Drosophila Mitochondrial Proteome Is Partially Corrected with Exercise

bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

The dysregulated Pink1- Drosophila mitochondrial proteome is partially corrected with exercise.

Brad Ebanks1*, Thomas L Ingram1*, Gunjan Katyal1, John R Ingram2, Nicoleta Moisoi3, Lisa Chakrabarti1,4#

1 School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington,

LE12 5RD, UK.

2 Ullswater Avenue, West End, Southampton

3 Leicester School of Pharmacy, Leicester Institute for Pharmaceutical Innovation, De

Montfort University, The Gateway, Leicester LE1 9BH

4 MRC Versus Arthritis Centre for Musculoskeletal Ageing Research, UK

* Equally contributing authors

# Corresponding author [email protected]

Running title: Pink1 fly mitochondrial proteomics and exercise Keywords: Mitochondria, PINK1/Pink1, Parkinson’s disease, exercise, proteomics bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Abstract One of the genes which has been linked to the onset of juvenile/early onset Parkinson’s disease (PD) is PINK1. There is evidence that supports the therapeutic potential of exercise in the alleviation of PD symptoms. It is possible that exercise may enhance synaptic plasticity, protect against neuro-inflammation and modulate L-Dopa regulated signalling pathways. We explored the effects of exercise on Pink1 deficient Drosophila melanogaster which undergo neurodegeneration and muscle degeneration. We used a ‘power-tower’ type exercise platform to deliver exercise activity to Pink1- and age matched wild-type flies. Mitochondrial proteomic profiles responding to exercise were obtained. Of the 516 proteins identified, 105 proteins had different levels between Pink1- and wild-type (WT) non-exercised D. melanogaster. Gene ontology enrichment analysis and STRING network analysis highlighted proteins and pathways with altered expression within the mitochondrial proteome. Comparison of the Pink1- exercised proteome to WT proteomes showed that exercising the Pink1- flies caused their proteomic profile to return towards wild-type levels. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Introduction Parkinson’s disease (PD) is a progressive, irreversible neurodegenerative condition which affects over 6 million people across the globe(Ray Dorsey et al, 2018). The greatest risk factor for PD is advancing age, and with the number of people over the age of 60 expected to exceed 2 billion by the year 2050 (currently estimated to be over 900 million) there will soon be significantly higher numbers of people living with PD(Storey, 2018). The classical physical symptoms are known to be resting tremor, rigidity and bradykinesia. It is now also known that symptoms of PD include reduced quality of sleep as well as both cognitive impairments and poor mental health. In terms of the pathophysiology of the disease, the death of pigmented dopaminergic neurons in the substantia nigra pars compacta in PD patients is critical(Hirsch et al, 1988). Molecular characteristics of PD include the aggregation of α-synuclein leading to Lewy body formation, alongside mitochondrial dysfunction(Schulz-Schaeffer, 2010; Park et al, 2018). Hereditary forms of PD can be either autosomal dominant or autosomal recessive, dependent upon the mutant gene involved. In both dominant and recessive forms of hereditary PD, autophagic and lysosomal pathways are both mechanistically implicated(Alegre-Abarrategui et al, 2009; Thomas et al, 2011; Zavodszky et al, 2014). One of the critical pathways which has been linked to the onset of juvenile/early onset PD is the PINK1/Parkin mitophagy pathway, a form of autophagy for the degradation of dysfunctional mitochondria. The role of mitophagy is to provide a quality control mechanism for the mitochondrial population within a cell, and this is a particularly crucial function in energetically demanding neuronal cells(Palikaras et al, 2018). Mechanistically, PINK1 localizes with outer mitochondrial membrane (OMM) of depolarized mitochondria and then recruits and activates the E3 ubiquitin-ligase activity of Parkin via phosphorylation of its Ser65 residue (Shiba-Fukushima et al, 2012; Kondapalli et al, 2012). PINK1 has also been shown to phosphorylate the Ser65 residue of Ubiquitin, which aids in the activation of Parkin’s E3 ligase activity (Koyano et al, 2014; Kazlauskaite et al, 2014). Ultimately these events lead to the ubiquitination of OMM proteins by Parkin, the autophagic machinery then degrades the ubiquitinated mitochondria (Ordureau et al, 2014). Mutations in PINK1 (and Parkin) can result in autosomal recessive juvenile onset PD, with onset before the age of 40 years old (Cookson, 2012). While this form of PD is rare, a comprehensive understanding of it can improve the outcomes of patients with PINK1 mutations and also those with idiopathic PD due to their shared pathophysiology (Pankratz & Foroud, 2007). A recent publication by Sliter et al. reported that Pink1 and Parkin mitigate STING induced inflammation, where both Pink1-/- and Prkn-/- mice under exhaustive exercise have a strong inflammatory phenotype that is rescued by the concurrent loss of the STING pathway(Sliter et al, 2018). In the same year, Zhong et al. reported that newly synthesised oxidised bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

mitochondrial DNA is exported to the cytosol and stimulates another of the innate immune responses, the NLRP3 inflammasome(Zhong et al, 2018). Given the overlapping biology between PINK1 loss of function in PD and other forms of genetic and sporadic PD, PINK1 null mutants of animal models are hugely useful in studies of PD. Pink1 loss of function D. melanogaster models were first developed in 2006, shortly after the first parkin loss of function mutant D. melanogaster models were being utilised in research(Park et al, 2006; Clark et al, 2006; Greene et al, 2003). These first studies found that Pink1 loss of function resulted in mitochondrial dysfunction, compromised fertility in males, indirect flight muscle degeneration and associated locomotor defects, increased sensitivity to oxidative stress, and dopaminergic degeneration (Park et al., 2006). Parkin overexpression rescued many of the defects observed in the Pink1 mutants, indicating the downstream function of Parkin in the now established PINK1/Parkin mitophagy pathway, reviewed here(Pickrell & Youle, 2015). More recent studies using Pink1 mutant D. melanogaster have found that their neurons exhibit decreased levels of synaptic transmission, defective fission and reduced ATP levels due to decreased COXI and COXIV activity as well as non-motor symptoms such as learning and memory deficits, weakened circadian rhythms and electrophysiological changes in clock-neurons(Morais et al, 2009; Liu et al, 2011; Julienne et al, 2017). It was first observed in 1992 that participation in exercise reduced the risk of the onset of PD in later years, while later data showed that this protection against PD risk is more obvious in males(Sasco et al, 1992; Yang et al, 2015). Many groups have presented data that show the therapeutic potential of exercise in the alleviation of patient symptoms(Ashburn et al, 2007; Goodwin et al, 2008; Murray et al, 2014). The biochemical mechanisms that would explain these observations are still unclear, but current evidence suggests that exercise may enhance synaptic plasticity, protect against neuroinflammation and modulate L-Dopa regulated signalling pathways(Shin et al, 2016; Jang et al, 2017; Klemann et al, 2018). In this study we aimed to analyse the biochemical changes induced by exercise in the mitochondrial proteome of the Pink1 loss of function mutant (Pink1-) D. melanogaster. As exercise is reported to both reduce the risk of onset and improve outcomes for Parkinson’s disease patients, we sought to characterise the biochemical changes that could underpin this improvement in our model of Parkinson’s disease. We focused on the mitochondria as their dysfunction is widely associated with Parkinson’s disease, and the Pink1- genetic model has a disrupted mitophagy pathway due to the absence of a functional PINK1 protein.

Methods D. melanogaster stocks Fly stocks were kindly provided to NM by Miguel Martins (MRC Toxicology Unit) and Alex Whitworth (MRC Mitochondrial Biology Unit). Fly stocks and crosses were maintained on standard cornmeal agar media at 25oC in a 12:12 light-dark cycle. The experiments were performed on males: WT (genotype w1118) and - Pink1 (genotype Pink1B9/Y).

D. melanogaster exercise - Approximately twenty wild type control (WT) or Pink1 flies, 1-4 days post- eclosion, were separated into glass vials filled with 5ml food. Exercised group vials were stoppered with cotton wool 6cm from the food; non-exercised group vials were stopped with cotton wool 1cm from the food, creating a physical barrier to activity. Both exercised and non-exercised groups were placed in racks on the ICE machine (Appendix) for 30 minutes per day for 7 days. The flies were exercised in the morning each day and were sacrificed by freezing at -80°C one hour after the final exercise bout. Comparison groups were exercised and non-exercised WT and Pink1- D. melanogaster.

Mitochondrial isolation Groups of twenty WT or Pink1- D. melanogaster were homogenised in 100-200µl mitochondrial extraction buffer (50mM Tris-Cl pH 7.4, 100mM KCL, 1.5mM MgCl2, 1mM EGTA, 50mM HEPES and 100mM sucrose) by 5 minutes of manual homogenisation using a 1.2-2ml Eppendorf micro-pestle (Sigma-Aldrich). The homogenate was centrifuged at 800g for 10 minutes, at 4°C, to remove the insoluble fraction. Supernatants from the first centrifugation were centrifuged at 1,000g for 10 minutes at 4°C to pellet the nuclear fraction. Supernatants from the second centrifugation were centrifuged at 13,200g for 30 minutes at 4°C to pellet the mitochondrial fraction. The protein content was determined by Bradford assay (µg/µl) and mitochondrial fractions were stored at -80°C.

2D-gel electrophoresis 50µg of the mitochondrial fraction were added to rehydration solution (8M urea, 2% CHAPS, 2% IPG Buffer, 0.1% bromophenol blue). 20mM DTT was added to an aliquot of rehydration solution directly before use. The standard protocol according to manufacturer instructions was followed(Life Technologies, 2012). Briefly, sample was applied to rehydrate ZOOM IPG strips for an hour at room temperature followed by iso-electric focussing using the ZOOM IPG (Life Technologies) system and pH 3-10 (non-linear) ZOOM IPG strips. Gels were stained (SimplyBlue™ SafeStain, Life Technologies) and imaged (ImageQuant bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

300, GE Healthcare Life Sciences). Analyses were performed using SameSpots software (Totallab) (one-way ANOVA). Three pooled biological replicates were included for each of the four groups. Samples were analysed by the Centre of Excellence in Mass Spectrometry at University of York(Centre for Excellence in Mass Spectrometry, 2019). Briefly, proteins were reduced and alkylated, followed by digestion in-gel with trypsin. Matrix Assisted Laser Desorption Ionization Tandem Time-of-Flight mass spectrometry (MALDI-TOF/MS) was used to analyse the samples. The generated tandem MS data was compared against the NCBI database using the MASCOT search programme to identify the proteins. De novo sequence interpretation for individual peptides were inferred from peptide matches.

Label-free proteomics 30µg/µl of each mitochondrial fraction was prepared with 4X LDS sample buffer and 4mM DTT. Samples were run in triplicate on a 4-12% Bis-Tris gel in 1X MES SDS running buffer for 40 minutes at 200V (all Invitrogen). Three biological replicates were run for each of the four groups. Individual gel lanes were excised and placed into separate Eppendorf tubes. Samples were analysed by the Centre of Excellence in Mass Spectrometry at University of York(Centre for Excellence in Mass Spectrometry, 2019). Briefly, protein was in-gel digested post-reduction and alkylation. The resulting extracted peptides were analysed over 1-hour LC-MS acquisitions with elution from a 50cm, C18 PepMap column onto a Thermo Orbitrap Fusion Tribrid mass spectrometer using a Waters mClass UPLC. Extracted tandem mass spectra were searched against the combined drosophila melanogaster and Saccharomyces cerevisiae subsets of the UniProt database. Protein identifications were filtered to achieve <1% false discovery rate as assessed against a reverse database. Identifications were further filtered to require a minimum of two unique peptides per protein group. For relative label-free quantification, extracted ion chromatograms for identified peptides were extracted and integrated for all samples. A maximum mass deviation of 3 ppm and retention time drift of 3 mins were set. Resulting quantifications were further filtered to an arbitrary PEAKS quality factor of 5 for feature mapping and required a minimum of two aligned features from a minimum of two unique peptides per protein quantification. Protein abundances were normalised between samples based on total identified peptide ion area.

Gene Ontology enrichment analysis gProfiler was used to undertake Gene Ontology (GO) enrichment analysis for the label-free mass spectrometry identified proteins with significant expression differences between each of the four experimental groups(Raudvere et al, 2019). bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

KEGG, Molecular Function (MF), Biological Process (BP) and Cellular Compartment (CC) enrichment analyses are generated by gProfiler are presented.

Protein-protein interaction network analysis Differences in expression of proteins between groups were further analysed using the STRING database v.11.0(Szklarczyk et al, 2019). The platform was used to create protein-protein interaction (PPI) networks based upon the differentially expressed proteins (DEPs) observed between groups. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Results & Discussion We subjected male Pink1 loss of function mutant (Pink1-) and WT D. melanogaster to a seven-day exercise regimen, whilst maintaining groups of unexercised Pink1- and WT as controls. The mitochondria of the four groups were then isolated and investigated using 2D gel electrophoresis and label-free mass spectrometry analyses to determine changes in their mitochondrial proteome. The 2D gel electrophoresis method allowed for a fast and simple separation of proteins, which served as a scoping method to identify some of the most significant changes in expression. Label-free mass spectrometry analyses generated proteomes that we used in network enrichment analysis. Previously we have used proteomic profiling to characterise mitochondrial populations in both mice and long-lived pipistrelle bats, and here we apply this to better understand the PD phenotype as well as possible changes due to exercise(Pollard et al, 2016, 2019).

Table 1 - Expression changes between exercised Pink1- flies and non-exercised Pink1- flies. Changes in expression were determined by 2DE-MS.

2DE-MS identified a general reduction in protein expression with exercise in the Pink1 mutant flies We isolated mitochondria from exercised Pink1- D. melanogaster and performed 2DE-MS on these fractions. All proteins identified as changed with our exercise intervention were reduced in expression (Table 1). PINK1 is recognised as having a central role in mitophagy, ensuring a healthy pool of mitochondria are maintained(Clark et al, 2006; Park et al, 2006). Dysfunctional mitophagy results in an accumulation of damaged and dysfunctional mitochondria and their contents(Anzell et al, 2018). It is possible that alternate mitophagy pathways can compensate for PINK1 and are upregulated during exercise, this could account for the sweeping reductions in protein levels observed here. Indeed, alternative proteins have been identified which promote PINK1/PARKIN-independent mitophagy: AMBRA1, HUWE1 and IKKα(Di Rita et al, 2018). However, it could also be suggested that the energetic demands of the exercise result in less energy available to protein synthesis pathways. In this instance, exercise would likely affect proteostasis more broadly, beyond just the mitochondrial proteome. We proceeded to pursue the directionally homologous 2DE-MS results by obtaining a global topology of the mitochondrial protein changes that occur to Pink1- D. melanogaster due to exercise intervention using a label-free proteomics method. .

