdh2 binds kinase Nnk1, which interacts with TORC1
  • Gdh3 binds PKA subunit Bcy1
  • Glt1 binds Pho85 and Yak1

ROS may play a role in nitrogen starvation induced mitophagy

The fact, that mitochondria are degraded upon nitrogen starvation, suggests, that mitochondria become disadvantageous. From what we know, the only real disadvantage, mitochondria can represent, is the generation of reactive oxygen species (ROS). Assuming, the ROS production increases upon nitrogen starvation, it would be reasonable to induce mitophagy. However, as stated already above, we would be unable to explain, why ROS production should be expected to increase under conditions of nitrogen starvation.

It's probably the glutathione levels


Mitophagy depends on an actively directed mechanism

Intuitively, we would have expected, that quality control and starvation induced mitophagy function via distinct mechanisms at the mitochondria, since they respond to totally different upstream signals and fulfill a completely different purpose in the cell. However, this assumption seems to be wrong: In cells deleted for Atg32, mitophagy did not rise over 7% of wild-type mitophagy levels, even under stress. If we account this basal, low level of remaining mitophagy to non-specific autophagy, this suggests, that all types of mitophagy depend on a directed mechanism, which is confered only by Atg32 (see below). Most probably, due to their physical size, mitochondria are just too big for efficient bulk phagocytosis, wherefore we would not be surprised, if an additional mechanism would be found, actively recruiting PASs to marked mitochondria, and/or vice-versa. In any case, we conclude, that specifically as well as non-specifically induced autophagy of mitochondria relies on the selective mitophagy mechanism.

However, this remains a weak hypothesis, since we cannot rule out the possibility, that not bulk autophagy, but quality control induced mitophagy accounts for the basal 7% mitophagy in the Atg32 mutant.

  • how to test this?

Different stress signalling pathways control mitophagy

Atg32 anchors mitochondria to the selective autophagy machinery

If we account the remaining basal levels of mitophagy to an unspecific autophagy mechanism, this finding strongly suggests, that Atg32 is the only factor, directly confering mitophagy.

Atg33 conferres mitophagy on only a subset of the mitochondrial pool

Deletion of Atg33 blocks mitophagy to half the level of wild type yeast during nitrogen starvation, whereas it almost completely inhibits mitophagy in stationary phase. The specific requirement of Atg33 for mitophagy primarily in stationary phase confirms, that there are differences between the pathways of mitophagy during nitrogen starvation and stationary phase. Additionally, it was observed, that only a fraction of the total mitochondrial pool is degraded by mitophagy in stationary phase, wherefore it is reasonable to assume, that Atg33 confers the selective degradation of inefficient mitochondria.

Of course, also under conditions of nitrogen starvation, it would make sense, to degrade mitochondria not unspecifically but rather via a efficiency-selective mechanism, acting with an increased stringency.

Atg33 is essential in cells with deficient glycolysis

Menon et al report, that delta gcr1 delta atg33 double deletion is lethal. This goes together with the assumption, that Atg33 is required for mitochondrial quality control (and thus for sustained mitochondrial energy efficiency), since Gcr1 is a transcriptional activator, required for efficient glycolysis. Thereby abolished mitochondrial efficiency control combined with diminished glycolysis efficiency would well explain the double mutant's lethality.

It is unclear, whether Atg33 action depends on Atg11

The question remained, whether Atg33 acts via an Atg11-dependent mechanism.

  • we planned, to compare the protein sequence of Atg11-binding proteins in search for a consensus motif
  • what does BioGRID say about Atg33-Atg11-Atg32 bindings?
  • is there maybe a sequence similarity between Atg11 and Atg33?

Since we previously assumed, that specific mitophagy completely depends on Atg32, we expect Atg33 to act upstream of Atg32, while, the other way around, the action of Atg32 does not depend on Atg33 (see above: still 50% mitophagy observed in Atg33-deleted cells).

Atg32 is known to function via an Atg11-mediated autophagy adaptor system. Therefore it would be possible, that either Atg33 also acts as adaptor to Atg11, or Atg33 functions homolog to Atg11, by binding Atg32. The latter hypothesis we rule out, since we identified Atg33 to be a transmembrane protein (see below). Still, it is unclear, if Atg33 action depends on Atg11 or an entirely different mechanism.

An unidentified kinase may regulate Atg33's activity

PhosphoGrid lists two CK-1 phosphorylation motifs, found by Li X in a large-scale assay. Overexpression of CK-1 isoforms has been shown to rescue starved, stationary-phase cells, deleted for a Gcs1, the latter being required to re-enter mitotic cycle after stationary phase. This suggests, that there exists a CK-1, responding to nutrient availability, phosphorylating Atg33. Taken together with the above assumption, that Atg33 confers mitochondrial quality control, it seems reasonable to connect the underlying mechanism to the levels of available carbon sources. However, due to a lack of further experimental hints it is difficult to predict, whether phosphorylation or dephosphorylation of Atg33 confers up- respectively downregulation. We speculate, that increased nutrient availability increases CK-1 kinase activity, partially but not completely inhibiting Atg33, allowing for an enlarged pool of mitochondria and thus increased metabolization of newly available carbon sources.

