The pyruvate dehydrogenase complex regulates matrix protein phosphorylation and mitophagic selectivity

  1. Panagiota Kolitsida
  2. Vladimir Nolic
  3. Jianwen Zhou
  4. Michael Stumpe
  5. Natalie M. Niemi
  6. Jörn Dengjel
  7. Hagai Abeliovich

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Abstract

The mitophagic degradation of mitochondrial matrix proteins in S. cerevisiae was 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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    Reply to the reviewers

    We thank the reviewers for their constructive comments, which greatly helped us in steering the manuscript in the right direction. Below is our point to point response to their concerns:

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    *The Abeliovich lab has discovered differential degradation rates for mitochondrial proteins by mitophagy, a **selective form of autophagy of mitochondria. In previous publications, they identified two protein kinases **Pkp1/2 and the protein phosphatase Aup1(Pct6), which regulate phosphorylation patterns of mitochondrial **proteins and affect their degradation rates by mitophagy. In the current manuscript, the authors now analyze **the role of the pyruvate dehydrogenase complex (PDC) in mitochondria in the regulation of these regulatory **link between Pkp1/2 and Aup1 and their substrate proteins. They find that the deletion of pda1, a core **subunit of PDC, inhibits the degradation of previously identified mitophagy substrates Mdh1, Aco2, Qcr2, **Aco1, and Idp1, while the turnover of mtDHFR-GFP, a model substrate diffusely localized in the mitochondrial **matrix, is not affected. These data indicate that tested mitochondrial proteins are excluded from **mitochondrial turnover by mitophagy in the absence of Pda1 by as yet unknown sorting mechanisms. **The authors next show that the mutation of a known Pkp1/2 and Aup1 phosphorylation site within Pda1 **(S313A) drastically reduces mitophagic turnover of Mdh1, which is completely blocked by the additional **deletion of AUP1. A mutation blocking Pda1 activity (R322C) also almost completely blocked Mdh1 **degradation. However, PDC activity does not generally affect mitophagy, since an inactivating mutation in **Lat1 (K75R) promotes mitophagy of Mdh1. **Published work has shown that Pkp1/2 and Aup1, which affect the phosphorylation and degradation of Mdh1, **physically interact with the PDC. Thus, the authors tested whether the absence of Pda1 affected the **phosphorylation state of Mdh1 and thus its turnover. Indeed, Mdh1 showed globally reduced **phosphorylation in the absence of Pda1, which was rescued by the overexpression of Pkp1/2 promoting **degradation of Mdh1. Consistent with the effects on mitophagic turnover, Mdh1 was phosphorylated at **higher level in the presence of Lat1-K75R. **The authors analyzed the mitochondrial distribution of Mdh1 in dependence of Pda1. They provide **cytological data suggesting that Mdh1 might segregate from a diffuse matrix localized mtRFP reporter, which **is suppressed upon Pkp1/2 overexpression. **Finally, the authors perform a mitochondrial phospho-proteome analysis and found that the absence of Pda1 **affected phosphorylation sites in 8 mitochondrial matrix proteins. **From these data the authors propose a model in which the PDC complex controls the activity of the **associated protein kinase Pkp1/2 and the phosphates Aup1 by allosteric changes, which in turn regulates the **phosphorylation and differential turnover of mitochondrial proteins by mitophagy. They speculate that the *PDC and associated factors could resemble large protein kinase complexes as TORC.

    (1) The authors postulate a structural role for the PDC in regulating Pkp1/2 and Aup1 for controllingmitophagy turnover of certain substrates. While the physical association of Pkp1/2 and Aup1 with PDC has been shown previously, the authors need to critically test their model and assess (a) whether this physicalinteraction occurs under their experimental conditions and (b) whether the different mutants that affect*mitophagy of Mdh1 also affect the physical interaction of Pkp1/2 and Aup1 with PDC. Otherwise, their model, *although consistent, remains purely speculative.

    Response: We fully agree with the reviewer that this is an important point and we are currently trying to verify these interactions under our working conditions. Naturally, we will then test whether the documented Pdh1-Aup1 and the Pdh1-Pkp1/2 interactions are affected by mutants such as the lat1K75R mutation and the lat1Δ mutation, as insightfully suggested by the reviewer.

    *(2) It is important to test every mutant that affects Mdh1 turnover for effects on mitophagy in general using **the mtDHFR-GFP reporter to be able to conclude specific effects on Mdh1 mitophagy. For example, the **deletion of Lat1 has been shown to induce mitophagy of a mitochondrial matrix reporter during nitrogen **starvation (Bockler and Westermann, 2013). While the authors observe a complete block of Mdh1 turnover in **lat1 deletion cells, they do observe increased Mdh1 degradation in Lat1-K75R cells. **In line with this reasoning, the authors have identified a number of Mdh1 variants in a previous publication **(Kolitsida et al. 2019) that can be used to further explore the observed phenomenon. For example, the Mdh1- **T199A could be used to test whether the expression of Pkp1/2 in pda1 deficient cells has specific or general **effects on mitophagy. Furthermore, can Mdh1-T199D, which is turned over independent of Pkp1/2, be degraded in the absence of Pda1?