GO annotation of identified label-free proteins and proportion identified that are localised to mitochondria Non-gel-based label-free proteomic analyses identified 516 proteins from the - mitochondrial fractions of Pink1 and WT D. melanogaster (Supp. table 1). GO and KEGG analyses showed that these fractions were enriched for mitochondrial processes and pathways, confirming the efficacy of our fractionation methodology (Fig. 1). The top term of the GO cellular compartment analysis was cytoplasm, followed by mitochondrion and sub-mitochondrial compartments, many mitochondrial proteins are known to also localise to the cytoplasm(Yogev & Pines, 2011; Ben-Menachem et al, 2011). bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Figure 1 - Enrichment analysis of all proteins identified by label-free proteomics. Biological process, cellular compartment and KEGG enrichment analysis each presented processes associated with mitochondrial function and physiology. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pink1- flies have decreased levels of proteins in energy metabolism pathways Label-free proteomics highlighted 105 differently expressed proteins between non- exercised WT and Pink1- groups (Supp. Table 3). Ten proteins were shown to be reduced in expression in Pink1- compared to WT. We found that Pink1- flies have reductions in mitochondrial processes associated with energy metabolism, with the top GO biological process terms being oxidative phosphorylation, electron transport chain, ATP metabolic process and oxidation-reduction process (Fig 2). The deficiencies in mitochondrial oxidative phosphorylation, the electron transport chain and specifically in the activity of Complex I in Parkinson’s disease are well established, and this aligns with our findings from the GO analysis of the proteomics data(Parker et al, 1989; Schapira et al, 1990; Shoffner et al, 1991; Finsterer et al, 2001). Specific subunits of complexes within the electron transport chain that decreased in expression include NADH dehydrogenase (ubiquinone) 75 kDa subunit isoform B (complex I), GH01077p (complex III in D. melanogaster), HDC00331 (complex IV in D. melanogaster) and Levy isoform A (complex IV in D. melanogaster). While complex I and complex IV have been reported as dysfunctional in PD, reduced expression or decreased activity for complex III isn’t well documented(Keeney et al, 2006; Müftüoglu et al, 2004; Schapira et al, 1990). However, decreased Complex II/III activity has been shown in platelets of untreated Parkinson’s disease patients(Haas et al, 1995). It is interesting to note that most (95/105) of the differentially expressed proteins were more highly expressed in Pink1- D. melanogaster. GO biological process analysis showed these proteins to be enriched for redox processes, cytoplasmic translation, cellular amide metabolic processes and fatty acid derivative biosynthetic processes. GO cellular compartment analysis showed that the more highly expressed proteins in Pink1- were enriched for cytoplasmic ribosomes, organelle membranes and endoplasmic reticulum. KEGG pathway analysis paralleled these findings, highlighting the identified proteins as involved in fatty acid metabolism, ribosomes and one carbon pool by folate. There is evidence linking fatty acid metabolism and function to Parkinson’s disease, with proteins identified by GWAS studies, supressed β-oxidation, and physical interaction between α-synuclein and fatty acids potential being key factors(Chang et al, 2017; Li et al, 2018; Saiki et al, 2017; Sharon et al, 2001; Karube et al, 2008). Early studies into the effect of α-synuclein (SNCA) gene deletion on lipid metabolism in mice reported reduced palmate uptake and altered palmate metabolism in the brain, reduced acyl-CoA Synthetase activity that resulted in reduced arachidonic acid uptake and turnover, and increased docosahexaenoic acid brain mass, incorporation and turnover(Golovko et al, 2005, 2006, 2007). Our own work shows differences in arachidonic acid derivatives in Parkinson’s disease mitochondria(Ingram et al, 2020). The data presented here show an enrichment of the folate metabolic pathway, not reported previously. It may be that in Pink1- flies this is a compensatory mechanism. KEGG analysis highlighted the metabolism of folate (vitamin B9) as bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

- enriched in Pink1 D. melanogaster. B-vitamins, in particular folate, are well studied in the context of Parkinson’s disease due to the observation of homocysteine (a methionine cycle metabolite) having neurotoxic effects(Lipton et al, 1997; Müller et al, 1999; Duan et al, 2002; Martignoni et al, 2007). It is hypothesised that the administration of B-vitamins can drive the synthesis of methionine, thus reducing intracellular homocysteine(Mayer et al, 2002; Maron & Loscalzo, 2009; Shen, 2015). However the relationship between B -vitamins, neurotoxicity and Parkinson’s disease is complex and a consensus has yet to be established. Some data show either little correlation between homocysteine levels and B vitamins including B6, folate and B12 while others show contradictory results, including elevated homocysteine levels and decreased folate levels in Parkinson’s disease patients(Xie et al, 2017; Shen et al, 2019; Dong & Wu, 2020; Shen, 2015). bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Figure 2 - GO: Biological Process analysis for downregulated protein expression differences between Pink1- non-exercised and wild-type non-exercised flies. Pink1- flies have reduced expression of proteins involved in mitochondrial respiration and oxidative phosphorylation.

Exercise reduces measured protein levels in Pink1- flies 2DE analysis of Pink1- D. melanogaster revealed reductions in twelve proteins - with exercise (Table 1). Label-free proteomics showed a similar pattern in Pink1 exercised D. melanogaster; of the fifty-seven protein differences, fifty-five were reductions of protein expression with exercise (Supp. table 2). GO:MF and KEGG pathway analysis determined that the terms ribosomal and fatty acid metabolism were significantly represented in the proteins with reduced expression (Fig 3). Interestingly, these terms were shown to be increased in Pink1- compared to WT flies that had not been exercised. This suggests that in Pink1- D. melanogaster exercise returns levels of protein expression towards WT values. It has been shown that PINK1 interacts with the protein translation pathway and that increased protein translation in Pink1- D. melanogaster causes an exacerbated Pink1- phenotype. Taking this a step further, it was shown that 40S ribosomal subunit S6 (RpS6) RNAi was able to mitigate the Pink1- phenotype(Liu & Lu, 2010). These data suggest improper protein translation regulation is involved in the pathogenesis of PD and that inhibition of this pathway mitigates progression. Exercise appears to be able to reverse the upregulated protein translation pathways found in Pink1- D. melanogaster. KEGG analysis identified fatty acid metabolism from proteins reduced in expression due to exercise, while GO:MF analysis highlighted CoA Carboxylase activity from the same protein data set. This reduction in the metabolism, and in particular the synthesis, of fatty acids can be contrasted with the KEGG analysis described earlier which identified elevated expression of fatty acid metabolism associated proteins in Pink1- flies compared with wild type flies. It can be interpreted that exercise reverses the change in Pink1- flies and returns the fatty acid metabolic profile back towards WT flies. The two proteins upregulated with exercise in Pink1- D. melanogaster were OCIA domain-containing protein 1 (OCIAD1) and dihydroorotate dehydrogenase (quinone) mitochondrial (DHODH), neither have previously been connected with exercise. OCIAD1 has been shown to localise to both endosomes and mitochondria and regulate pathways such as JAK/STAT, Notch and PI3K/AKT(Morel et al, 2013; Khadilkar et al, 2014; Li et al, 2020). OCIAD1 has been shown to regulate mitochondrial ETC activity via control of complex I activity, which showed an inverse association with OCIAD1 overexpression(Shetty et al, 2018). Deregulated OCIAD1 levels have been linked to mitochondrial dysfunction, interaction with BCL-2 and Alzheimer’s disease(Li et al, 2020). DHODH is an inner mitochondrial membrane enzyme that catalyses the fourth step in de novo synthesis of pyrimidines(Cherwinski et al, 1995). A link between pyrimidine synthesis and mitochondrial morphology was shown with the addition of the drug leflunomide to muscle cells(Miret-Casals et al, 2018). The group showed that leflunomide inhibited DHODH by binding to its ubiquinone binding channel, thereby preventing the production of pyrimidine ribonucleotide uridine monophosphate (UMP). DHODH inhibition induced upregulation of mitofusions and subsequent mitochondrial elongation, by depleting the cellular pyrimidine bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

pool. As ubiquinone is reduced to ubiquinol in the DHODH-mediated catalysis of dihydroorotate to orotate, and as ubiquinol is a substrate of respiratory complex III, DHODH is important for the ETS. DHODH deficiency has been reported to partially inhibit complex III and increase ROS generation(Fang et al, 2013). - The same Pink1 D. melanogaster strain has previously been reported to have upregulated genes involved in nucleotide metabolism, which is also the case in brains of PD patients with PINK1 mutations(Tufi et al, 2014). Genetic and pharmacological upregulation of nucleotide metabolism and scavenging pathways restored mitochondrial function caused by PINK1 loss. Therefore, DHODH upregulation by exercise may act in a compensatory manner to manage metabolic stress due to the Pink1- phenotype. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Figure 3 - GO: Molecular Function and KEGG analysis for proteins reduced in expression in Pink1- exercised flies compared with Pink1- non-exercised flies. Both Molecular Function and KEGG enrichment analysis indicated a decrease in expression of fatty acid metabolic proteins in the exercised Pink1- flies.

Exercise reduces the difference in levels of protein expression between Pink1- and WT flies Of the 516 proteins identified, 105 protein had different levels between Pink1- and WT non-exercised D. melanogaster (Supp. Table 3). Comparing the Pink1- exercised group to either WT non-exercised or exercised halved the number of differentially expressed proteins (55 and 56 proteins, respectively) (Supp. table 2). Qualitatively the heatmap of protein expression for Pink1- exercised D. melanogaster more closely resembles WT D. melanogaster (Fig 4). This suggests exercise can ameliorate the aberrant protein profile of Pink1- D. melanogaster towards a more WT profile. In agreement, gene expression data from an exercised mouse model of arrhythmogenic cardiomyopathy, that originally showed a dysregulation of near 800 genes, showed partial restoration of gene expression with regular exercise, with greatest remedial effects on proteins involved in inflammation and oxidative phosphorylation(Cheedipudi et al, 2020). bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Figure 4 - Heat map of protein expression levels (for proteins identified among all groups), determined by label-free mass spectrometry of mitochondrial fractions. Qualitatively, the identified Pink1- exercised fly proteome more closely resembles the two WT fly proteomes than does the Pink1- non-exercised fly proteome. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A .

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Figure 5 - Network Analysis of differentially expressed proteins. A) Pink1- exercised vs WT exercised; B) Pink1- non- exercised vs WT non-exercised. Networks analysed using STRINGdb. The nodes are coloured according to the processes (legend) that the proteins are involved in by using GO Terms for Biological Processes. The edge shows type of interactions, experimentally determined interactions are pink and those obtained from databases are sky blue. Predicted interactions such as gene neighbourhood are blue, green and red for gene co-occurrence, gene neighbourhood and gene fusions. Co-expression interactions are shown in black, text-mining interactions are shown in light green and protein homology edges are purple. Network analyses of differentially expressed proteins bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

STRING database network analysis complemented the results seen with gProfiler. The protein-protein interaction (PPI) networks were generated using STRING, and (Fig 5A) shows that Pink1- exercised compared with the WT exercised flies have a differentially expressed proteins (DEPs) network that consisted of 49 nodes and 200 edges with average node degree 8.16 and PPI enrichment p-value of (P < 1.0e-16).

Most of the proteins in the network have downregulated expression, with the following proteins found to be upregulated: endoplasmic reticulum chaperone BiP, enoyl-CoA hydratase short chain, glutamine synthetase, protein disulphide isomerase, methylmalonate-semialdehyde dehydrogenase, polyadenylate-binding protein, poly(U)-specific endoribonuclease, glutamine synthetase, flotillin-1, heat shock protein 22, phosphotidate cytidylyltransferase, fatty acyl-CoA reductase and dihydroorotate dehydrogenase. The DEPs were found to be part of the ETC and OXPHOS processes, transmembrane transport and oxidative-reduction processes. The highly connected network consisted of proteins from complex I: ND-18, ND-MLRQ, ND- ASHI, ND-B14.5B, ND-B14, ND-B16.6, ND-49, ND-51, ND-B17.2, ND-B14.7; complex IV: COX6B, COX4, mt: COII, COX5A; complex III: OX, and other proteins CYPE, UQCR-6.4, UQCR-14, levy and 40S ribosomal proteins S18. This cluster of proteins are downregulated in the PINK1- exercised. (Fig 5B) shows the Pink1- non-exercised compared with the WT non-exercised PPI network consisted of 86 nodes and 121 edges with average node degree 2.81 and PPI enrichment p-value (P < 1.0e-16). In contrast to Pink1- exercised versus WT exercised all DEPs were upregulated, except for cytochrome c oxidase subunit 4, cyclope isoform A, flightin isoform B, cytochrome b-c1 complex subunit 7, NADH dehydrogenase 1 alpha subcomplex 12, Troponin 1, NADH dehydrogenase 18, cytochrome c oxidase subunit, cytochrome P450, NADH dehydrogenase 1 beta subcomplex subunit 8, cytochrome c oxidase subunit 5A, NADH dehydrogenase B14 and levy isoform A. The DEPs were found to be a part of oxidation-reduction processes, fatty-acyl- CoA metabolic processes, translation and protein folding. The identification of DEPs from fatty-acyl-CoA metabolic process is concurrent with the enrichment analysis presented in (Fig. 3). The highly connecting nodes in this network is that of ribosomal proteins, RPS18, RPS23, RPS23, RPS7, RPS14b, RPL6, RPL5, RPL10AB and RPL9 involved in translation that are upregulated. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A . P I N K 1 - e x

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Figure 6 - Network Analysis of differentially expressed proteins. A) Pink1- exercised vs Pink1- non-exercised; B) Pink1- non-exercised vs WT exercised; C) Pink1- exercised vs WT non-exercised. Networks analysed using STRINGdb. The nodes are coloured according to the processes (legend) that the proteins are involved in by using GO Terms for Biological Processes. The edge shows type of interactions, experimentally determined interactions are pink and the one obtained from databases are sky blue. Predicted interactions such as gene neighbourhood are blue, green and red for gene co- occurrence, gene neighbourhood and gene fusions. Co-expression interactions are shown in black, text-mining interactions are shown in light green and protein homology edges are purple. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Similarly, for (Fig 6A-C), three additional PPI networks were generated for DEPs in pairwise group comparisons. For Pink1- exercised flies compared with Pink1- non-exercised flies the PPI network consisted of 43 nodes and 29 edges with average node degree 1.35 and PPI enrichment p-value <1.62e-06 (Fig 6A). For Pink1- non-exercised flies compared with WT exercised flies the network has 107 nodes and 341 edges with average node degree 6.37 and PPI enrichment p-value (P <1.0e-16) (Fig 6B). For the final PPI network, Pink1- exercised flies compared with WT non-exercised flies, there are 44 nodes and 40 edges with average node degree 1.82 and PPI enrichment p-value 6.79e-11 (Fig 6C). For each of the three PPI networks, the number of edges is larger than expected and the nodes were more connected than for a random PPI network of the same size. In Pink1- exercised flies versus Pink1- non-exercised flies all DEPs were downregulated except for OCIA domain containing protein 1 and dihydroorotate dehydrogenase (Fig 6A). The Pink1- exercised flies and Pink1- non-exercised flies DEPs were found to be involved in formation of 40S ribosomal subunit, oxidation reduction processes, synthesis of ketone bodies, translation and cellular lipid catabolic processes. Pink1- non-exercised flies and WT exercised flies DEPs were found to be involved in ETC, translation, peroxisome, formation of 40S subunits and protein folding (Fig 6B). The Pink1- exercised versus WT non- exercised were involved in purine ribonucleotide triphosphate metabolic processes, ETC and peroxisomes (Fig 6C). PINK1- non-exercised compared with wildtype exercised showed three highly connected clusters (Fig 6B). Cluster 1 had proteins from complex I and complex IV proteins that are downregulated like that of Pink1- exercised compared with the WT exercised flies and Cluster 2 has ribosomal proteins that are upregulated, similar to that of Pink1- non-exercised compared with the WT non-exercised PPI network. Cluster 3 has proteins UGT, HSP22, HSP60C, CABP1, GP93, HSC70-5, HSC70Cb, HSP60A, RTNL1 and CNX99A, proteins involved in protein control and folding which are found to be upregulated.

Conclusion A picture of global proteomic changes made in response to exercise was obtained. By using two methodologies to assess the mitochondrial proteome we were able to measure changes in an organelle whose function in exercise, and dysfunction in Parkinson’s disease, is crucial. 2D-GE MS analysis used to compare WT exercised flies and control flies identified five out of seven proteins with altered expression, the majority of which have roles in metabolism and bioenergetics. 2D-GE MS data comparison between Pink1- exercised and Pink1- non-exercised flies revealed several proteins with decreased levels of expression in response to exercise. GO and KEGG analyses validated our mitochondrial isolation methodology by identifying the enrichment of mitochondrial processes and pathways. In the four comparisons that we made; proteins involved in bioenergetics had changed expression. GO, KEGG and STRING network analysis of these proteome comparisons identified enrichment of bioenergetic pathways. The most significant pathways included oxidation-reduction, fatty acid metabolism, and folate metabolism which are associated with PD. Our data point to exercise aiding normalisation of these pathways. Specific proteins in the pathways may be candidates to develop therapeutic approaches in PD.

Author contributions

BE performed data analysis, assisted with experiments, and wrote the manuscript, TLI performed experimental work and helped prepare the manuscript, GK performed STRING analyses and helped prepare the manuscript, JRI developed and constructed the ICE machine for fly exercise, NM generated the flies used for these experiments and was consulted on all aspects of the fly work, LC directed the research, supervised experiments, provided reagents and prepared the manuscript.