Atg33's membrane integration may depend on signal peptide cleavage

According to protein sequence analysis, Atg33, just like Atg32, contains transmembrane motifs, in fact, several of them, suggesting localization to the mitochondrial outer membrane (23 aa mitochondrial outer membrane anchor, see below). Additionally, Atg33 contains a signal peptide subsequence, irreversible cleaved by a signal peptidase I.

Signal peptides have previously been shown to be responsible for allocation of proteins to their designated target organelles. It may therefore be possible, that Atg33's signal peptide is controlling it's integration into the mitochondrial membrane. Considering the role of Atg33 for mitochondrial quality control, this cleavage would most probably not occur spantaneously, but in dependence of mitochondrial quality signals. However, due to a lack of experimental data, this remains a weak hypothesis.

Atg33 may be target to mitochondrial rhomboid protease cleavage

As we explained above, mitochondrial quality is measured in the cell via membrane potential dependent cleavage of Mgm1. The fact, that Mgm1 as well as Atg33 contain an amino-terminal signal peptide sequence (amino acids 1-24 in Mgm1 vs amino acids 1-26 in Atg33), lead us to the question, whether Atg33 may also be a target of a mitochondrial rhomboid protease.

Two such proteases are mentioned in the context of mitochondrial quality controlled mitophagy: Pcp1 and Rbd2. Mgm1 actually is target of Pcp1, therefore we searched for similarities between Atg33 and Mgm1. In fact, protein sequence comprison of Atg33 with the much larger Mgm1 (EMBOSS needle), aligns Atg33 to the amino-terminal end of Mgm1. Although the local sequence similarity, also due to several gaps, is "only" at 31%, considering the promiscousness of Pcp1 we believe, that it is actually likely, that Atg33's integration into the mitochondrial membrane depends on membrane potential dependent peptide cleavage. We suggest experimental studies in this direction.

Atg33 is unlikely to fulfill an active function

Atg33 is comparably small protein (197 aa, 20,4 kDa). After peptide cleavage, only the tiny region between aa 102-171 (70 aa) remains to potentially fulfill an active function. The rest encodes four transmembrane domains:

  • aa 13 - 35 = 23 aa
  • aa 55 - 72 = 18 aa
  • aa 79 - 101 = 23 aa
  • aa 172 - 194 = 23 aa

Mgm1, for comparison, is five times heavier (881 aa, 99,2 kDa), apparently not fulfilling any active function. Notably Atg32 contains only one transmembrane domain (23 aa; aa 389-411), rendering Atg33 a rather strongly anchored transmembrane protein. Besides it's transmembrane domain, Atg32 contains one 388 aa cytosolic and one 119 aa intra-mitochondrial membrane space domain (N-terminal respectively C-terminal), the first one acting as Atg11 adaptor region, the latter not having any apparent function. Therefore it would be cryptic, how the comparably tiny Atg33 region could become active in induction of mitophagy. We rather believe, that there exists a currently unidentified Atg33-binding protein, which is conferring this activity.

In this context it is noteworthy, that Atg33's phosphorylation sites, mentioned above, indeed both ly on this tiny 70 aa region. We predict, that this region exhibits a regulatory function, after the protein's membrane integration.

Atg33's homologs (BLASTP)

NCBI BlastP Atg33 homolog alignment reveals:

  • N-terminal aa 1 - approx. 23 are conserved in
    • Naumovozyma castellii
    • Torulaspora delbrueckii
    • Zygosaccharomyces rouxii
    • Naumovozyma dairenensis
    • Kazachstania africana
    • Vanderwaltozyma polyspora
    • Tetrapisispora phaffii
    • Tetrapisispora blattae
    • Lachancea thermotolerans
    • Candida glabrata
  • C-terminal approx 22 aa
    • same species
  • HPYLL region at approx 80 aa
  • first one, very nicely, corresponding to the signal peptide
  • the latter two to transmembrane domains
  • the phosphorylation sites is not conserved at all

Inhibition of Mitophagy

Cells measuring mitochondrial quality

Fusion and fission from the mitochondrial network

  • mitophagy dependent on fusion/fission ? no! ->Reichert

(Nadine Mendl)

Fission factors

  • Dnm1, Fis1, Mdv1, Caf4

ATP-dependent peptide cleavage

Further references

Paper.svg Ishihara (2009)
A Receptor for Eating Mitochondria
PubMed (title) PubMed (ID) Google Vorlage:Paper
Paper.svg Kanki (2009)
Atg32 is a mitochondrial protein that confers selectivity during mitophagy
PubMed (title) PubMed (ID) Google Vorlage:Paper
Paper.svg Okamoto (2009)
Mitochondria-Anchored Receptor Atg32 Mediates Degradation of Mitochondria via Selective Autophagy
PubMed (title) PubMed (ID) Google Vorlage:Paper
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