    Response: We completely agree with the reviewer, and we have tested the effect of the lat1Δ mutations on mtDHFR-GFP (Supplemental Figure 2). It is important to note, however, that while the Bockler and Westermann paper does report an increased red/green fluorescence ratio in lat1Δ cells using mtRosella as a mitophagy reporter, this is part of a general screen, and was never further investigated or validated with additional, more rigorous methods (that paper focused on the role of the HERMES complex in mitophagy). In vivo fluorescence can be affected by a multitude of physical intracellular and intra-organellar factors including local pH, ionic strength, and molecular crowding. Therefore, the result from the screen conducted by Bockler and Westermann - although intriguing- is somewhat preliminary and requires more scrutiny (e.g. validation with a GFP release assay, complementation, etc) before we can make conclusions regarding the effect of the lat1Δ mutation on mitophagy, under their experimental conditions.

    *Furthermore, can Mdh1-T199D, which is turned over independent of Pkp1/2, be degraded in the absence of Pda1?

    This is now addressed in Figure 3E and 3F. The results are consistent with our hypothesis, namely showing complete or near complete suppression of the phenotype when tested using the phosphomimetic reporter.

    *(3) The cytological data are not convincing. The imaging quality is low and it is very difficult for the reader to **appreciate the suggested segregation of Mdh1-GFP and mtRFP. Thus, it is important to provide cortical **sections in order to visualize mitochondrial tubules/networks and Mdh1 distribution. This is particularly **important, because the authors mention effects of Pda1 on mitochondrial morphology, which appears to be *rescued by Pkp1/2 overexpression.

    We are sorry to hear that the reviewer is not convinced by the microscopy data. We agree with the comment that it is not trivial to observe the segregation by eye. We do not intend to say that the segregation is absolute, and the differences we observe in the distributions of the two signals are relative. By that, we mean that a prominent green dot in the cell, which is brighter than other green dots in the same cell, has a red channel counterpart which is not brighter than the other red dots in the cell, or may even be dimmer than the other red dots in the same cell. However, we do provide illustrative examples of segregation with specific arrows pointing to loci of segregation between the two channels. This was not pointed out in the original figure legend and this oversight has now been corrected. More importantly, we performed a mathematical analysis of the overlap between the two signals, and demonstrate a statistically (very) significant difference between the endogenous mitochondrial protein and the artificial mitochondrially-targeted GFP chimera. We would also like to stress that under our conditions (gluconeogenic medium, stationary phase) yeast mitochondria (at least in our genetic background) are not normally found in tubules (this pattern is specifically observed in cells growing in glucose-based medium). Nonetheless, we will attempt to carry out a 3-d reconstruction using our available z-sections, as per the reviewer’s request.

    *(3 continued) In this context it would be very informative to follow the localization of PDC in mitochondria. Previous work **has shown that Pda1 forms punctate structures in mitochondria in proximity to ER-mitochondria contact **sites marked by ERMES (Cohen et al. 2014). Is Pda1 itself degraded by mitophagy or is it excluded? Could **the physical interaction of Mdh1 with Pda1 explain mitophagy phenotypes if Pda1 is excluded from *mitophagy? In other words, *could the PDC form a physical unit to segregate and prevent proteins from *mitophagy turnover?

    Response: It was shown by others that Pda1-GFP localizes to mitochondrial puncta, and we also observe this in our system. We have also briefly looked at the mitophagic efficiency of Pda1-GFP. While it is inefficiently delivered to the vacuole (5% free GFP after 5 days, vs 20% for Mdh1), it is not excluded from mitophagic targeting. We are not sure what the reviewer is referring to as a “physical interaction between Pdh1 and Mdh1”. We do not demonstrate such an interaction, and to our knowledge such an interaction has not been reported and validated in any publication. Even if such an interaction existed, it cannot explain the rescue of the pda1Δ phenotype by the co-overexpression of Pkp1 and 2, nor the effect of the pda1Δ mutation on Mdh1 phosphorylation, or the effect of the pda1Δ mutation on other mitophagy reporters which we used, such as Aco1, Qcr2, Idp1, and Aco2. We agree with the reviewer that there is a possibility that an organizing center, be it the PDC or another complex, may regulate intra-matrix segregation and mitophagic trafficking. However, addressing this question would require significant additional time and resources, and we would beg the referee to defer this question to future investigations, as it is not central to the claims made in the current manuscript.