The authors declare they have no conflict of interest.

Funding

This work was supported by the Biotechnology and Biological Sciences Research Council [grant number BB/J014508/1], via awards to BE and TLI. GK is supported by a University of Nottingham Vice-Chancellors International Scholarship award. NM and LC are funded by HEFCE. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Appendix

Ingram counter-balanced exerciser

Our bespoke Ingram Counter-balanced Exercise (ICE) machine was adapted from the PT design of Piazza et al., by Mr. John Ingram (TI’s father)(Piazza et al, 2009). The exerciser fits into an incubator with internal dimensions of 48 x 48 x 35cm and provides a vertically moving tray measuring 30 x 34cm. A solenoid-powered counterbalanced lever causes the tray to be lifted 3cm. The tray is lifted and immediately dropped every 15 seconds. Counterbalancing the lever are a series of springs, which can be adjusted to allow lifts of up to 3.0kg. Springs efficiently store and release energy enabling a more rapid drop than would be the case if weights were used. They also reduce the size and overall weight of the device. Lift is provided by a solenoid wound onto a nylon core and fixed to the body of the exerciser. The steel armature passing through the solenoid is attached to the counterbalanced lever. Activation of the solenoid causes the armature to rise which lifts the lever. Solenoid activation is controlled by an astable timer. This triggers a relay to pulse the applied AC voltage. Two capacitors acting as a loss-less resistor allow the voltage to be reduced without producing excess heat, before it is rectified, smoothed and finally applied to the solenoid. Powering with DC current causes less vibration and heat generation in the solenoid but, as the capacitors and the solenoids both work more efficiently at lower temperatures, any excess heat is subsequently dissipated by proximate fans. Using a pulse of current as described bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

rather than the discharge from a large capacitor to activate the solenoid saves space, it is also safer as there is far less stored energy.

Bibliography Alegre-Abarrategui J, Christian H, Lufino MMP, Mutihac R, Venda LL, Ansorge O & Wade-Martins R (2009) LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model. Hum Mol Genet 18: 4022–4034 Anzell AR, Maizy R, Przyklenk K & Sanderson TH (2018) Mitochondrial Quality Control and Disease: Insights into Ischemia-Reperfusion Injury. Mol Neurobiol 55: 2547–2564 Ashburn A, Fazakarley L, Ballinger C, Pickering R, McLellan LD & Fitton C (2007) A randomised controlled trial of a home based exercise programme to reduce the risk of falling among people with Parkinson’s disease. J Neurol Neurosurg Psychiatry 78: 678–684 Ben-Menachem R, Tal M, Shadur T & Pines O (2011) A third of the yeast mitochondrial proteome is dual localized: A question of evolution. Proteomics 11: 4468–4476 Centre for Excellence in Mass Spectrometry (2019) FAQs - Biology, The University of York. Chang D, Nalls MA, Hallgrímsdóttir IB, Hunkapiller J, Brug M van der, Cai F, Kerchner GA, Ayalon G, Bingol B, Sheng M, et al (2017) A meta-analysis of genome-wide association studies identifies 17 new Parkinson’s disease risk loci. Nat Genet 49: 1511–1516 Cheedipudi SM, Hu J, Fan S, Yuan P, Karmouch J, Czernuszewicz G, Robertson MJ, Coarfa C, Hong K, Yao Y, et al (2020) Exercise restores dysregulated gene expression in a mouse model of arrhythmogenic cardiomyopathy. Cardiovasc Res 116: 1199–1213 Cherwinski HM, Cohn RG, Cheung P, Webster DJ, Xu YZ, Caulfield JP, Young JM, Nakano G & Ransom JT (1995) The immunosuppressant leflunomide inhibits lymphocyte proliferation by inhibiting pyrimidine biosynthesis. J Pharmacol Exp Ther 275: 1043–1049 Clark IE, Dodson MW, Jiang C, Cao JH, Huh JR, Seol JH, Yoo SJ, Hay BA & Guo M (2006) Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441: 1162–1166 Cookson MR (2012) Parkinsonism due to mutations in PINK1, Parkin, and DJ-1 and oxidative stress and mitochondrial pathways. Cold Spring Harb Perspect Med 2 Dong B & Wu R (2020) Plasma homocysteine, folate and vitamin B12 levels in Parkinson’s disease in China: A meta-analysis. Clin Neurol Neurosurg 188: 105587 Duan W, Ladenheim B, Cutler RG, Kruman II, Cadet JL & Mattson MP (2002) bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson’s disease. J Neurochem 80: 101–110 Fang JX, Uchiumi T, Yagi M, Matsumoto S, Amamoto R, Takazaki S, Yamaza H, Nonaka K & Kang D (2013) Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction. Biosci Rep 33: 217–227 Finsterer J, Jarius C & Baumgartner M (2001) Parkinson’s disease associated with impaired oxidative phosphorylation. Neuroradiology 43: 997–1000 Golovko MY, Faergeman NJ, Cole NB, Castagnet PI, Nussbaum RL & Murphy EJ (2005) Α-Synuclein Gene Deletion Decreases Brain Palmitate Uptake and Alters the Palmitate Metabolism in the Absence of Α-Synuclein Palmitate Binding. Biochemistry 44: 8251–8259 Golovko MY, Rosenberger TA, Færgeman NJ, Feddersen S, Cole NB, Pribill I, Berger J, Nussbaum RL & Murphy EJ (2006) Acyl-CoA synthetase activity links wild-type but not mutant α-synuclein to brain arachidonate metabolism. Biochemistry 45: 6956–6966 Golovko MY, Rosenberger TA, Feddersen S, Færgeman NJ & Murphy EJ (2007) α-Synuclein gene ablation increases docosahexaenoic acid incorporation and turnover in brain phospholipids. J Neurochem 101: 201–211 Goodwin VA, Richards SH, Taylor RS, Taylor AH & Campbell JL (2008) The effectiveness of exercise interventions for people with Parkinson’s disease: A systematic review and meta-analysis. Mov Disord 23: 631–640 Greene JC, Whitworth AJ, Kuo I, Andrews LA, Feany MB & Pallanck LJ (2003) Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. Proc Natl Acad Sci 100: 4078–4083 Haas RH, Nasirian F, Nakano K, Ward D, Pay M, Hill R & Shults CW (1995) Low platelet mitochondrial complex I and complex II/III activity in early untreated parkinson’s disease. Ann Neurol 37: 714–722 Hirsch EC, Graybiel AM & Agid Y (1988) Melanized dopaminergic neurons are differentially affected in Parkinson’s disease. Nature 334: 345--348. Ingram TL, Shephard F, Sarmad S, Ortori CA, Barrett DA & Chakrabarti L (2020) Sex specific inflammatory profiles of cerebellar mitochondria are attenuated in Parkinson’s disease. Aging (Albany NY) 12: 17713–17737 Jang Y, Koo JH, Kwon I, Kang EB, Um HS, Soya H, Lee Y & Cho JY (2017) Neuroprotective effects of endurance exercise against neuroinflammation in MPTP-induced Parkinson’s disease mice. Brain Res 1655: 186–193 Julienne H, Buhl E, Leslie DS & Hodge JJL (2017) Drosophila PINK1 and parkin loss-of-function mutants display a range of non-motor Parkinson’s disease phenotypes. Neurobiol Dis 104: 15–23 Karube H, Sakamoto M, Arawaka S, Hara S, Sato H, Ren CH, Goto S, Koyama S, Wada M, Kawanami T, et al (2008) N-terminal region of α-synuclein is essential for the fatty acid-induced oligomerization of the molecules. FEBS bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Lett 582: 3693–3700 Kazlauskaite A, Kondapalli C, Gourlay R, Campbell DG, Ritorto MS, Hofmann K, Alessi DR, Knebel A, Trost M & Muqit MMK (2014) Accelerated publication: Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem J 460: 127–139 Keeney PM, Xie J, Capaldi RA & Bennett JP (2006) Parkinson’s disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci 26: 5256–5264 Khadilkar RJ, Rodrigues D, Mote RD, Sinha AR, Kulkarni V, Magadi SS & Inamdar MS (2014) ARF1-GTP regulates Asrij to provide endocytic control of Drosophila blood cell homeostasis. Proc Natl Acad Sci U S A 111: 4898–4903 Klemann CJHM, Xicoy H, Poelmans G, Bloem BR, Martens GJM & Visser JE (2018) Physical Exercise Modulates L-DOPA-Regulated Molecular Pathways in the MPTP Mouse Model of Parkinson’s Disease. Mol Neurobiol 55: 5639– 5657 Kondapalli C, Kazlauskaite A, Zhang N, Woodroof HI, Campbell DG, Gourlay R, Burchell L, Walden H, MacArtney TJ, Deak M, et al (2012) PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biol 2 Koyano F, Okatsu K, Kosako H, Tamura Y, Go E, Kimura M, Kimura Y, Tsuchiya H, Yoshihara H, Hirokawa T, et al (2014) Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 510: 162–166 Li G, Cui S, Du J, Liu J, Zhang P, Fu Y, He Y, Zhou H, Ma J & Chen S (2018) Association of GALC, ZNF184, IL1R2 and ELOVL7 With Parkinson’s Disease in Southern Chinese. Front Aging Neurosci 10: 1–6 Li X, Wang L, Cykowski M, He T, Liu T, Chakranarayan J, Rivera A, Zhao H, Powell S, Xia W, et al (2020) OCIAD1 contributes to neurodegeneration in Alzheimer’s disease by inducing mitochondria dysfunction, neuronal vulnerability and synaptic damages. EBioMedicine 51 Life Technologies (2012) ZOOM ® IPGRunner TM System For isoelectric focusing of ZOOM ® Strips Catalog Numbers ZM0001, ZM0002, ZM0004 Lipton SA, Kim WK, Choi YB, Kumar S, D’Emilia DM, Rayudu P V., Arnelle DR & Stamler JS (1997) Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor. Proc Natl Acad Sci U S A 94: 5923– 5928 Liu S & Lu B (2010) Reduction of Protein Translation and Activation of Autophagy Protect against PINK1 Pathogenesis in Drosophila melanogaster. PLoS Genet 6: e1001237 Liu W, Acin-Perez R, Geghman KD, Manfredi G, Lu B & Li C (2011) Pink1 regulates the oxidative phosphorylation machinery via mitochondrial fission. Proc Natl Acad Sci 108: 12920–12924 Maron BA & Loscalzo J (2009) The treatment of hyperhomocysteinemia. Annu Rev Med 60: 39–54 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Martignoni E, Tassorelli C, Nappi G, Zangaglia R, Pacchetti C & Blandini F (2007) Homocysteine and Parkinson’s disease: A dangerous liaison? J Neurol Sci 257: 31–37 Mayer O, Filipovský J, Hromádka M, Svobodová V, Racek J, Mayer O, Stehlík P, Trefil L & Zárybnická M (2002) Treatment of hyperhomocysteinemia with folic acid: Effects on homocysteine levels, coagulation status, and oxidative stress markers. J Cardiovasc Pharmacol 39: 851–857 Miret-Casals L, Sebastiá D, Brea J, Loza MI, Albericio F, Zorzano A, Rico-Leo EM, Palacín M & Ferná Ndez-Salguero PM (2018) Identification of New Activators of Mitochondrial Fusion Reveals a Link between Mitochondrial Morphology and Pyrimidine Metabolism. Cell Chem Biol 25: 268–278 Morais VA, Verstreken P, Roethig A, Smet J, Snellinx A, Vanbrabant M, Haddad D, Frezza C, Mandemakers W, Vogt-Weisenhorn D, et al (2009) Parkinson’s disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function. EMBO Mol Med 1: 99–111 Morel E, Chamoun Z, Lasiecka ZM, Chan RB, Williamson RL, Vetanovetz C, Dall’Armi C, Simoes S, Point Du Jour KS, McCabe BD, et al (2013) Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system. Nat Commun 4: 2250 Müftüoglu M, Elibol B, Dalmizrak Ö, Ercan A, Kulaksiz G, Ögüs H, Dalkara T & Özer N (2004) Mitochondrial complex I and IV activities in leukocytes from patients with parkin mutations. Mov Disord 19: 544–548 Müller T, Werne B, Fowler B & Kuhn W (1999) Nigral endothelial dysfunction, homocysteine, and Parkinson’s disease. Lancet 354: 126–127 Murray DK, Sacheli MA, Eng JJ & Stoessl AJ (2014) The effects of exercise on cognition in Parkinson’s disease: A systematic review. Transl Neurodegener 3 doi:10.1186/2047-9158-3-5 [PREPRINT] Ordureau A, Sarraf SA, Duda DM, Heo JM, Jedrychowski MP, Sviderskiy VO, Olszewski JL, Koerber JT, Xie T, Beausoleil SA, et al (2014) Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol Cell 56: 360–375 Palikaras K, Lionaki E & Tavernarakis N (2018) Mechanisms of mitophagy in cellular homeostasis, physiology and pathology. Nat Cell Biol 20: 1013–1022 Pankratz N & Foroud T (2007) Genetics of Parkinson disease. Genet Med 9: 801– 811 doi:10.1097/GIM.0b013e31815bf97c [PREPRINT] Park J, Lee SB, Lee S, Kim Y, Song S, Kim S, Bae E, Kim J, Shong M, Kim JM, et al (2006) Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature 441: 1157–1161 Park JS, Davis RL & Sue CM (2018) Mitochondrial Dysfunction in Parkinson’s Disease: New Mechanistic Insights and Therapeutic Perspectives. Curr Neurol Neurosci Rep 18 doi:10.1007/s11910-018-0829-3 [PREPRINT] Parker WD, Boyson SJ & Parks JK (1989) Abnormalities of the electron transport chain in idiopathic parkinson’s disease. Ann Neurol 26: 719–723 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Piazza N, Gosangi B, Devilla S, Arking R & Wessells R (2009) Exercise-Training in Young Drosophila melanogaster Reduces Age-Related Decline in Mobility and Cardiac Performance. PLoS One 4: e5886 Pickrell AM & Youle RJ (2015) The roles of PINK1, Parkin, and mitochondrial fidelity in parkinson’s disease. Neuron 85: 257–273 Pollard A, Shephard F, Freed J, Liddell S & Chakrabarti L (2016) Mitochondrial proteomic profiling reveals increased carbonic anhydrase II in aging and neurodegeneration. Aging (Albany NY) Pollard AK, Ingram TL, Ortori CA, Shephard F, Brown M, Liddell S, Barrett DA & Chakrabarti L (2019) A comparison of the mitochondrial proteome and lipidome in the mouse and long-lived Pipistrelle bats. Aging (Albany NY) 11: 1664–1685 Raudvere U, Kolberg L, Kuzmin I, Arak T, Adler P, Peterson H & Vilo J (2019) G:Profiler: A web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res 47: W191–W198 Ray Dorsey E, Elbaz A, Nichols E, Abd-Allah F, Abdelalim A, Adsuar JC, Ansha MG, Brayne C, Choi JYJ, Collado-Mateo D, et al (2018) Global, regional, and national burden of Parkinson’s disease, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol 17: 939–953 Di Rita A, Peschiaroli A, D Acunzo P, Strobbe D, Hu Z, Gruber J, Nygaard M, Lambrughi M, Melino G, Papaleo E, et al (2018) HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKKα. Nat Commun 9: 3755 Saiki S, Hatano T, Fujimaki M, Ishikawa KI, Mori A, Oji Y, Okuzumi A, Fukuhara T, Koinuma T, Imamichi Y, et al (2017) Decreased long-chain acylcarnitines from insufficient β-oxidation as potential early diagnostic markers for Parkinson’s disease. Sci Rep 7: 1–15 Sasco AJ, Paffenbarger RS, Gendre I & Wing AL (1992) The Role of Physical Exercise in the Occurrence of Parkinson’s Disease. Arch Neurol 49: 360–365 Schapira AHV, Cooper JM, Dexter D, Clark JB, Jenner P & Marsden CD (1990) Mitochondrial Complex I Deficiency in Parkinson’s Disease. J Neurochem 54: 823–827 Schulz-Schaeffer WJ (2010) The synaptic pathology of α-synuclein aggregation in dementia with Lewy bodies, Parkinson’s disease and Parkinson’s disease dementia. Acta Neuropathol 120: 131–143 Sharon R, Goldberg MS, Bar-Josef I, Betensky RA, Shen J & Selkoe DJ (2001) Α- Synuclein Occurs in Lipid-Rich High Molecular Weight Complexes, Binds Fatty Acids, and Shows Homology To the Fatty Acid-Binding Proteins. Proc Natl Acad Sci U S A 98: 9110–9115 Shen L (2015) Associations between B vitamins and Parkinson’s disease. Nutrients 7: 7197–7208 Shen Y, Dong Z feng, Pan PL, Xu G, Huang JY & Liu CF (2019) Association of homocysteine, folate, and white matter hyperintensities in Parkinson’s bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