    (4) The conclusions that can be drawn from the analysis of the mitochondrial phospho-proteome independence of Pda1 are rather limited. First, the experimental setup does not distinguish between direct*effects of PDC on Pkp1/2 or Aup1 activity and the effects of simply PDC dysfunction on mitochondrial **proteins. Along those lines, it is unclear whether the few identified proteins with altered phosphorylation **states are indeed targets of Pkp1/2 or Aup1. Thus, to be able to support their conclusion, the authors need to *include a number of additional controls/strains.

    Response: We agree with the reviewer that the phosphoproteomic analysis is not comprehensive. However, it was the best that we were able to do at the time, and it is unlikely that we could distinguish direct from indirect effects using this approach. The purpose of the experiment was to test whether we could identify more global effects of Pda1 on mitochondrial protein phosphorylation. This was in no way intended to imply a direct effect. Rather, it provides an unbiased map for potentially identifying the signaling network(s) involved. This may also include downstream events outside the mitochondrial matrix and even in the cytoplasm. We previously showed a connection of the Aup1-dependent signaling pathway to the RTG retrograde signaling pathway (Journo et al, 2009), and such a global analysis may allow a future understanding of how intra-matrix events can signal to the cytoplasm. However, we do not make any specific claims regarding which effects are direct and which are not. To address the reviewer’s concern, we have now modified the text to clarify these points (Page 9 line 28-Page 10 line 1).In an effort to improve the evidence for PDC- dependent mitochondrial matrix phosphorylation, we will carry out a phosphoproteomic analysis comparing WT cells to cells expressing the lat1K75R mutation. Since this mutation is expected to increase kinase activity, we expect to obtain a clearer picture that will complement the results obtained with the pda1Δ deletion mutant.

    Reviewer #1 (Significance (Required)):

    *The authors continue to explore the mechanisms underlying their initial observation of differential turnover **rates for mitochondrial proteins by mitophagy. This current work specifically builds on the previous **publication identifying Pkp1/2 and Aup1 as regulators of the phosphorylation state of specific substrate **proteins (Kolitsida et al. 2019). Here the authors now explore how these regulators might be organized and **controlled by PDC. Thus, the study adds another layer of regulation. However, the molecular mechanisms of **how PDC might regulate Pkp1/2 and Aup1 is not directly addressed. In conclusion, the current study opens **up new lines of research that need to be explored to critically test the proposed model. A key question to me **is of course how mitochondrial proteins can be excluded from mitophagy and whether the proteins in this *study may play a direct role in these mechanisms.

    To further address the mechanism by which the PDC affects mitophagy via Aup1, Pkp1, and Pkp2, we will carry out the following experiments: 1) We will verify the Pda1-Pkp1/2 interaction and test for effects of PDC mutants on this interaction, with an emphasis on the lat1K75R mutation 3) We will analyze the effect of the lat1K75R mutation on mitochondrial phosphoproteomics 4) we will analyze the effect of the pda1Δ mutation on the phosphorylation state of additional proteins which exhibit defective mitophagy in the *pda1Δ *background (Figure 1B). Our basic hypothesis regarding the reviewers’ question, namely the molecular mechanism behind the observed regulation is based on mitochondrial heterogeneity. We suggest that mitochondrial heterogeneity underlies mitophagic selectivity and that factors which affect heterogeneity also affect mitophagic selectivity.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Kolitsida et al. unravels an interesting link between mitochondrial pyruvate*dehydrogenase complex (PDC) and protein sorting for mitophagy-mediated clearance. Using yeast genetics **combined with immunoblotting-based mitophagy protein sorting reporter assay, authors show that targeting **components of PDC and associated regulatory factors (kinases and phosphatases) interferes with **phosphorylation status of at least some mitochondrial matrix proteins and prevents their degradation by **autophagic machinery. Experimental design is sound, and statistic evaluation seems appropriate. Although I **find this work important and overall valuable, some of the conclusions made by the authors are still *preliminary and could require additional experimental testing.

    We thank the reviewer for these supportive comments

    *In this regard, some of my major suggestions include: *

    *1. Authors propose that the interaction of dedicated kinases and phosphatases with pyruvate **dehydrogenase complex changes their affinity/activity toward the non-PDC substrates (possibly by allosteric **regulation). Yet, alternatively, it can also be considered that the PDC complex acts as a docking station that **stabilizes the associated regulatory factors inside the mitochondria and allows their additional functions. **Indeed, simultaneous overexpression of Pkp1/2 increases the free GFP signal and Mdh1 phosphorylation **even in the absence of core E1 PDC component (pda1, figure 3 C, D and figure 4 A, B), suggesting that the **interaction of phosphatases and kinases with pda1 is not essential to mediate the phosphorylation of other **mitoproteins. Are the protein levels of regulatory PDC factors (Pkp1, Pkp2, Aup1) somewhat altered by **depletion of their target E1a subunit (pda1)? Could depletion of pda1 trigger destabilization of these factors **and decrease their half-life? This can be tested by western-blotting and combined with cycloheximide chase *assay.

    We agree with the reviewer that, theoretically, the PDC could be necessary for the stability of Pkp1, Pkp2 and Aup1. However, have now tested this, and found that deletion of PDA1 has no effect on the expression levels of Aup1, Pkp1 and Pkp2 under our conditions (see Supplementary Figure 2).

    *2. Allosteric regulation of specificity of PDC kinases and phosphatases is an intriguing yet still slightly **unbacked hypothesis. To provide further experimental evidence for this explanation, authors could develop **the classical in vitro kinase assay for Pkp1/2 on their model substrate (Mdh1) in the absence or presence of **the Pda1 subunit. Even more excitingly, authors could isolate the Pkp1/2 from wild-type and Pda1- **compromised cells and see whether they are able to phosphorylate their model substrate in vitro. **Additionally, does specific inhibition of pyruvate dehydrogenase kinases (e.g., with dichloroacetate) affect *the Mdh1 phosphorylation levels?

    We present the hypothesis that the PDC allosterically regulates Aup1 and Pkp1/2 , as an explanation for the data. We agree with the reviewer that further experimental work will be necessary to verify this hypothesis. While the Pkp1/2 kinase assays could be useful in this regard, several issues temper our enthusiasm for this experiment:

    i) This assay is far from “classical” with respect to this study. Neither of the two yeast mitochondrial kinases (Pkp1 and Pkp2) was previously characterized using in vitro kinase assays. While the mammalian assay conditions may work here, this is not guaranteed.

    ii) A negative result will have no value

    iii) A positive result (e.g. phosphorylation of recombinant Mdh1 by recombinant Pkp1/2) could arise due to low stringency conditions in the assay

    iv) The nature of the kinase activity is unclear. We do not know if it is Pkp1, Pkp2 or a heterodimer of Pkp1 and Pkp2, or whether currently unknown ancillary factors are necessary for activity. This greatly complicates the analysis.

    In lieu of the direct in vitro kinase assay, we have carried out an experiment demonstrating that an Mdh1-GFP variant with a phosphomimetic threonine to aspartate mutation at position 199, which we previously demonstrated to suppress both the aup1Δ and Pkp2Δ phenotypes (Kolitsida et al, 2019), can also suppress the *pda1Δ *phenotype (New Figure 3E, F). We suggest that this result provides sufficient evidence of a phosphorylation cascade linking the PDC, Pkp1/2 and Mdh1 mitophagic trafficking.

    In addition, we will also attempt to further support the allosteric regulation hypothesis, by testing whether mutations in LAT1 such as lat1K75R modulate the known Aup1-Pda1 and Pkp1/2-Pda1 interactions (also see response to referee #1).

    We greatly appreciate the suggestion to test the effect of DCA on our assays, and this experiment will be carried out in the near future. However DCA has not been shown to affect Pda1 phosphorylation in yeast or to modulate kinase activity of Pkp1/2, and it is not clear whether this approach will work. Many reagents and inhibitors which act in mammalian cells, are unable to cross the yeast cell wall.

    *3. Are the Pkp1/Pkp2 changing their interactome upon PDC alternation (e.g., Pda1 depletion)? Do their **interactome alters upon stimulation of mitophagy? Co-immunoprecipitation or bioID assay could shed light **on how the partitioning of the Pkp1/Pkp2/Aup1 functions between the metabolic regulation and protein *quality control is maintained.

    We agree with the referee that this is an excellent question. Our current hypothesis posits that the established physical interaction between Pkp1/2 and Pda1 is crucial for activity of the kinases towards "third party" clients. To test this we will assay whether the catalytically inactive lat1K75R mutation affects the Pkp1/2 - Pda1 interaction. If we cannot find evidence for such a direct mechanism using these straightforward hypothesis-driven experiments, then we will also analyze effects the* pda1Δ* mutation on the general interactomes of Pkp1 and Pkp2.

    Minor points:

    *1. The paper would benefit from a brief explanation of the principles of the mitophagy reporter assay used in *this study.

    Thank you for the suggestion. We have now expanded our explanation of the GFP release assay (see Methods section, Page 13 lines 8-16).

    2. MS phosphoproteomics is indeed a great approach to tackle many questions in this study. Surprisingly,*the authors did not discuss these data extensively. Although changes in the phosphorylation status of some **proposed targets are apparent (as QCR2), many others were not detected (including Aco1/2, Idp1, and model *substrate - Mdh1; Figure 1).

    In our experiment, the coverage of the mitochondrial phosphoproteome was not complete. Some proteins, such as Mdh1, were observed only in a subset of the replicates, and therefore are not included in the figure. In the revision, we plan to add an analysis of the mitochondrial phosphoproteome in the lat1K75R mutation, and hopefully this will provide further insights.