patients with different motor phenotypes. Neurol Sci 40: 1855–1863 Shetty DK, Kalamkar KP & Inamdar MS (2018) Stem Cell Reports Article OCIAD1 Controls Electron Transport Chain Complex I Activity to Regulate Energy Metabolism in Human Pluripotent Stem Cells. Shiba-Fukushima K, Imai Y, Yoshida S, Ishihama Y, Kanao T, Sato S & Hattori N (2012) PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy. Sci Rep 2 Shin MS, Jeong HY, An DI, Lee HY & Sung YH (2016) Treadmill exercise facilitates synaptic plasticity on dopaminergic neurons and fibers in the mouse model with Parkinson’s disease. Neurosci Lett 621: 28–33 Shoffner JM, Watts RL, Juncos JL, Torroni A & Wallace DC (1991) Mitochondrial oxidative phosphorylation defects in parkinson’s disease. Ann Neurol 30: 332–339 Sliter DA, Martinez J, Hao L, Chen X, Sun N, Fischer TD, Burman JL, Li Y, Zhang Z, Narendra DP, et al (2018) Parkin and PINK1 mitigate STING-induced inflammation Storey A (2018) Living longer - how our population is changing and why it matters. Off Natl Stat: 1–53 Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, Simonovic M, Doncheva NT, Morris JH, Bork P, et al (2019) STRING v11: Protein- protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 47: D607–D613 Thomas KJ, McCoy MK, Blackinton J, Beilina A, van der Brug M, Sandebring A, Miller D, Maric D, Cedazo-Minguez A & Cookson MR (2011) DJ-1 acts in parallel to the PINK1/parkin pathway to control mitochondrial function and autophagy. Hum Mol Genet 20: 40–50 Tufi R, Gandhi S, de Castro IP, Lehmann S, Angelova PR, Dinsdale D, Deas E, Plun-Favreau H, Nicotera P, Abramov AY, et al (2014) Enhancing nucleotide metabolism protects against mitochondrial dysfunction and neurodegeneration in a PINK1 model of Parkinson’s disease. Nat Cell Biol 16: 157–166 Xie Y, Feng H, Peng S, Xiao J & Zhang J (2017) Association of plasma homocysteine, vitamin B12 and folate levels with cognitive function in Parkinson’s disease: A meta-analysis. Neurosci Lett 636: 190–195 Yang F, Lagerros YT, Bellocco R, Adami HO, Fang F, Pedersen NL & Wirdefeldt K (2015) Physical activity and risk of Parkinson’s disease in the Swedish National March Cohort. Brain 138: 269–275 Yogev O & Pines O (2011) Dual targeting of mitochondrial proteins: Mechanism, regulation and function. Biochim Biophys Acta - Biomembr 1808: 1012–1020 doi:10.1016/j.bbamem.2010.07.004 [PREPRINT] Zavodszky E, Seaman MNJ, Moreau K, Jimenez-Sanchez M, Breusegem SY, bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Harbour ME & Rubinsztein DC (2014) Mutation in VPS35 associated with Parkinson’s disease impairs WASH complex association and inhibits autophagy. Nat Commun 5 Zhong Z, Liang S, Sanchez-Lopez E, He F, Shalapour S, Lin X jia, Wong J, Ding S, Seki E, Schnabl B, et al (2018) New mitochondrial DNA synthesis enables NLRP3 inflammasome activation. Nature 560: 198–203 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Pink1 manuscript supplemental data

Supplemental table 1 – Label-free MS identified 516 proteins from the mitochondrial fractions of Pink1- and WT D. melanogaster

Identifier Protein name Q94523 Succinate dehydrogenase [ubiquinone] flavoprotein subunit mitochondrial OS=Drosophila melanogaster OX=7227 GN=SdhA PE=2 SV=3 P29844 Endoplasmic reticulum chaperone BiP OS=Drosophila melanogaster OX=7227 GN=Hsc70-3 PE=1 SV=2 Q7JUS9 MIP16332p OS=Drosophila melanogaster OX=7227 GN=Mpcp1 PE=1 SV=1 Q27597 NADPH--cytochrome P450 reductase OS=Drosophila melanogaster OX=7227 GN=Cpr PE=2 SV=2 Q9W3L4 GH20802p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG2233 PE=1 SV=1 Q9VIQ8 Cytochrome c oxidase subunit 4 isoform A OS=Drosophila melanogaster OX=7227 GN=COX4 PE=1 SV=1 Q9VWV6 Transferrin OS=Drosophila melanogaster OX=7227 GN=Tsf1 PE=1 SV=1 Q9VMS1 Cyclope isoform A OS=Drosophila melanogaster OX=7227 GN=cype PE=1 SV=3 A0A0B4KH Chaoptin isoform C OS=Drosophila melanogaster OX=7227 GN=chp PE=1 G5 SV=1 Q9VLJ7 Fatty acyl-CoA reductase OS=Drosophila melanogaster OX=7227 GN=Sgp PE=2 SV=2 O02649 Heat shock protein 60A OS=Drosophila melanogaster OX=7227 GN=Hsp60A PE=1 SV=3 A1Z6V5 FI01422p OS=Drosophila melanogaster OX=7227 GN=Spn43Ab PE=1 SV=1 Q9V4E0 LD47962p OS=Drosophila melanogaster OX=7227 GN=ND-49 PE=1 SV=2 Q9VMI3 NADH dehydrogenase [ubiquinone] flavoprotein 1 mitochondrial OS=Drosophila melanogaster OX=7227 GN=ND-51 PE=1 SV=1 Q9W303 Chitinase-like protein Idgf4 OS=Drosophila melanogaster OX=7227 GN=Idgf4 PE=2 SV=1 Q7JR58 Enoyl-CoA hydratase short chain 1 isoform A OS=Drosophila melanogaster OX=7227 GN=Echs1 PE=1 SV=1 Q3YMU0 Protein disulfide-isomerase (Fragment) OS=Drosophila melanogaster OX=7227 GN=ERp60 PE=1 SV=1 Q9VAY2 Glycoprotein 93 OS=Drosophila melanogaster OX=7227 GN=Gp93 PE=1 SV=1 Q9W402 NADH dehydrogenase (Ubiquinone) B16.6 subunit isoform A OS=Drosophila melanogaster OX=7227 GN=ND-B16.6 PE=1 SV=1 A0A0B4K71 Ryanodine receptor isoform J OS=Drosophila melanogaster OX=7227 5 GN=RyR PE=1 SV=1 Q9VG86 Fatty acyl-CoA reductase OS=Drosophila melanogaster OX=7227 GN=anon- WO0140519.58 PE=1 SV=3 M9PD14 Flightin isoform B OS=Drosophila melanogaster OX=7227 GN=fln PE=4 SV=1 Q9VXI6 Cytochrome b-c1 complex subunit 7 OS=Drosophila melanogaster OX=7227 GN=UQCR-14 PE=1 SV=1 Q9VMN5 60 kDa heat shock protein homolog 2 mitochondrial OS=Drosophila melanogaster OX=7227 GN=Hsp60C PE=2 SV=2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Q7KUB1 Isocitrate dehydrogenase [NADP] OS=Drosophila melanogaster OX=7227 GN=Idh PE=1 SV=1 X2JFD6 Peroxisomal multifunctional enzyme type 2 isoform B OS=Drosophila melanogaster OX=7227 GN=Mfe2 PE=4 SV=1 Q9VN21 LD30155p OS=Drosophila melanogaster OX=7227 GN=lost PE=1 SV=1 Q9W3X6 FI05334p OS=Drosophila melanogaster OX=7227 GN=Fum1 PE=1 SV=3 Q9VQD7 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 OS=Drosophila melanogaster OX=7227 GN=ND-B17.2 PE=1 SV=1 E2QCS7 AB hydrolase-1 domain-containing protein OS=Drosophila melanogaster OX=7227 GN=Dmel\CG17097 PE=1 SV=1 Q9VVU1 FI09602p OS=Drosophila melanogaster OX=7227 GN=GH07925p PE=1 SV=1 Q9VMV9 Reticulon-like protein OS=Drosophila melanogaster OX=7227 GN=Rtnl1 PE=1 SV=2 Q9VG81 RH49330p OS=Drosophila melanogaster OX=7227 GN=Sccpdh2 PE=1 SV=1 Q9VZL3 Sc2 OS=Drosophila melanogaster OX=7227 GN=Sc2 PE=1 SV=1 P00408 Cytochrome c oxidase subunit 2 OS=Drosophila melanogaster OX=7227 GN=mt:CoII PE=3 SV=1 P36188 Troponin I OS=Drosophila melanogaster OX=7227 GN=wupA PE=2 SV=3 X2JJG8 Glutamine synthetase OS=Drosophila melanogaster OX=7227 GN=Gs2 PE=1 SV=1 Q7K511 CG3835-RA OS=Drosophila melanogaster OX=7227 GN=D2hgdh PE=1 SV=1 Q7KRU8 Ferritin OS=Drosophila melanogaster OX=7227 GN=Fer1HCH PE=1 SV=1 Q7KN62 Transitional endoplasmic reticulum ATPase TER94 OS=Drosophila melanogaster OX=7227 GN=TER94 PE=1 SV=1 Q9VN13 Sideroflexin-1-3 OS=Drosophila melanogaster OX=7227 GN=Sfxn1-3 PE=2 SV=1 Q9W1F7 IP15825p OS=Drosophila melanogaster OX=7227 GN=Stoml2 PE=1 SV=2 P06002 Opsin Rh1 OS=Drosophila melanogaster OX=7227 GN=ninaE PE=1 SV=1 Q9W0M4 LP10861p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG13887 PE=1 SV=1 Q9VS34 60S ribosomal protein L18 OS=Drosophila melanogaster OX=7227 GN=RpL18 PE=1 SV=1 Q9VW68 Gamma-aminobutyric acid transaminase isoform A OS=Drosophila melanogaster OX=7227 GN=Gabat PE=1 SV=1 Q9VWI0 NADH dehydrogenase (Ubiquinone) 18 kDa subunit OS=Drosophila melanogaster OX=7227 GN=ND-18 PE=1 SV=2 Q9U1L2 EG:BACR7A4.14 protein OS=Drosophila melanogaster OX=7227 GN=EG:BACR7A4.14 PE=1 SV=1 Q9VQR2 CG8844 protein OS=Drosophila melanogaster OX=7227 GN=ND-PDSW PE=1 SV=1 Q9VLL3 A-kinase anchor protein 200 OS=Drosophila melanogaster OX=7227 GN=Akap200 PE=1 SV=3 Q9V9W3 RE08669p OS=Drosophila melanogaster OX=7227 GN=RpL6 PE=1 SV=1 Q9V4N3 Cytochrome b5 OS=Drosophila melanogaster OX=7227 GN=Cyt-b5 PE=2 SV=1 Q27606 Cytochrome P450 4e2 OS=Drosophila melanogaster OX=7227 GN=Cyp4e2 PE=2 SV=2 Q8IQW2 Cytochrome c oxidase subunit OS=Drosophila melanogaster OX=7227 GN=COX6B PE=1 SV=1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Q9W457 Serine hydroxymethyltransferase OS=Drosophila melanogaster OX=7227 GN=Shmt PE=1 SV=1 Q9VN86 AT14148p OS=Drosophila melanogaster OX=7227 GN=Sccpdh1 PE=1 SV=1 P29845 Heat shock 70 kDa protein cognate 5 OS=Drosophila melanogaster OX=7227 GN=Hsc70-5 PE=1 SV=2 P20432 Glutathione S-transferase D1 OS=Drosophila melanogaster OX=7227 GN=GstD1 PE=1 SV=1 Q9VY04 Fezzik isoform A OS=Drosophila melanogaster OX=7227 GN=fiz PE=3 SV=1 M9NEQ9 Ribosomal protein S10b isoform D OS=Drosophila melanogaster OX=7227 GN=RpS10b PE=1 SV=1 Q9V7Y2 Sphingosine-1-phosphate lyase OS=Drosophila melanogaster OX=7227 GN=Sply PE=1 SV=1 Q9VEY5 MICOS complex subunit OS=Drosophila melanogaster OX=7227 GN=Dmel\CG5903 PE=1 SV=1 A0A0B4LHS Myosin alkali light chain 1 isoform D OS=Drosophila melanogaster OX=7227 1 GN=Mlc1 PE=1 SV=1 E1JJ68 Rm62 isoform H OS=Drosophila melanogaster OX=7227 GN=Rm62 PE=1 SV=1 C0HKA1 40S ribosomal protein S14b OS=Drosophila melanogaster OX=7227 GN=RpS14b PE=2 SV=1 Q9VXM4 LD12946p OS=Drosophila melanogaster OX=7227 GN=MSBP PE=1 SV=1 Q9W5R8 60S ribosomal protein L5 OS=Drosophila melanogaster OX=7227 GN=RpL5 PE=1 SV=2 Q9VG82 Probable cytochrome P450 9f2 OS=Drosophila melanogaster OX=7227 GN=Cyp9f2 PE=2 SV=1 P29327 40S ribosomal protein S6 OS=Drosophila melanogaster OX=7227 GN=RpS6 PE=1 SV=1 Q6IHY5 HDC00331 OS=Drosophila melanogaster OX=7227 GN=Dmel\CG34172 PE=1 SV=1 Q9VRJ4 SD02021p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG10672 PE=1 SV=1 Q24046 Sodium/potassium-transporting ATPase subunit beta-1 OS=Drosophila melanogaster OX=7227 GN=nrv1 PE=1 SV=2 M9PCC1 Receptor of activated protein kinase C 1 isoform C OS=Drosophila melanogaster OX=7227 GN=Rack1 PE=4 SV=1 Q7YTZ4 GH22187p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG10737 PE=1 SV=1 Q9V419 Probable cytochrome P450 28a5 OS=Drosophila melanogaster OX=7227 GN=Cyp28a5 PE=2 SV=1 Q9V438 Protein disulfide-isomerase A6 homolog OS=Drosophila melanogaster OX=7227 GN=CaBP1 PE=1 SV=1 Q7KW39 Probable methylmalonate-semialdehyde dehydrogenase [acylating] mitochondrial OS=Drosophila melanogaster OX=7227 GN=CG17896 PE=2 SV=1 Q9VQM2 NADH dehydrogenase [ubiquinone] 1 subunit C2 OS=Drosophila melanogaster OX=7227 GN=ND-B14.5B PE=1 SV=1 Q9VC18 RE29555p OS=Drosophila melanogaster OX=7227 GN=BEST:GH11240 PE=1 SV=1 Q9VY05 GH11762p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG9512 PE=1 SV=1 P21187 Polyadenylate-binding protein OS=Drosophila melanogaster OX=7227 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