    *Furthermore, many significant changes occur in the proteins that do not belong **to the mitochondrial matrix compartment (as TOMM20 or YAT1), questioning the direct involvement of *Pkp1/Pkp2/Aup1. How do authors interpret these data?

    We previously showed that the Aup1 signaling pathway converges with the RTG retrograde signaling pathway, which signals from the mitochondrial matrix to the nucleus (Journo et al, 2009). We would like to suggest that these effects on non-matrix proteins may indicate a possible role in transducing this signal. We have now added this comment to the discussion on Page 9 lines 28- Page 10 line 1.

    3. Minor spelling mistakes should be reviewed (e.g., "mutophagic" pg. 7).

    Thank you. Fixed.

    *Reviewer #2 (Significance (Required)): *

    *This work further expands on the previous observation by the authors (Kolitsida et al., 2019) and provides a novel and interesting hypothesis that may potentially impact our understanding of mitophagy selectivity toward particular mito proteome content. Furthermore, it unravels the additional function of PDC kinases and phosphatases behind the well-established regulation of energy metabolism. Therefore, it could interest the broad field of researchers interested in mitochondrial quality control and its interplay with cellular metabolism. *

    We thank the reviewer for these encouraging comments.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    *In this elegant and creative study, Dr. Abeliovich and his collaborators describe a novel pathway for the **selective elimination of specific proteins. The flow of the experiments is logical and the writing is clear. The **topic is very timely as the subject of mitochondrial quality control is of great interest. **The main strength of this study is the creative ideas and the very out-of-the-box concept of selective **elimination of specific proteins. The main shortcoming of this study is the incomplete direct evidence for a **mechanism and conclusions that exceed the results. I believe these are fixable and I am sure this study will *be well received after these issues are addressed.

    We thank the reviewer for this positive assessment of the manuscript.

    Specific comments:

    Major comments:

    *1. It is not clear what is the mechanism for the recovery of pkp1 and pkp2 shown in figure 4. Supposedly, it is *downstream of mitophagy, by how? Is there any data on that?

    We presume the referee is referring to Figure 4A and B, where we demonstrate suppression of the pda1Δ phosphorylation phenotype by co-overexpression of Pkp1 and Pkp2. As indicated in the manuscript, we interpret these results as indicating that Pkp1 and Pkp2 function downstream of the PDC in the regulation of mitophagic trafficking. To further bolster this conclusion we now also show that a phosphomimetic mutation in Mdh1 that suppresses the pkp2Δ mutation as well as the aup1Δ mutation (Kolitsida et al, 2019), is also able to suppress the pda1Δ mutation. In addition, we will use co-IPs to test whether mutations in Lat1, and specifically the lat1K75R mutation which increases mitophagy and Mdh1 phosphorylation, can affect the known Pkp1/2-Pda1 interaction.

    *2. Pertaining to the experiment shown in figure 4, it is not clear if this was conducted in the presence of low *glucose and if this was required for the effect.

    As pointed out in the legend to Figure 4, the cells were grown in SL medium. As per the “Methods” section, SL contains, in addition to 2% lactate, 0.1% glucose. This small amount of glucose is always added to gluconeogenic media because initial growth in the total absence of glucose is very sluggish. 0.1% glucose does not induce the crabtree effect (does not inhibit respiration) and is consumed during the initial growth phase.

    *3. It is not clear if the effect is directly mediated by pct 5&7 acting on MDH1 or if this is an indirect effect. Can *this be stated and then addressed experimentally? Are pct5 and pct7 the only phosphatases in the matrix?

    The purpose of this experiment was to untangle the apparent paradox wherein Aup1/Ptc6 is the opposing phosphatase countering Pkp1/2 on Pda1, but this is clearly not the case for the signaling pathway uncovered in this study. This finding further differentiates our signaling pathway from the conventional PDC regulation cascade, and provides a previously unidentified function for Ptc5. We do not claim that these effects are direct. There are 3 documented phosphatases in yeast mitochondria: Ptc5, Ptc7 and Ptc6/Aup1. Since we have previously shown that the aup1Δ phenotype is very similar to that of pkp2Δ, it disqualifies Aup1 from being a candidate for the opposing phosphatase acting on Mdh1-GFP. The experiment shown in Figure 4D and E indicates that Ptc5 is the prime candidate for a phosphatase that targets Mdh1-GFP. We now clarify this point in the discussion (Page 10, lines 14-24), and we suggest that a more definitive identification will be made in future studies.

    *4. Figure 6 uses deletion of PDA1 as a loss of function experiment. However in this case, this is a rather **crude approach since the point the authors are trying to make is that it is the regulation rather than the **expression levels that is critical. If possible, the authors should try and affect the regulatory site by mutation *rather than delete the gene.

    We fully agree with this suggestion. We will now test the effect of the lat1K75R mutation on the mitochondrial phosphoproteome, in order to address this deficiency.

    Minor comments:

    *1. The title should include that this was done in yeast. Similarly, yeast should be mentioned in the abstract *too.

    We have now added this information to the abstract. We do not wish to do the same for the title, as most journals have strict limits on the number of characters in the title.

    *2. In the introduction, it is stated that mitophagy is a process degrading dysfunctional mitochondria. It will be **more accurate to say that it is degrading dysfunctional mitochondria as well as removing mitochondria in *cells that shrink or rebuild their mitochondrial proteome.

    Thank you. We have now made these changes (Page 3, lines 8-11)

    3.Are pkp1 and pkp2 found only in the mitochondria?

    As per the literature, Pkp1 and 2 are exclusively mitochondrial

    4. In figure 1A/B only the blots of MDH1 are shown. It will be informative to show the blots of the other

    proteins (currently in the supplementary)

    We have added these blots to Figure 1

    5. It is not clear how the reporter in fig 1C is different from the reporter in 1A.

    This has now been clarified in the text (Page 4 line 28- Page 5 line 2)

    Figure 4C. Please add an image of the colonies.

    Thank you. This will be added to Figure 4.

    *6. It is not clear what the author defines as "increased segregation of Mdh1-GFP relative *to generic mtRFP". Please clarify "generic mtRFP" and explain how the relativeness was deducted.

    Thank you. We have now dropped the qualification “generic” and explain the difference. We now also explain the protocol for quantifying the overlap between the two signals (Page 8 lines 9-12, Page 16 lines 19-21 and 27-31).

    Reviewer #3 (Significance (Required)):

    *This is a very hot topic and this lab is at the front of it *It is for broad cell biology and biochemistry audience

    We thank the referee for his/her generous and supportive comments

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    Referee #3

    Evidence, reproducibility and clarity

    In this elegant and creative study, Dr. Abeliovich and his collaborators describe a novel pathway for the selective elimination of specific proteins. The flow of the experiments is logical and the writing is clear. The topic is very timely as the subject of mitochondrial quality control is of great interest. The main strength of this study is the creative ideas and the very out-of-the-box concept of selective elimination of specific proteins. The main shortcoming of this study is the incomplete direct evidence for a mechanism and conclusions that exceed the results. I believe these are fixable and I am sure this study will be well received after these issues are addressed.

    Specific comments:

    Major comments:

    1. It is not clear what is the mechanism for the recovery of pkp1 and pkp2 shown in figure 4. Supposedly, it is downstream of mitophagy, by how? Is there any data on that?
    2. Pertaining to the experiment shown in figure 4, it is not clear if this was conducted in the presence of low glucose and if this was required for the effect.
    3. It is not clear if the effect is directly mediated by pct 5&7 acting on MDH1 or if this is an indirect effect. Can this be stated and then addressed experimentally? Are pct5 and pct7 the only phosphatases in the matrix?
    4. Figure 6 uses deletion of PDA1 as a loss of function experiment. However in this case, this is a rather crude approach since the point the authors are trying to make is that it is the regulation rather than the expression levels that is critical. If possible, the authors should try and affect the regulatory site by mutation rather than delete the gene.

    Minor comments:

    1. The title should include that this was done in yeast. Similarly, yeast should be mentioned in the abstract too.
    2. In the introduction, it is stated that mitophagy is a process degrading dysfunctional mitochondria. It will be more accurate to say that it is degrading dysfunctional mitochondria as well as removing mitochondria in cells that shrink or rebuild their mitochondrial proteome. 3.Are pkp1 and pkp2 found only in the mitochondria?
    3. In figure 1A/B only the blots of MDH1 are shown. It will be informative to show the blots of the other proteins (currently in the supplementary)
    4. It is not clear how the reporter in fig 1C is different from the reporter in 1A. Figure 4C. Please add an image of the colonies.
    5. It is not clear what the author defines as "increased segregation of Mdh1-GFP relative to generic mtRFP". Please clarify "generic mtRFP" and explain how the relativeness was deducted.

    Significance

    This is a very hot topic and this lab is at the front of it It is for broad cell biology and biochemistry audience

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    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Kolitsida et al. unravels an interesting link between mitochondrial pyruvate dehydrogenase complex (PDC) and protein sorting for mitophagy-mediated clearance. Using yeast genetics combined with immunoblotting-based mitophagy protein sorting reporter assay, authors show that targeting components of PDC and associated regulatory factors (kinases and phosphatases) interferes with phosphorylation status of at least some mitochondrial matrix proteins and prevents their degradation by autophagic machinery. Experimental design is sound, and statistic evaluation seems appropriate. Although I find this work important and overall valuable, some of the conclusions made by the authors are still preliminary and could require additional experimental testing.