GN=pAbp PE=1 SV=3 Q9V4I1 Cytochrome P450 9b2 OS=Drosophila melanogaster OX=7227 GN=Cyp9b2 PE=2 SV=1 A0A0B4LFR Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 4 OS=Drosophila melanogaster OX=7227 GN=OstDelta PE=1 SV=1 Q9VG87 Fatty acyl-CoA reductase OS=Drosophila melanogaster OX=7227 GN=CG32919 PE=2 SV=2 Q95SI7 GH23390p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG6028 PE=1 SV=1 Q9W4N8 LD30122p OS=Drosophila melanogaster OX=7227 GN=Vap33 PE=1 SV=1 Q9VCR9 RH48101p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG17121 PE=1 SV=1 Q8T3U2 40S ribosomal protein S23 OS=Drosophila melanogaster OX=7227 GN=RpS23 PE=1 SV=1 Q86BQ3 Uncharacterized protein isoform A OS=Drosophila melanogaster OX=7227 GN=Dmel\CG13284 PE=3 SV=1 Q9VZ49 Poly(U)-specific endoribonuclease homolog OS=Drosophila melanogaster OX=7227 GN=CG2145 PE=2 SV=1 A0A0B4JD9 Zipper isoform H OS=Drosophila melanogaster OX=7227 GN=zip PE=1 5 SV=1 Q9VE00 Probable cytochrome P450 12a4 mitochondrial OS=Drosophila melanogaster OX=7227 GN=Cyp12a4 PE=2 SV=2 E1JHQ1 Glutamine synthetase OS=Drosophila melanogaster OX=7227 GN=Gs1 PE=1 SV=1 A0A0B4KHJ Glycogen [starch] synthase OS=Drosophila melanogaster OX=7227 5 GN=GlyS PE=1 SV=1 Q9VU35 RH34413p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG11267 PE=1 SV=1 Q7KTC7 Multidrug-Resistance like protein 1 isoform C OS=Drosophila melanogaster OX=7227 GN=MRP PE=1 SV=1 A0A0B4LGZ Ribosomal protein L11 isoform B OS=Drosophila melanogaster OX=7227 5 GN=RpL11 PE=1 SV=1 Q7KVB1 ATP binding cassette subfamily B member 7 isoform B OS=Drosophila melanogaster OX=7227 GN=ABCB7 PE=1 SV=1 Q9VCC6 GM05240p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG6178 PE=1 SV=1 Q9V3W0 RutC family protein UK114 OS=Drosophila melanogaster OX=7227 GN=UK114 PE=2 SV=1 Q8SXQ1 Aldehyde dehydrogenase 7 family member A1 OS=Drosophila melanogaster OX=7227 GN=Aldh7A1 PE=1 SV=1 Q9VZS5 60S ribosomal protein L28 OS=Drosophila melanogaster OX=7227 GN=RpL28 PE=1 SV=1 M9NFR5 Ypsilon schachtel isoform B OS=Drosophila melanogaster OX=7227 GN=yps PE=1 SV=1 Q9Y114 BcDNA.GH10229 OS=Drosophila melanogaster OX=7227 GN=anon- WO0172774.24 PE=1 SV=1 Q8MSU3 Putative ferric-chelate reductase 1 homolog OS=Drosophila melanogaster OX=7227 GN=CG8399 PE=2 SV=1 Q9W1G0 Probable transaldolase OS=Drosophila melanogaster OX=7227 GN=Taldo PE=2 SV=2 Q7K3N4 GH26015p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG8888 PE=1 SV=1 A0A140SRF Uncharacterized protein isoform C OS=Drosophila melanogaster OX=7227 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

8 GN=Dmel\CG11857 PE=1 SV=1 E1JJM9 GEO07866p1 OS=Drosophila melanogaster OX=7227 GN=RpS15Aa PE=1 SV=1 Q7JYW9 Phosphotransferase OS=Drosophila melanogaster OX=7227 GN=Hex-C PE=1 SV=1 O61491 Flotillin-1 OS=Drosophila melanogaster OX=7227 GN=Flo1 PE=2 SV=1 E1JJF9 Neuroglian isoform D OS=Drosophila melanogaster OX=7227 GN=Nrg PE=1 SV=1 P02515 Heat shock protein 22 OS=Drosophila melanogaster OX=7227 GN=Hsp22 PE=1 SV=4 Q7JXC4 CG6459 protein OS=Drosophila melanogaster OX=7227 GN=P32 PE=1 SV=1 Q7K485 CathD isoform A OS=Drosophila melanogaster OX=7227 GN=cathD PE=1 SV=1 X2JC55 Phosphatidate cytidylyltransferase OS=Drosophila melanogaster OX=7227 GN=Cds PE=1 SV=1 Q9W4P5 V-type proton ATPase subunit d 1 OS=Drosophila melanogaster OX=7227 GN=VhaAC39-1 PE=2 SV=1 P50882 60S ribosomal protein L9 OS=Drosophila melanogaster OX=7227 GN=RpL9 PE=1 SV=2 A4V449 NADH dehydrogenase (Ubiquinone) 75 kDa subunit isoform B OS=Drosophila melanogaster OX=7227 GN=ND-75 PE=1 SV=1 Q9VHW0 AT07710p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG7910 PE=2 SV=1 Q9VA83 Ferritin OS=Drosophila melanogaster OX=7227 GN=Fer2LCH PE=1 SV=1 Q9VLB7 Rab GDP dissociation inhibitor OS=Drosophila melanogaster OX=7227 GN=Gdi PE=1 SV=1 Q8IMI7 40S ribosomal protein S7 OS=Drosophila melanogaster OX=7227 GN=RpS7 PE=1 SV=2 C7LA75 Heat shock protein cognate 4 isoform G OS=Drosophila melanogaster OX=7227 GN=Hsc70-4 PE=1 SV=1 Q9VL70 HL08109p OS=Drosophila melanogaster OX=7227 GN=yip2 PE=1 SV=1 O16797 60S ribosomal protein L3 OS=Drosophila melanogaster OX=7227 GN=RpL3 PE=1 SV=3 Q9W3N9 LD24105p OS=Drosophila melanogaster OX=7227 GN=154229_at PE=1 SV=1 A0A0B4K7L ATP-dependent 6-phosphofructokinase OS=Drosophila melanogaster 1 OX=7227 GN=Pfk PE=1 SV=1 P19107 Phosrestin-1 OS=Drosophila melanogaster OX=7227 GN=Arr2 PE=1 SV=2 Q7PLB8 Fatty acid synthase 3 OS=Drosophila melanogaster OX=7227 GN=FASN3 PE=1 SV=3 Q9VKT1 GH11711p OS=Drosophila melanogaster OX=7227 GN=CG17093 PE=1 SV=2 Q9VFP1 Probable cytochrome P450 6d5 OS=Drosophila melanogaster OX=7227 GN=Cyp6d5 PE=2 SV=1 Q960W6 Putative fatty acyl-CoA reductase CG8306 OS=Drosophila melanogaster OX=7227 GN=CG8306 PE=2 SV=1 Q9VGF7 Glutamate carrier 1 isoform A OS=Drosophila melanogaster OX=7227 GN=GC1 PE=1 SV=1 Q9VQL6 Fatty acid synthase 2 isoform A OS=Drosophila melanogaster OX=7227 GN=FASN2 PE=1 SV=1 Q24319 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

subunit OS=Drosophila melanogaster OX=7227 GN=Ost48 PE=2 SV=2 Q8SY19 Microsomal glutathione S-transferase-like isoform A OS=Drosophila melanogaster OX=7227 GN=Mgstl PE=1 SV=1 Q7KV27 Uncharacterized protein isoform A OS=Drosophila melanogaster OX=7227 GN=ALT PE=1 SV=1 Q9VVW3 Sideroflexin-2 OS=Drosophila melanogaster OX=7227 GN=Sfxn2 PE=2 SV=2 A0A0B4KH3 Annexin OS=Drosophila melanogaster OX=7227 GN=AnxB9 PE=1 SV=1 4 Q9W125 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 OS=Drosophila melanogaster OX=7227 GN=ND-19 PE=1 SV=1 Q9VSA3 Probable medium-chain specific acyl-CoA dehydrogenase mitochondrial OS=Drosophila melanogaster OX=7227 GN=CG12262 PE=2 SV=1 P20228 Glutamate decarboxylase OS=Drosophila melanogaster OX=7227 GN=Gad1 PE=2 SV=2 Q9VZF6 GH04863p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG14997 PE=1 SV=1 Q9VES7 Fatty acyl-CoA reductase OS=Drosophila melanogaster OX=7227 GN=Dmel\CG17562 PE=2 SV=2 Q9VMB9 Cytochrome c oxidase subunit 5B isoform A OS=Drosophila melanogaster OX=7227 GN=COX5B PE=1 SV=1 Q9V771 Probable cytochrome P450 6a23 OS=Drosophila melanogaster OX=7227 GN=Cyp6a23 PE=2 SV=2 H8F4T6 FI19911p1 OS=Drosophila melanogaster OX=7227 GN=CG16935-RA PE=1 SV=1 Q23997 Imaginal disk growth factor 6 OS=Drosophila melanogaster OX=7227 GN=Idgf6 PE=1 SV=2 Q9VAL7 Calnexin 99A isoform A OS=Drosophila melanogaster OX=7227 GN=Cnx99A PE=1 SV=2 O96553 C-1-tetrahydrofolate synthase cytoplasmic OS=Drosophila melanogaster OX=7227 GN=pug PE=2 SV=4 Q9VXI1 Beta hydroxy acid dehydrogenase 1 isoform A OS=Drosophila melanogaster OX=7227 GN=Had1 PE=1 SV=2 Q9VV75 AT02348p OS=Drosophila melanogaster OX=7227 GN=UQCR-C2 PE=1 SV=1 O46199 Accessory gland protein Acp53Ea OS=Drosophila melanogaster OX=7227 GN=Acp53Ea PE=2 SV=1 P07486 Glyceraldehyde-3-phosphate dehydrogenase 1 OS=Drosophila melanogaster OX=7227 GN=Gapdh1 PE=2 SV=2 Q9W3X7 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 mitochondrial OS=Drosophila melanogaster OX=7227 GN=ND-ASHI PE=1 SV=1 Q9W227 Peptidyl-prolyl cis-trans isomerase OS=Drosophila melanogaster OX=7227 GN=Dmel\CG2852 PE=1 SV=1 Q7K2P3 GH20817p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG1648 PE=1 SV=1 Q9VKU1 Fatty acid transport protein 1 isoform A OS=Drosophila melanogaster OX=7227 GN=Fatp1 PE=1 SV=2 Q0E8X7 Reduction of Rh1 isoform A OS=Drosophila melanogaster OX=7227 GN=roh PE=1 SV=1 Q9VFF0 GH01077p OS=Drosophila melanogaster OX=7227 GN=UQCR-C1 PE=1 SV=2 Q7KB18 Epoxide hydrolase OS=Drosophila melanogaster OX=7227 GN=Jheh2 PE=1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

SV=1 Q7JV23 Acetyl-CoA carboxylase isoform B OS=Drosophila melanogaster OX=7227 GN=ACC PE=1 SV=1 Q9VVH5 AT13736p OS=Drosophila melanogaster OX=7227 GN=UQCR-Q PE=1 SV=1 Q9V8M5 Probable 3-hydroxyisobutyrate dehydrogenase mitochondrial OS=Drosophila melanogaster OX=7227 GN=CG15093 PE=2 SV=2 Q9I7S8 Multifunctional protein ADE2 OS=Drosophila melanogaster OX=7227 GN=ade5 PE=2 SV=2 P17336 Catalase OS=Drosophila melanogaster OX=7227 GN=Cat PE=1 SV=2 Q9VU17 HL01062p OS=Drosophila melanogaster OX=7227 GN=CT30701 PE=1 SV=1 A0A0B4KHJ Tropomyosin 2 isoform E OS=Drosophila melanogaster OX=7227 GN=Tm2 9 PE=1 SV=1 Q0E8E8 MIP08013p1 OS=Drosophila melanogaster OX=7227 GN=Mpcp2 PE=1 SV=1 Q9VJ28 FI05204p OS=Drosophila melanogaster OX=7227 GN=L2HGDH PE=1 SV=1 X2JGP4 Protein disulfide-isomerase OS=Drosophila melanogaster OX=7227 GN=Pdi PE=3 SV=1 M9PH10 Comatose isoform B OS=Drosophila melanogaster OX=7227 GN=comt PE=1 SV=1 P48602 V-type proton ATPase catalytic subunit A isoform 1 OS=Drosophila melanogaster OX=7227 GN=Vha68-1 PE=2 SV=2 Q8STG9 DSec61alpha OS=Drosophila melanogaster OX=7227 GN=Sec61alpha PE=1 SV=1 Q7KTJ7 Basigin isoform G OS=Drosophila melanogaster OX=7227 GN=Bsg PE=1 SV=1 P50887 60S ribosomal protein L22 OS=Drosophila melanogaster OX=7227 GN=RpL22 PE=1 SV=2 A0A0B4LGS Vacuolar H[+]-ATPase 26kD subunit isoform C OS=Drosophila 4 melanogaster OX=7227 GN=Vha26 PE=3 SV=1 P07190 Maltase A1 OS=Drosophila melanogaster OX=7227 GN=Mal-A1 PE=2 SV=2 Q9VGQ4 GH25284p OS=Drosophila melanogaster OX=7227 GN=scpr-B PE=2 SV=2 Q8MKK5 GH09271p OS=Drosophila melanogaster OX=7227 GN=kcc PE=1 SV=1 Q9VKM3 ATP synthase subunit OS=Drosophila melanogaster OX=7227 GN=ATPsynG PE=1 SV=1 Q0E9B6 40S ribosomal protein S11 OS=Drosophila melanogaster OX=7227 GN=RpS11 PE=1 SV=1 Q9VE75 V-type proton ATPase subunit a OS=Drosophila melanogaster OX=7227 GN=Vha100-2 PE=1 SV=2 D4G7B1 Imaginal disc growth factor 2 isoform B OS=Drosophila melanogaster OX=7227 GN=Idgf2 PE=1 SV=1 P91929 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10 mitochondrial OS=Drosophila melanogaster OX=7227 GN=ND-42 PE=2 SV=2 P46223 60S ribosomal protein L7a OS=Drosophila melanogaster OX=7227 GN=RpL7A PE=1 SV=2 Q7K188 Protein quiver OS=Drosophila melanogaster OX=7227 GN=Dmel\CG6329 PE=2 SV=1 A0A0B4KFE Acyl-CoA synthetase long-chain isoform J OS=Drosophila melanogaster 4 OX=7227 GN=Acsl PE=1 SV=1 Q9V3A8 Ergic53 isoform A OS=Drosophila melanogaster OX=7227 GN=ergic53 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

PE=1 SV=1 Q9V3P0 Peroxiredoxin 1 OS=Drosophila melanogaster OX=7227 GN=Jafrac1 PE=1 SV=1 E1JJA4 Shibire isoform L OS=Drosophila melanogaster OX=7227 GN=shi PE=1 SV=1 Q94514 Cytochrome c oxidase subunit 5A mitochondrial OS=Drosophila melanogaster OX=7227 GN=COX5A PE=2 SV=2 C9QP21 Carboxylic ester hydrolase OS=Drosophila melanogaster OX=7227 GN=Est- 6 PE=1 SV=1 A8DYI6 Prohibitin 2 isoform E OS=Drosophila melanogaster OX=7227 GN=Phb2 PE=1 SV=1 P21914 Succinate dehydrogenase [ubiquinone] iron-sulfur subunit mitochondrial OS=Drosophila melanogaster OX=7227 GN=SdhB PE=2 SV=2 A8Y535 Cytochrome b-c1 complex subunit 6 OS=Drosophila melanogaster OX=7227 GN=UQCR-11 PE=1 SV=2 Q9VQ61 Aspartate aminotransferase OS=Drosophila melanogaster OX=7227 GN=Got2 PE=1 SV=1 P16378 G protein alpha o subunit OS=Drosophila melanogaster OX=7227 GN=Galphao PE=1 SV=1 Q9VNX4 Multifunctional fusion protein OS=Drosophila melanogaster OX=7227 GN=P5CDh1 PE=1 SV=1 A1Z8N1 Facilitated trehalose transporter Tret1-1 OS=Drosophila melanogaster OX=7227 GN=Tret1-1 PE=1 SV=1 Q7KLX3 GEO05407p1 OS=Drosophila melanogaster OX=7227 GN=Tapdelta PE=1 SV=1 Q9VAJ9 SD09259p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG1907 PE=1 SV=1 Q9W3F3 RH03540p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG15347 PE=1 SV=2 Q76NQ0 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 OS=Drosophila melanogaster OX=7227 GN=CG5364 PE=1 SV=1 O76742 CG5915 protein OS=Drosophila melanogaster OX=7227 GN=Rab7 PE=1 SV=1 B7YZQ3 Prominin isoform D OS=Drosophila melanogaster OX=7227 GN=prom PE=1 SV=1 E2QD65 Ribosomal protein S19a isoform C OS=Drosophila melanogaster OX=7227 GN=RpS19a PE=1 SV=1 B7Z0L1 Fasciclin 1 isoform E OS=Drosophila melanogaster OX=7227 GN=Fas1 PE=1 SV=1 Q9Y112 BcDNA.GH10614 OS=Drosophila melanogaster OX=7227 GN=dm1 PE=1 SV=1 Q500Y7 GEO11443p1 OS=Drosophila melanogaster OX=7227 GN=UQCR-6.4 PE=2 SV=1 P41073 Zinc finger protein on ecdysone puffs OS=Drosophila melanogaster OX=7227 GN=Pep PE=1 SV=1 E1JI91 UTP--glucose-1-phosphate uridylyltransferase OS=Drosophila melanogaster OX=7227 GN=UGP PE=1 SV=1 P09180 60S ribosomal protein L4 OS=Drosophila melanogaster OX=7227 GN=RpL4 PE=1 SV=2 Q7KSQ0 LD46175p OS=Drosophila melanogaster OX=7227 GN=sea PE=1 SV=1 Q9W2H8 Uncharacterized protein isoform B OS=Drosophila melanogaster OX=7227 GN=BEST:GH22856 PE=1 SV=3 C0PDF4 MIP06482p OS=Drosophila melanogaster OX=7227 GN=CG18815-RA PE=1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