    In this regard, some of my major suggestions include:

    1. Authors propose that the interaction of dedicated kinases and phosphatases with pyruvate dehydrogenase complex changes their affinity/activity toward the non-PDC substrates (possibly by allosteric regulation). Yet, alternatively, it can also be considered that the PDC complex acts as a docking station that stabilizes the associated regulatory factors inside the mitochondria and allows their additional functions. Indeed, simultaneous overexpression of Pkp1/2 increases the free GFP signal and Mdh1 phosphorylation even in the absence of core E1 PDC component (pda1, figure 3 C, D and figure 4 A, B), suggesting that the interaction of phosphatases and kinases with pda1 is not essential to mediate the phosphorylation of other mitoproteins. Are the protein levels of regulatory PDC factors (Pkp1, Pkp2, Aup1) somewhat altered by depletion of their target E1a subunit (pda1)? Could depletion of pda1 trigger destabilization of these factors and decrease their half-life? This can be tested by western-blotting and combined with cycloheximide chase assay.
    2. Allosteric regulation of specificity of PDC kinases and phosphatases is an intriguing yet still slightly unbacked hypothesis. To provide further experimental evidence for this explanation, authors could develop the classical in vitro kinase assay for Pkp1/2 on their model substrate (Mdh1) in the absence or presence of the Pda1 subunit. Even more excitingly, authors could isolate the Pkp1/2 from wild-type and Pda1-compromised cells and see whether they are able to phosphorylate their model substrate in vitro. Additionally, does specific inhibition of pyruvate dehydrogenase kinases (e.g., with dichloroacetate) affect the Mdh1 phosphorylation levels?
    3. Are the Pkp1/Pkp2 changing their interactome upon PDC alternation (e.g., Pda1 depletion)? Do their interactome alters upon stimulation of mitophagy? Co-immunoprecipitation or bioID assay could shed light on how the partitioning of the Pkp1/Pkp2/Aup1 functions between the metabolic regulation and protein quality control is maintained.

    Minor points:

    1. The paper would benefit from a brief explanation of the principles of the mitophagy reporter assay used in this study.
    2. MS phosphoproteomics is indeed a great approach to tackle many questions in this study. Surprisingly, the authors did not discuss these data extensively. Although changes in the phosphorylation status of some proposed targets are apparent (as QCR2), many others were not detected (including Aco1/2, Idp1, and model substrate - Mdh1; Figure 1). Furthermore, many significant changes occur in the proteins that do not belong to the mitochondrial matrix compartment (as TOMM20 or YAT1), questioning the direct involvement of Pkp1/Pkp2/Aup1. How do authors interpret these data?
    3. Minor spelling mistakes should be reviewed (e.g., "mutophagic" pg. 7).

    Significance

    This work further expands on the previous observation by the authors (Kolitsida et al., 2019) and provides a novel and interesting hypothesis that may potentially impact our understanding of mitophagy selectivity toward particular mito proteome content. Furthermore, it unravels the additional function of PDC kinases and phosphatases behind the well-established regulation of energy metabolism. Therefore, it could interest the broad field of researchers interested in mitochondrial quality control and its interplay with cellular metabolism.

  12. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The Abeliovich lab has discovered differential degradation rates for mitochondrial proteins by mitophagy, a selective form of autophagy of mitochondria. In previous publications, they identified two protein kinases Pkp1/2 and the protein phosphatase Aup1(Pct6), which regulate phosphorylation patterns of mitochondrial proteins and affect their degradation rates by mitophagy. In the current manuscript, the authors now analyze the role of the pyruvate dehydrogenase complex (PDC) in mitochondria in the regulation of these regulatory link between Pkp1/2 and Aup1 and their substrate proteins. They find that the deletion of pda1, a core subunit of PDC, inhibits the degradation of previously identified mitophagy substrates Mdh1, Aco2, Qcr2, Aco1, and Idp1, while the turnover of mtDHFR-GFP, a model substrate diffusely localized in the mitochondrial matrix, is not affected. These data indicate that tested mitochondrial proteins are excluded from mitochondrial turnover by mitophagy in the absence of Pda1 by as yet unknown sorting mechanisms.