SV=1 Q9VIB5 Carboxylic ester hydrolase OS=Drosophila melanogaster OX=7227 GN=alpha-Est7 PE=1 SV=1 Q9VPR1 GH02075p OS=Drosophila melanogaster OX=7227 GN=Tspo PE=1 SV=1 P38979 40S ribosomal protein SA OS=Drosophila melanogaster OX=7227 GN=sta PE=1 SV=3 C0HK92 Uncharacterized protein CG45076 OS=Drosophila melanogaster OX=7227 GN=CG45076 PE=1 SV=1 E2QCF1 ATP-citrate synthase OS=Drosophila melanogaster OX=7227 GN=ATPCL PE=1 SV=1 Q9W141 Putative ATP synthase subunit f mitochondrial OS=Drosophila melanogaster OX=7227 GN=CG4692 PE=3 SV=1 Q9V3S9 Very long-chain-fatty-acid--CoA ligase bubblegum OS=Drosophila melanogaster OX=7227 GN=bgm PE=1 SV=1 M9PGL7 Translocase of outer membrane 40 isoform C OS=Drosophila melanogaster OX=7227 GN=Tom40 PE=4 SV=1 A0A0B4LFL Eukaryotic translation initiation factor 3 subunit C OS=Drosophila 2 melanogaster OX=7227 GN=eIF3c PE=3 SV=1 A0A0B4KEL Aldehyde dehydrogenase OS=Drosophila melanogaster OX=7227 GN=Aldh- 0 III PE=1 SV=1 M9PJP0 Nipsnap isoform F OS=Drosophila melanogaster OX=7227 GN=Nipsnap PE=4 SV=2 M9PDK5 V-type proton ATPase subunit H OS=Drosophila melanogaster OX=7227 GN=VhaSFD PE=3 SV=1 M9PG76 60S acidic ribosomal protein P0 OS=Drosophila melanogaster OX=7227 GN=RpLP0 PE=3 SV=1 P08736 Elongation factor 1-alpha 1 OS=Drosophila melanogaster OX=7227 GN=eEF1alpha1 PE=1 SV=2 Q9VD29 RE74312p OS=Drosophila melanogaster OX=7227 GN=Sar1 PE=1 SV=1 Q9W1B9 GEO07602p1 OS=Drosophila melanogaster OX=7227 GN=RpL12 PE=1 SV=1 A0A0B4LG5 GEO08239p1 OS=Drosophila melanogaster OX=7227 GN=RpS16 PE=2 2 SV=1 C8VV14 Fructose-bisphosphate aldolase OS=Drosophila melanogaster OX=7227 GN=Ald1 PE=1 SV=1 Q0KHZ6 Uncharacterized protein isoform A OS=Drosophila melanogaster OX=7227 GN=ETFB PE=1 SV=1 Q8IN25 Aminopeptidase OS=Drosophila melanogaster OX=7227 GN=Dmel\CG31198 PE=1 SV=1 P55841 60S ribosomal protein L14 OS=Drosophila melanogaster OX=7227 GN=RpL14 PE=1 SV=1 Q06559 40S ribosomal protein S3 OS=Drosophila melanogaster OX=7227 GN=RpS3 PE=1 SV=1 M9MSJ3 Uncharacterized protein isoform B OS=Drosophila melanogaster OX=7227 GN=HDC03337 PE=4 SV=1 Q7K569 Glycerol-3-phosphate dehydrogenase OS=Drosophila melanogaster OX=7227 GN=Gpo1 PE=1 SV=1 Q9W2E8 NADH dehydrogenase (Ubiquinone) B12 subunit isoform A OS=Drosophila melanogaster OX=7227 GN=ND-B12 PE=2 SV=1 P13008 40S ribosomal protein S26 OS=Drosophila melanogaster OX=7227 GN=RpS26 PE=1 SV=1 Q9VA18 Coiled-coil-helix-coiled-coil-helix domain containing 3 OS=Drosophila melanogaster OX=7227 GN=Chchd3 PE=1 SV=1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Q9W095 FI03690p OS=Drosophila melanogaster OX=7227 GN=Gk2 PE=2 SV=2 Q7JYH3 NADH dehydrogenase (Ubiquinone) B14.7 subunit OS=Drosophila melanogaster OX=7227 GN=ND-B14.7 PE=1 SV=1 Q9W1H8 GH13256p OS=Drosophila melanogaster OX=7227 GN=Mtpbeta PE=1 SV=1 Q9VGT8 UDP-glucuronosyltransferase OS=Drosophila melanogaster OX=7227 GN=Ugt35C1 PE=3 SV=2 Q9VTU2 NADH dehydrogenase (Ubiquinone) SGDH subunit isoform A OS=Drosophila melanogaster OX=7227 GN=ND-SGDH PE=1 SV=1 Q9VM14 AT21758p OS=Drosophila melanogaster OX=7227 GN=muc PE=1 SV=1 A0A0C4DH B52 isoform O OS=Drosophila melanogaster OX=7227 GN=B52 PE=4 G5 SV=1 P35381 ATP synthase subunit alpha mitochondrial OS=Drosophila melanogaster OX=7227 GN=blw PE=1 SV=2 P35415 Paramyosin long form OS=Drosophila melanogaster OX=7227 GN=Prm PE=1 SV=1 P29613 Triosephosphate isomerase OS=Drosophila melanogaster OX=7227 GN=Tpi PE=1 SV=3 M9PBL3 Heat shock protein 83 isoform B OS=Drosophila melanogaster OX=7227 GN=Hsp83 PE=3 SV=1 Q6GUR9 ACP53C14B OS=Drosophila melanogaster OX=7227 GN=Acp53C14b PE=1 SV=1 P55830 40S ribosomal protein S3a OS=Drosophila melanogaster OX=7227 GN=RpS3A PE=1 SV=4 Q9VEX6 ATPase family AAA domain-containing protein 3A homolog OS=Drosophila melanogaster OX=7227 GN=bor PE=1 SV=2 Q9VHS2 Cytochrome c oxidase subunit 7A mitochondrial OS=Drosophila melanogaster OX=7227 GN=COX7A PE=3 SV=1 Q9VMT5 Probable cytochrome P450 28d1 OS=Drosophila melanogaster OX=7227 GN=Cyp28d1 PE=2 SV=1 X2J8Y6 Accessory gland protein 36DE isoform B OS=Drosophila melanogaster OX=7227 GN=Acp36DE PE=4 SV=1 Q9VXK7 LD31474p OS=Drosophila melanogaster OX=7227 GN=ND-20 PE=2 SV=1 L0MPS3 Apolipophorin isoform B OS=Drosophila melanogaster OX=7227 GN=apolpp PE=1 SV=1 Q9VQ29 Cytochrome b-c1 complex subunit Rieske mitochondrial OS=Drosophila melanogaster OX=7227 GN=RFeSP PE=1 SV=3 B7YZI0 Vacuolar H[+] ATPase 44kD subunit isoform F OS=Drosophila melanogaster OX=7227 GN=Vha44 PE=1 SV=1 Q9VTB4 NADH dehydrogenase (Ubiquinone) 13 kDa B subunit OS=Drosophila melanogaster OX=7227 GN=ND-13B PE=1 SV=2 P24156 Protein l(2)37Cc OS=Drosophila melanogaster OX=7227 GN=l(2)37Cc PE=2 SV=2 Q9VBP6 Succinate-semialdehyde dehydrogenase OS=Drosophila melanogaster OX=7227 GN=Ssadh PE=1 SV=1 Q9VKI8 GH03305p OS=Drosophila melanogaster OX=7227 GN=GH26 PE=1 SV=1 Q9VII1 FI02856p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG9336 PE=1 SV=1 Q9VZU4 LD25561p OS=Drosophila melanogaster OX=7227 GN=ND-30 PE=1 SV=1 Q9Y166 BcDNA.GH02431 OS=Drosophila melanogaster OX=7227 GN=Dic1 PE=1 SV=1 O18404 3-hydroxyacyl-CoA dehydrogenase type-2 OS=Drosophila melanogaster bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

OX=7227 GN=scu PE=1 SV=1 B6IDY5 CYP6G1 OS=Drosophila melanogaster OX=7227 GN=Cyp6g1 PE=2 SV=1 Q8I0D4 RE20510p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG9297 PE=1 SV=1 Q7JQH9 LD31742p OS=Drosophila melanogaster OX=7227 GN=whd PE=1 SV=1 Q24186 40S ribosomal protein S5a OS=Drosophila melanogaster OX=7227 GN=RpS5a PE=1 SV=1 Q24251 ATP synthase subunit d mitochondrial OS=Drosophila melanogaster OX=7227 GN=ATPsynD PE=2 SV=2 O77460 Inorganic pyrophosphatase OS=Drosophila melanogaster OX=7227 GN=Nurf-38 PE=1 SV=3 Q9W3K9 FI02989p OS=Drosophila melanogaster OX=7227 GN=Ldsdh1 PE=1 SV=1 A0A0B4K7C Aspartyl beta-hydroxylase isoform H OS=Drosophila melanogaster 1 OX=7227 GN=Asph PE=1 SV=1 P10676 Neither inactivation nor afterpotential protein C OS=Drosophila melanogaster OX=7227 GN=ninaC PE=1 SV=2 Q9VM18 Trehalose 6-phosphate phosphatase OS=Drosophila melanogaster OX=7227 GN=21430192 PE=1 SV=1 P52029 Glucose-6-phosphate isomerase OS=Drosophila melanogaster OX=7227 GN=Pgi PE=2 SV=2 A4V4F2 Flotillin 2 isoform F OS=Drosophila melanogaster OX=7227 GN=Flo2 PE=1 SV=1 Q7K8X7 Glutathione S transferase E9 OS=Drosophila melanogaster OX=7227 GN=GstE9 PE=1 SV=1 Q9VJZ4 AT12494p OS=Drosophila melanogaster OX=7227 GN=ND-B22 PE=1 SV=1 C6SUW3 LD13662p OS=Drosophila melanogaster OX=7227 GN=RpS9 PE=1 SV=1 Q9VVH3 MICOS complex subunit MIC13 homolog QIL1 OS=Drosophila melanogaster OX=7227 GN=QIL1 PE=2 SV=1 Q0E9N2 Lethal (2) 01289 isoform K OS=Drosophila melanogaster OX=7227 GN=l(2)01289 PE=1 SV=3 Q2QBM1 Malic enzyme OS=Drosophila melanogaster OX=7227 GN=Men PE=1 SV=1 Q9W2J4 UDP-glucuronosyltransferase OS=Drosophila melanogaster OX=7227 GN=Ugt49B1 PE=1 SV=2 Q7KE33 Odorant binding protein c OS=Drosophila melanogaster OX=7227 GN=Obp51a PE=2 SV=1 Q9W2M4 Uncharacterized protein OS=Drosophila melanogaster OX=7227 GN=Dmel\CG10527 PE=1 SV=1 P54385 Glutamate dehydrogenase mitochondrial OS=Drosophila melanogaster OX=7227 GN=Gdh PE=1 SV=2 Q9VUC1 Hsc70Cb isoform A OS=Drosophila melanogaster OX=7227 GN=Hsc70Cb PE=1 SV=1 A0A0B4KEX Uncharacterized protein OS=Drosophila melanogaster OX=7227 7 GN=Dmel\CG43788 PE=4 SV=1 A0A0B4K6N 40S ribosomal protein S8 OS=Drosophila melanogaster OX=7227 GN=RpS8 1 PE=3 SV=1 Q9VRL0 Cytochrome c1 isoform A OS=Drosophila melanogaster OX=7227 GN=Cyt- c1 PE=1 SV=1 Q9VX36 NADH dehydrogenase (Ubiquinone) 24 kDa subunit isoform A OS=Drosophila melanogaster OX=7227 GN=ND-24 PE=1 SV=1 Q9VB10 GH01709p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG5590 PE=1 SV=1 Q9VXG9 GH25683p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG4239 PE=2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

SV=1 M9PI17 Uncharacterized protein isoform D OS=Drosophila melanogaster OX=7227 GN=Dmel\CG6084 PE=1 SV=1 Q7KN94 Walrus isoform A OS=Drosophila melanogaster OX=7227 GN=wal PE=1 SV=1 X2JGM9 40S ribosomal protein S4 OS=Drosophila melanogaster OX=7227 GN=RpS4 PE=3 SV=1 Q7JS69 FI04632p OS=Drosophila melanogaster OX=7227 GN=nrv3 PE=1 SV=1 P02574 Actin larval muscle OS=Drosophila melanogaster OX=7227 GN=Act79B PE=1 SV=2 Q9W1X9 OCIA domain-containing protein 1 OS=Drosophila melanogaster OX=7227 GN=asrij PE=1 SV=1 P29829 Guanine nucleotide-binding protein subunit beta-2 OS=Drosophila melanogaster OX=7227 GN=Gbeta76C PE=1 SV=3 P31009 40S ribosomal protein S2 OS=Drosophila melanogaster OX=7227 GN=RpS2 PE=1 SV=2 Q9XY35 Cytochrome b-c1 complex subunit 9 OS=Drosophila melanogaster OX=7227 GN=ox PE=3 SV=1 Q9VAC1 GM14349p OS=Drosophila melanogaster OX=7227 GN=anon- WO0153538.36 PE=1 SV=1 O18335 Drab11 OS=Drosophila melanogaster OX=7227 GN=Rab11 PE=1 SV=1 Q9VGS3 RH44771p OS=Drosophila melanogaster OX=7227 GN=SdhC PE=1 SV=2 P83967 Actin indirect flight muscle OS=Drosophila melanogaster OX=7227 GN=Act88F PE=1 SV=1 Q9VB69 Malic enzyme OS=Drosophila melanogaster OX=7227 GN=Men-b PE=1 SV=1 Q24439 ATP synthase subunit O mitochondrial OS=Drosophila melanogaster OX=7227 GN=ATPsynO PE=2 SV=2 P26308 Guanine nucleotide-binding protein subunit beta-1 OS=Drosophila melanogaster OX=7227 GN=Gbeta13F PE=1 SV=1 A4UZZ4 Alpha-1 4 glucan phosphorylase OS=Drosophila melanogaster OX=7227 GN=GlyP PE=1 SV=1 Q9VKX2 Malate dehydrogenase OS=Drosophila melanogaster OX=7227 GN=Mdh1 PE=1 SV=2 Q7K3D4 Peptidylprolyl isomerase OS=Drosophila melanogaster OX=7227 GN=zda PE=1 SV=1 Q9Y119 BcDNA.GH08860 OS=Drosophila melanogaster OX=7227 GN=Tps1 PE=1 SV=1 P41093 60S ribosomal protein L18a OS=Drosophila melanogaster OX=7227 GN=RpL18A PE=1 SV=1 Q9V397 BcDNA.GH12558 OS=Drosophila melanogaster OX=7227 GN=Mtpalpha PE=1 SV=1 Q9VZW7 Carnitine palmitoyltransferase 2 OS=Drosophila melanogaster OX=7227 GN=CPT2 PE=1 SV=1 Q8IRD3 Glutathione peroxidase OS=Drosophila melanogaster OX=7227 GN=PHGPx PE=1 SV=1 A0A0B4KHD Mitochondrial pyruvate carrier OS=Drosophila melanogaster OX=7227 3 GN=Mpc1 PE=2 SV=1 Q9VPE2 NADH dehydrogenase (Ubiquinone) 39 kDa subunit isoform A OS=Drosophila melanogaster OX=7227 GN=ND-39 PE=1 SV=1 Q09332 UDP-glucose:glycoprotein glucosyltransferase OS=Drosophila melanogaster OX=7227 GN=Ugt PE=1 SV=2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Q9VJ19 RE25263p OS=Drosophila melanogaster OX=7227 GN=RpL30 PE=1 SV=1 P92177 14-3-3 protein epsilon OS=Drosophila melanogaster OX=7227 GN=14-3- 3epsilon PE=1 SV=2 P13060 Elongation factor 2 OS=Drosophila melanogaster OX=7227 GN=EF2 PE=1 SV=4 Q94516 ATP synthase subunit b mitochondrial OS=Drosophila melanogaster OX=7227 GN=ATPsynB PE=2 SV=2 Q9VUZ0 Translocon-associated protein subunit beta OS=Drosophila melanogaster OX=7227 GN=SsRbeta PE=1 SV=2 Q6GUT7 ACP53C14A OS=Drosophila melanogaster OX=7227 GN=Acp53C14a PE=2 SV=1 A1Z9E3 Elongation factor Tu OS=Drosophila melanogaster OX=7227 GN=mEFTu1 PE=1 SV=1 Q8T4G5 SD01613p OS=Drosophila melanogaster OX=7227 GN=AFG3L2 PE=1 SV=1 Q02645 Protein hu-li tai shao OS=Drosophila melanogaster OX=7227 GN=hts PE=1 SV=2 X2JAJ8 Serpin 38F isoform B OS=Drosophila melanogaster OX=7227 GN=Spn38F PE=3 SV=1 Q9VBR6 RE74917p OS=Drosophila melanogaster OX=7227 GN=tobi PE=2 SV=1 P41572 6-phosphogluconate dehydrogenase decarboxylating OS=Drosophila melanogaster OX=7227 GN=Pgd PE=2 SV=1 P07251 ATP synthase subunit alpha mitochondrial OS=Saccharomyces cerevisiae (strain ATCC 204508 / S288c) OX=559292 GN=ATP1 PE=1 SV=5 Q7JZK1 NADH dehydrogenase (Ubiquinone) B14 subunit isoform A OS=Drosophila melanogaster OX=7227 GN=ND-B14 PE=1 SV=1 A1Z7S3 Rab32 isoform B OS=Drosophila melanogaster OX=7227 GN=Rab32 PE=1 SV=1 Q9V3S0 Cytochrome P450 4g1 OS=Drosophila melanogaster OX=7227 GN=Cyp4g1 PE=2 SV=1 P22465 Annexin B10 OS=Drosophila melanogaster OX=7227 GN=AnxB10 PE=2 SV=3 A4V0N4 MIP16230p OS=Drosophila melanogaster OX=7227 GN=Vha68-2 PE=1 SV=1 P84051 Histone H2A OS=Drosophila melanogaster OX=7227 GN=His2A PE=1 SV=2 O46067 EG:25E8.1 protein OS=Drosophila melanogaster OX=7227 GN=GRP170 PE=1 SV=1 Q9VWP2 RH57257p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG7322 PE=1 SV=1 P41126 60S ribosomal protein L13 OS=Drosophila melanogaster OX=7227 GN=RpL13 PE=1 SV=1 Q9V9T5 GM14617p OS=Drosophila melanogaster OX=7227 GN=Mccc1 PE=1 SV=2 Q9W1N3 Levy isoform A OS=Drosophila melanogaster OX=7227 GN=levy PE=1 SV=1 O18332 FI01544p OS=Drosophila melanogaster OX=7227 GN=Rab1 PE=1 SV=1 M9PBV2 NTPase isoform F OS=Drosophila melanogaster OX=7227 GN=NTPase PE=1 SV=1 Q9VWH4 Probable isocitrate dehydrogenase [NAD] subunit alpha mitochondrial OS=Drosophila melanogaster OX=7227 GN=l(1)G0156 PE=2 SV=1 A0A0B4KG Extended synaptotagmin-like protein 2 isoform D OS=Drosophila U9 melanogaster OX=7227 GN=Esyt2 PE=1 SV=1 P84040 Histone H4 OS=Drosophila melanogaster OX=7227 GN=His4 PE=1 SV=2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A4V0B5 Nervana 2 isoform F OS=Drosophila melanogaster OX=7227 GN=nrv2 PE=1 SV=1 A0A0B4LHE V-type proton ATPase subunit G OS=Drosophila melanogaster OX=7227 7 GN=Vha13 PE=3 SV=1 Q9VV42 HL08057p OS=Drosophila melanogaster OX=7227 GN=Pdh PE=1 SV=1 Q9VY92 GEO07753p1 OS=Drosophila melanogaster OX=7227 GN=Dmel\CG11151 PE=1 SV=1 Q9VQG4 Congested-like trachea protein OS=Drosophila melanogaster OX=7227 GN=colt PE=2 SV=1 Q9W229 40S ribosomal protein S24 OS=Drosophila melanogaster OX=7227 GN=RpS24 PE=1 SV=1 Q9V9S8 Ferrochelatase mitochondrial OS=Drosophila melanogaster OX=7227 GN=FeCh PE=2 SV=1 Q9U6L5 Ejaculatory bulb-specific protein 1 OS=Drosophila melanogaster OX=7227 GN=Ebp PE=1 SV=1 Q9W2L6 RH02475p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG9394 PE=1 SV=1 Q9W401 Probable citrate synthase mitochondrial OS=Drosophila melanogaster OX=7227 GN=kdn PE=2 SV=1 Q9VB46 FI18644p1 OS=Drosophila melanogaster OX=7227 GN=Hmu PE=1 SV=1 P29310 14-3-3 protein zeta OS=Drosophila melanogaster OX=7227 GN=14-3-3zeta PE=1 SV=1 Q9VEJ3 Pyrroline-5-carboxylate reductase OS=Drosophila melanogaster OX=7227 GN=P5cr-2 PE=1 SV=1 Q9VLC5 Aldehyde dehydrogenase OS=Drosophila melanogaster OX=7227 GN=Aldh PE=1 SV=1 P84029 Cytochrome c-2 OS=Drosophila melanogaster OX=7227 GN=Cyt-c-p PE=1 SV=2 Q9VGZ3 Iron regulatory protein 1B OS=Drosophila melanogaster OX=7227 GN=Irp- 1B PE=1 SV=1 E1JIJ5 Vacuolar proton pump subunit B OS=Drosophila melanogaster OX=7227 GN=Vha55 PE=1 SV=1 A8JRB8 MIP04243p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG5028 PE=1 SV=1 O01666 ATP synthase subunit gamma mitochondrial OS=Drosophila melanogaster OX=7227 GN=ATPsyngamma PE=2 SV=2 A4UZR3 Trehalase OS=Drosophila melanogaster OX=7227 GN=Treh PE=1 SV=1 Q76NR6 Regucalcin isoform D OS=Drosophila melanogaster OX=7227 GN=regucalcin PE=1 SV=1 Q6IDF5 NADH dehydrogenase (Ubiquinone) B15 subunit OS=Drosophila melanogaster OX=7227 GN=ND-B15 PE=1 SV=1 Q9W3N1 RH59310p OS=Drosophila melanogaster OX=7227 GN=spidey PE=1 SV=1 Q8MSI2 GH15296p OS=Drosophila melanogaster OX=7227 GN=Scp1 PE=1 SV=1 P41094 40S ribosomal protein S18 OS=Drosophila melanogaster OX=7227 GN=RpS18 PE=1 SV=1 Q7JWF1 Electron transfer flavoprotein-ubiquinone oxidoreductase isoform A OS=Drosophila melanogaster OX=7227 GN=Etf-QO PE=1 SV=1 Q8SWX4 Aminopeptidase OS=Drosophila melanogaster OX=7227 GN=CG5839 PE=1 SV=1 Q9VHN7 LP07963p OS=Drosophila melanogaster OX=7227 GN=anon- WO0118547.344 PE=1 SV=3 M9MRC9 GEO07462p1 OS=Drosophila melanogaster OX=7227 GN=RpL27A PE=2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

SV=1 Q95U15 GH14252p OS=Drosophila melanogaster OX=7227 GN=Mf PE=1 SV=1 Q7K332 GH17623p OS=Drosophila melanogaster OX=7227 GN=CG3364 PE=2 SV=1 X2JEA2 Uncharacterized protein isoform B OS=Drosophila melanogaster OX=7227 GN=Dmel\CG4666 PE=4 SV=1 X2JB48 Stress-sensitive B isoform E OS=Drosophila melanogaster OX=7227 GN=sesB PE=1 SV=1 Q9VKR4 Aminomethyltransferase OS=Drosophila melanogaster OX=7227 GN=Dmel\CG6415 PE=1 SV=1 M9MS06 Alpha actinin isoform D OS=Drosophila melanogaster OX=7227 GN=Actn PE=1 SV=1 Q9VGQ1 GM01350p OS=Drosophila melanogaster OX=7227 GN=alpha-KGDHC PE=1 SV=1 Q8IPP8 Uncharacterized protein OS=Drosophila melanogaster OX=7227 GN=Dmel\CG31548 PE=1 SV=1 Q8IQQ0 Neural conserved at 73EF isoform F OS=Drosophila melanogaster OX=7227 GN=Nc73EF PE=1 SV=1 A1ZA73 Stretchin-Mlck isoform R OS=Drosophila melanogaster OX=7227 GN=Strn- Mlck PE=1 SV=2 B4ZJ91 Seminal fluid protein 24Bb OS=Drosophila melanogaster OX=7227 GN=Sfp24Bb PE=2 SV=1 A0A0B4LFB Optic atrophy 1 isoform D OS=Drosophila melanogaster OX=7227 8 GN=Opa1 PE=1 SV=1 Q9VAM6 CDGSH iron-sulfur domain-containing protein 2 homolog OS=Drosophila melanogaster OX=7227 GN=Cisd2 PE=2 SV=1 Q9VZF1 GH07444p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG1309 PE=1 SV=1 Q8T6I0 EH domain containing protein OS=Drosophila melanogaster OX=7227 GN=Past1 PE=1 SV=1 X2J4W8 Kruppel homolog 2 isoform C OS=Drosophila melanogaster OX=7227 GN=Kr-h2 PE=1 SV=1 Q9V9U2 FI19428p1 OS=Drosophila melanogaster OX=7227 GN=salt PE=2 SV=2 Q6NP72 GEO09659p1 OS=Drosophila melanogaster OX=7227 GN=Dmel\CG13220 PE=1 SV=1 Q9VGM2 Fatty acid binding protein isoform B OS=Drosophila melanogaster OX=7227 GN=fabp PE=1 SV=1 A1Z729 Uncharacterized protein OS=Drosophila melanogaster OX=7227 GN=Dmel\CG2064 PE=1 SV=1 M9PJN8 Glyceraldehyde-3-phosphate dehydrogenase OS=Drosophila melanogaster OX=7227 GN=Gapdh2 PE=3 SV=1 O97418 EG:152A3.7 protein OS=Drosophila melanogaster OX=7227 GN=ND-B14.5A PE=1 SV=1 G4LTX1 GH14535p2 OS=Drosophila melanogaster OX=7227 GN=Pmi PE=1 SV=1 Q9W4H6 Pyruvate dehydrogenase E1 component subunit alpha OS=Drosophila melanogaster OX=7227 GN=Pdha PE=1 SV=2 A0A0B4KG MICOS complex subunit MIC60 OS=Drosophila melanogaster OX=7227 N2 GN=Mitofilin PE=3 SV=1 Q9V4E7 Transporter OS=Drosophila melanogaster OX=7227 GN=Gat PE=1 SV=4 M9NEX3 Uncharacterized protein isoform D OS=Drosophila melanogaster OX=7227 GN=BcDNA:RH45308 PE=1 SV=1 Q8SYJ2 GEO09626p1 OS=Drosophila melanogaster OX=7227 GN=ND-MLRQ PE=1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