    The authors next show that the mutation of a known Pkp1/2 and Aup1 phosphorylation site within Pda1 (S313A) drastically reduces mitophagic turnover of Mdh1, which is completely blocked by the additional deletion of AUP1. A mutation blocking Pda1 activity (R322C) also almost completely blocked Mdh1 degradation. However, PDC activity does not generally affect mitophagy, since an inactivating mutation in Lat1 (K75R) promotes mitophagy of Mdh1. Published work has shown that Pkp1/2 and Aup1, which affect the phosphorylation and degradation of Mdh1, physically interact with the PDC. Thus, the authors tested whether the absence of Pda1 affected the phosphorylation state of Mdh1 and thus its turnover. Indeed, Mdh1 showed globally reduced phosphorylation in the absence of Pda1, which was rescued by the overexpression of Pkp1/2 promoting degradation of Mdh1. Consistent with the effects on mitophagic turnover, Mdh1 was phosphorylated at higher level in the presence of Lat1-K75R. The authors analyzed the mitochondrial distribution of Mdh1 in dependence of Pda1. They provide cytological data suggesting that Mdh1 might segregate from a diffuse matrix localized mtRFP reporter, which is suppressed upon Pkp1/2 overexpression.

    Finally, the authors perform a mitochondrial phospho-proteome analysis and found that the absence of Pda1 affected phosphorylation sites in 8 mitochondrial matrix proteins.

    From these data the authors propose a model in which the PDC complex controls the activity of the associated protein kinase Pkp1/2 and the phosphates Aup1 by allosteric changes, which in turn regulates the phosphorylation and differential turnover of mitochondrial proteins by mitophagy. They speculate that the PDC and associated factors could resemble large protein kinase complexes as TORC.

    Critical points:

    1. The authors postulate a structural role for the PDC in regulating Pkp1/2 and Aup1 for controlling mitophagy turnover of certain substrates. While the physical association of Pkp1/2 and Aup1 with PDC has been shown previously, the authors need to critically test their model and assess (a) whether this physical interaction occurs under their experimental conditions and (b) whether the different mutants that affect mitophagy of Mdh1 also affect the physical interaction of Pkp1/2 and Aup1 with PDC. Otherwise, their model, although consistent, remains purely speculative.
    2. It is important to test every mutant that affects Mdh1 turnover for effects on mitophagy in general using the mtDHFR-GFP reporter to be able to conclude specific effects on Mdh1 mitophagy. For example, the deletion of Lat1 has been shown to induce mitophagy of a mitochondrial matrix reporter during nitrogen starvation (Bockler and Westermann, 2013). While the authors observe a complete block of Mdh1 turnover in lat1 deletion cells, they do observe increased Mdh1 degradation in Lat1-K75R cells. In line with this reasoning, the authors have identified a number of Mdh1 variants in a previous publication (Kolitsida et al. 2019) that can be used to further explore the observed phenomenon. For example, the Mdh1-T199A could be used to test whether the expression of Pkp1/2 in pda1 deficient cells has specific or general effects on mitophagy. Furthermore, can Mdh1-T199D, which is turned over independent of Pkp1/2, be degraded in the absence of Pda1?
    3. The cytological data are not convincing. The imaging quality is low and it is very difficult for the reader to appreciate the suggested segregation of Mdh1-GFP and mtRFP. Thus, it is important to provide cortical sections in order to visualize mitochondrial tubules/networks and Mdh1 distribution. This is particularly important, because the authors mention effects of Pda1 on mitochondrial morphology, which appears to be rescued by Pkp1/2 overexpression. In this context it would be very informative to follow the localization of PDC in mitochondria. Previous work has shown that Pda1 forms punctate structures in mitochondria in proximity to ER-mitochondria contact sites marked by ERMES (Cohen et al. 2014). Is Pda1 itself degraded by mitophagy or is it excluded? Could the physical interaction of Mdh1 with Pda1 explain mitophagy phenotypes if Pda1 is excluded from mitophagy? In other words, could the PDC form a physical unit to segregate and prevent proteins from mitophagy turnover?
    4. The conclusions that can be drawn from the analysis of the mitochondrial phospho-proteome in dependence of Pda1 are rather limited. First, the experimental setup does not distinguish between direct effects of PDC on Pkp1/2 or Aup1 activity and the effects of simply PDC dysfunction on mitochondrial proteins. Along those lines, it is unclear whether the few identified proteins with altered phosphorylation states are indeed targets of Pkp1/2 or Aup1. Thus, to be able to support their conclusion, the authors need to include a number of additional controls/strains.

    Significance

    The authors continue to explore the mechanisms underlying their initial observation of differential turnover rates for mitochondrial proteins by mitophagy. This current work specifically builds on the previous publication identifying Pkp1/2 and Aup1 as regulators of the phosphorylation state of specific substrate proteins (Kolitsida et al. 2019). Here the authors now explore how these regulators might be organized and controlled by PDC. Thus, the study adds another layer of regulation. However, the molecular mechanisms of how PDC might regulate Pkp1/2 and Aup1 is not directly addressed. In conclusion, the current study opens up new lines of research that need to be explored to critically test the proposed model. A key question to me is of course how mitochondrial proteins can be excluded from mitophagy and whether the proteins in this study may play a direct role in these mechanisms.

  13. Version published to 10.1101/2022.03.16.484611 on bioRxiv