SV=1 O18333 GH01619p OS=Drosophila melanogaster OX=7227 GN=Rab2 PE=1 SV=1 Q6IHT7 HDC01001 OS=Drosophila melanogaster OX=7227 GN=Sfp26Ad PE=4 SV=1 Q9V3Y4 LD43650p OS=Drosophila melanogaster OX=7227 GN=Mtch PE=1 SV=1 Q01604 Phosphoglycerate kinase OS=Drosophila melanogaster OX=7227 GN=Pgk PE=2 SV=2 Q9U915 Adenylate kinase OS=Drosophila melanogaster OX=7227 GN=Adk2 PE=1 SV=1 Q9V3L7 GEO04710p1 OS=Drosophila melanogaster OX=7227 GN=NP15.6 PE=1 SV=1 Q8IQH0 FI12817p OS=Drosophila melanogaster OX=7227 GN=Nrx-IV PE=1 SV=2 Q9VES6 Fatty acyl-CoA reductase OS=Drosophila melanogaster OX=7227 GN=Dmel\CG17560 PE=3 SV=2 Q9XYZ9 Glutathione S transferase E12 isoform A OS=Drosophila melanogaster OX=7227 GN=GstE12 PE=1 SV=1 Q8IMJ0 Uncharacterized protein isoform A OS=Drosophila melanogaster OX=7227 GN=anon-EST:Posey81 PE=1 SV=1 C8VV60 FI03659p OS=Drosophila melanogaster OX=7227 GN=CG10616-RA PE=1 SV=1 Q960M4 Peroxiredoxin OS=Drosophila melanogaster OX=7227 GN=Prx5 PE=1 SV=1 Q9VF20 Heavy metal tolerance factor 1 OS=Drosophila melanogaster OX=7227 GN=Hmt-1 PE=1 SV=1 Q9VD58 GH26270p OS=Drosophila melanogaster OX=7227 GN=Idh3b PE=1 SV=1 Q8SZ28 RE21371p OS=Drosophila melanogaster OX=7227 GN=CG3884 PE=1 SV=1 P18432 Myosin regulatory light chain 2 OS=Drosophila melanogaster OX=7227 GN=Mlc2 PE=1 SV=2 A0A0B4LGB Calcium-transporting ATPase OS=Drosophila melanogaster OX=7227 7 GN=SERCA PE=1 SV=1 P15007 Enolase OS=Drosophila melanogaster OX=7227 GN=Eno PE=1 SV=2 Q9VV72 Multiple inositol polyphosphate phosphatase 1 OS=Drosophila melanogaster OX=7227 GN=Mipp1 PE=1 SV=2 Q7K5K3 Pyruvate dehydrogenase E1 component subunit beta OS=Drosophila melanogaster OX=7227 GN=Pdhb PE=1 SV=1 Q59E30 Failed axon connections isoform C OS=Drosophila melanogaster OX=7227 GN=fax PE=1 SV=1 O77062 Amino acid transporter OS=Drosophila melanogaster OX=7227 GN=Eaat1 PE=1 SV=1 Q7K221 Glutamate oxaloacetate transaminase 1 isoform A OS=Drosophila melanogaster OX=7227 GN=Got1 PE=1 SV=1 Q94915 Rhythmically expressed gene 2 protein OS=Drosophila melanogaster OX=7227 GN=Reg-2 PE=2 SV=1 Q9V7D2 V-type proton ATPase subunit D 1 OS=Drosophila melanogaster OX=7227 GN=Vha36-1 PE=2 SV=1 Q9VVL7 Dihydrolipoyl dehydrogenase OS=Drosophila melanogaster OX=7227 GN=E3 PE=1 SV=1 P48148 Ras-like GTP-binding protein Rho1 OS=Drosophila melanogaster OX=7227 GN=Rho1 PE=1 SV=1 Q00637 Superoxide dismutase [Mn] mitochondrial OS=Drosophila melanogaster OX=7227 GN=Sod2 PE=2 SV=3 P53501 Actin-57B OS=Drosophila melanogaster OX=7227 GN=Act57B PE=1 SV=1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Q23982 Ejaculatory bulb-specific protein 2 OS=Drosophila melanogaster OX=7227 GN=EbpII PE=2 SV=2 Q9VTP4 60S ribosomal protein L10a-2 OS=Drosophila melanogaster OX=7227 GN=RpL10Ab PE=1 SV=2 Q95TZ7 GH19182p OS=Drosophila melanogaster OX=7227 GN=Zasp66 PE=1 SV=1 Q9I7R1 NADH-cytochrome b5 reductase OS=Drosophila melanogaster OX=7227 GN=Dmel\CG5946 PE=1 SV=2 X2JEM4 GEO07185p1 OS=Drosophila melanogaster OX=7227 GN=RpS28b PE=2 SV=1 Q9W2X6 ATP synthase delta subunit isoform A OS=Drosophila melanogaster OX=7227 GN=ATPsyndelta PE=1 SV=1 Q9VNW6 Delta-1-pyrroline-5-carboxylate synthase OS=Drosophila melanogaster OX=7227 GN=Dmel\CG7470 PE=1 SV=1 X2JE06 GEO07624p1 OS=Drosophila melanogaster OX=7227 GN=RpL24 PE=1 SV=1 Q9VUY9 Phosphoglucomutase OS=Drosophila melanogaster OX=7227 GN=Pgm PE=1 SV=1 Q9W1I7 GH07506p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG5554 PE=1 SV=1 A0A0B4JC Succinate--CoA ligase [ADP-forming] subunit beta mitochondrial W4 OS=Drosophila melanogaster OX=7227 GN=ScsbetaA PE=1 SV=1 Q961R9 GH09241p OS=Drosophila melanogaster OX=7227 GN=CT19169 PE=1 SV=1 Q9Y115 UNC93-like protein OS=Drosophila melanogaster OX=7227 GN=CG4928 PE=2 SV=1 P91927 Mitochondrial proton/calcium exchanger protein OS=Drosophila melanogaster OX=7227 GN=Letm1 PE=2 SV=2 O77266 Uncharacterized protein isoform A OS=Drosophila melanogaster OX=7227 GN=Dmel\CG4199 PE=1 SV=3 Q9VKP2 GH26960p OS=Drosophila melanogaster OX=7227 GN=Porin2 PE=2 SV=1 Q9VI17 SCP domain-containing protein OS=Drosophila melanogaster OX=7227 GN=Dmel\CG42564 PE=4 SV=2 Q9VFQ9 Dipeptidase B isoform A OS=Drosophila melanogaster OX=7227 GN=Dip-B PE=1 SV=2 P41043 Glutathione S-transferase S1 OS=Drosophila melanogaster OX=7227 GN=GstS1 PE=1 SV=2 A1ZB79 Uncharacterized protein isoform M OS=Drosophila melanogaster OX=7227 GN=Dmel\CG5174 PE=1 SV=2 Q7JRC3 Epoxide hydrolase OS=Drosophila melanogaster OX=7227 GN=Jheh1 PE=2 SV=1 Q9VIL5 GEO08953p1 OS=Drosophila melanogaster OX=7227 GN=Dmel\CG17472 PE=2 SV=1 Q94522 Succinate--CoA ligase [ADP/GDP-forming] subunit alpha mitochondrial OS=Drosophila melanogaster OX=7227 GN=Scsalpha1 PE=2 SV=3 Q9VEB1 IP09655p OS=Drosophila melanogaster OX=7227 GN=Mdh2 PE=1 SV=1 Q9VLQ7 Serpin 28F OS=Drosophila melanogaster OX=7227 GN=Spn28F PE=3 SV=2 Q9VD61 Sulfhydryl oxidase OS=Drosophila melanogaster OX=7227 GN=Qsox2 PE=2 SV=1 Q9VM12 MIP26555p1 OS=Drosophila melanogaster OX=7227 GN=Dmel\CG5958 PE=1 SV=1 Q7K084 Odorant-binding protein 44a isoform A OS=Drosophila melanogaster OX=7227 GN=Obp44a PE=1 SV=1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Q9W4W8 Paraplegin isoform A OS=Drosophila melanogaster OX=7227 GN=Spg7 PE=1 SV=1 Q94920 Voltage-dependent anion-selective channel OS=Drosophila melanogaster OX=7227 GN=porin PE=1 SV=3 Q9VQB4 LD06553p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG3609 PE=1 SV=1 Q9VVA4 GH26789p OS=Drosophila melanogaster OX=7227 GN=GS PE=1 SV=2 Q9VIH9 GEO04151p1 OS=Drosophila melanogaster OX=7227 GN=BcDNA:GH07967 PE=2 SV=1 D1Z3A2 ATP synthase-coupling factor 6 mitochondrial OS=Drosophila melanogaster OX=7227 GN=ATPsynCF6 PE=1 SV=1 Q9VAX7 Tubulin beta chain OS=Drosophila melanogaster OX=7227 GN=betaTub97EF PE=1 SV=3 Q9VXZ0 GEO07190p1 OS=Drosophila melanogaster OX=7227 GN=ND-B18 PE=1 SV=1 Q7K5N8 GH01592p OS=Drosophila melanogaster OX=7227 GN=BEST:GH19547 PE=1 SV=1 B5RIM9 Glycerol-3-phosphate dehydrogenase [NAD(+)] OS=Drosophila melanogaster OX=7227 GN=Gpdh1 PE=1 SV=1 Q9VAN7 Phosphoglycerate mutase OS=Drosophila melanogaster OX=7227 GN=Pglym78 PE=1 SV=2 Q9VZL1 LP07226p OS=Drosophila melanogaster OX=7227 GN=mge PE=1 SV=1 Q9V427 Innexin inx2 OS=Drosophila melanogaster OX=7227 GN=Inx2 PE=1 SV=1 P45594 Cofilin/actin-depolymerizing factor homolog OS=Drosophila melanogaster OX=7227 GN=tsr PE=1 SV=1 X2J979 Synaptotagmin 1 isoform H OS=Drosophila melanogaster OX=7227 GN=Syt1 PE=4 SV=1 Q9W306 GEO08256p1 OS=Drosophila melanogaster OX=7227 GN=CT27394 PE=1 SV=1 Q8SXY6 Transmembrane emp24 domain-containing protein bai OS=Drosophila melanogaster OX=7227 GN=bai PE=2 SV=1 P08879 Nucleoside diphosphate kinase OS=Drosophila melanogaster OX=7227 GN=awd PE=1 SV=3 P14318 Muscle-specific protein 20 OS=Drosophila melanogaster OX=7227 GN=Mp20 PE=2 SV=2 Q9VHP0 ATP-dependent RNA helicase bel OS=Drosophila melanogaster OX=7227 GN=bel PE=1 SV=1 Q9VPJ9 Uncharacterized protein isoform B OS=Drosophila melanogaster OX=7227 GN=CK02656 PE=1 SV=2 O62619 Pyruvate kinase OS=Drosophila melanogaster OX=7227 GN=PyK PE=2 SV=2 P32748 Dihydroorotate dehydrogenase (quinone) mitochondrial OS=Drosophila melanogaster OX=7227 GN=Dhod PE=2 SV=2 P17704 40S ribosomal protein S17 OS=Drosophila melanogaster OX=7227 GN=RpS17 PE=1 SV=2 A1ZBJ2 Uncharacterized protein isoform B OS=Drosophila melanogaster OX=7227 GN=cg7461 PE=1 SV=2 Q9W1H6 GH04238p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG5597 PE=1 SV=1 Q9V3W2 GM23292p OS=Drosophila melanogaster OX=7227 GN=ND-B17 PE=1 SV=1 Q9V3E7 LD24793p OS=Drosophila melanogaster OX=7227 GN=Ref1 PE=1 SV=1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

P15372 Phosrestin-2 OS=Drosophila melanogaster OX=7227 GN=Arr1 PE=1 SV=1 M9NGK3 Terribly reduced optic lobes isoform AL OS=Drosophila melanogaster OX=7227 GN=trol PE=1 SV=2 Q9VF27 GM02062p OS=Drosophila melanogaster OX=7227 GN=ND-23 PE=1 SV=1 E1JH55 Aquaporin OS=Drosophila melanogaster OX=7227 GN=AQP PE=3 SV=1 A1ZA47 PDZ and LIM domain protein Zasp OS=Drosophila melanogaster OX=7227 GN=Zasp52 PE=1 SV=2 Q9VX69 FI01450p OS=Drosophila melanogaster OX=7227 GN=Dmel\CG5162 PE=1 SV=1 P48588 40S ribosomal protein S25 OS=Drosophila melanogaster OX=7227 GN=RpS25 PE=1 SV=3 P06603 Tubulin alpha-1 chain OS=Drosophila melanogaster OX=7227 GN=alphaTub84B PE=1 SV=1 M9PJQ5 Wings up A isoform K OS=Drosophila melanogaster OX=7227 GN=wupA PE=1 SV=1 Q8SXR1 Neural lazarillo isoform A OS=Drosophila melanogaster OX=7227 GN=NLaz PE=1 SV=1 Q9VIE8 Aconitate hydratase mitochondrial OS=Drosophila melanogaster OX=7227 GN=mAcon1 PE=1 SV=2 Q8T4C4 ATP synthase subunit beta OS=Drosophila melanogaster OX=7227 GN=ATPsynbetaL PE=2 SV=1 R9PY16 Uncharacterized protein OS=Drosophila melanogaster OX=7227 GN=Ubi- p5E5 PE=4 SV=1 Q95U34 GH11113p OS=Drosophila melanogaster OX=7227 GN=Galk PE=1 SV=1 Q9VQA3 Uncharacterized protein isoform A OS=Drosophila melanogaster OX=7227 GN=anon-SAGE:Wang-110 PE=4 SV=1 P00334 Alcohol dehydrogenase OS=Drosophila melanogaster OX=7227 GN=Adh PE=1 SV=2

Supplemental table 2 – label-free MS identified 57 proteins with altered abundance between exercised and non-exercised Pink1- flies, 55 of which had decreased abundance.

Protein Heat-map Database Mean 95.00% CI Adjust Chan Row identifier Diff. of diff. ed p- ge /w value exerc ise Peroxisomal 26 X2JFD6 9.333 0.3787 to 0.0372 * ↓ multifunction 18.29 al enzyme type 2 isoform B Sc2 34 Q9VZL3 10.67 1.712 to 0.0119 * ↓ 19.62 EG:BACR7A 48 Q9U1L2 11 2.045 to 0.0087 ** ↓ 4.14 protein 19.95 Ribosomal 60 M9NEQ9 9.333 0.3787 to 0.0372 * ↓ protein S10b 18.29 isoform D Rm62 64 E1JJ68 11 2.045 to 0.0087 ** ↓ isoform H 19.95 Receptor of 73 M9PCC1 11.33 2.379 to 0.0063 ** ↓ activated 20.29 protein kinase C 1 isoform C Probable 75 Q9V419 23.67 14.71 to <0.000 ** ↓ cytochrome 32.62 1 ** P450 28a5 RE29555p 79 Q9VC18 9 0.04537 to 0.0483 * ↓ 17.95 Cytochrome 82 Q9V4I1 10.67 1.712 to 0.0119 * ↓ P450 9b2 19.62 GH23390p 85 Q95SI7 11.33 2.379 to 0.0063 ** ↓ 20.29 Zipper 91 A0A0B4J 12.33 3.379 to 0.0023 ** ↓ isoform H D95 21.29 Probable 92 Q9VE00 9.333 0.3787 to 0.0372 * ↓ cytochrome 18.29 P450 12a4 mitochondrial Ribosomal 97 A0A0B4L 10.33 1.379 to 0.0161 * ↓ protein L11 GZ5 19.29 isoform B ATP binding 98 Q7KVB1 14.33 5.379 to 0.0002 ** ↓ cassette 23.29 * subfamily B member 7 isoform B GM05240p 99 Q9VCC6 10.67 1.712 to 0.0119 * ↓ 19.62 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

60S 102 Q9VZS5 11 2.045 to 0.0087 ** ↓ ribosomal 19.95 protein L28 BcDNA.GH1 104 Q9Y114 10.67 1.712 to 0.0119 * ↓ 0229 19.62 GH26015p 107 Q7K3N4 15.33 6.379 to <0.000 ** ↓ 24.29 1 ** CathD 115 Q7K485 17.33 8.379 to <0.000 ** ↓ isoform A 26.29 1 ** LD24105p 127 Q9W3N9 11.67 2.712 to 0.0046 ** ↓ 20.62 Annexin 140 A0A0B4K 10 1.045 to 0.0215 * ↓ H34 18.95 Imaginal disk 149 Q23997 12 3.045 to 0.0032 ** ↓ growth factor 20.95 6 Acetyl-CoA 163 Q7JV23 9.667 0.7120 to 0.0284 * ↓ carboxylase 18.62 isoform B GH02075p 219 Q9VPR1 9.667 0.7120 to 0.0284 * ↓ 18.62 Eukaryotic 226 A0A0B4L 14.33 5.379 to 0.0002 ** ↓ translation FL2 23.29 * initiation factor 3 subunit C 60S acidic 230 M9PG76 9.333 0.3787 to 0.0372 * ↓ ribosomal 18.29 protein P0 RE74312p 232 Q9VD29 12.33 3.379 to 0.0023 ** ↓ 21.29 Aminopeptid 237 Q8IN25 9.667 0.7120 to 0.0284 * ↓ ase 18.62 FI02856p 270 Q9VII1 9.333 0.3787 to 0.0372 * ↓ 18.29 LD31742p 276 Q7JQH9 14.33 5.379 to 0.0002 ** ↓ 23.29 * Neither 282 P10676 10.33 1.379 to 0.0161 * ↓ inactivation 19.29 nor afterpotential protein C Glutathione S 286 Q7K8X7 11 2.045 to 0.0087 ** ↓ transferase 19.95 E9 Hsc70Cb 296 Q9VUC1 9.333 0.3787 to 0.0372 * ↓ isoform A 18.29 OCIA 308 Q9W1X9 -9.667 -18.62 to - 0.0284 * ↑ domain- 0.7120 containing protein 1 UDP- 329 Q09332 12 3.045 to 0.0032 ** ↓ glucose:glyco 20.95 protein bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

glucosyltrans ferase Translocon- 334 Q9VUZ0 10.33 1.379 to 0.0161 * ↓ associated 19.29 protein subunit beta 6- 341 P41572 9 0.04537 to 0.0483 * ↓ phosphogluc 17.95 onate dehydrogena se decarboxylati ng GM14617p 352 Q9V9T5 11 2.045 to 0.0087 ** ↓ 19.95 Extended 357 A0A0B4K 18.67 9.712 to <0.000 ** ↓ synaptotagmi GU9 27.62 1 ** n-like protein 2 isoform D GEO07753p 362 Q9VY92 9.333 0.3787 to 0.0372 * ↓ 1 18.29 40S 364 Q9W229 10.33 1.379 to 0.0161 * ↓ ribosomal 19.29 protein S24 40S 383 P41094 10.33 1.379 to 0.0161 * ↓ ribosomal 19.29 protein S18 GEO07462p 387 M9MRC9 17.33 8.379 to <0.000 ** ↓ 1 26.29 1 ** Aminomethylt 392 Q9VKR4 19 10.05 to <0.000 ** ↓ ransferase 27.95 1 ** FI19428p1 404 Q9V9U2 13.67 4.712 to 0.0005 ** ↓ 22.62 * GH14535p2 410 G4LTX1 10.33 1.379 to 0.0161 * ↓ 19.29 Uncharacteri 414 M9NEX3 9.333 0.3787 to 0.0372 * ↓ zed protein 18.29 isoform D HDC01001 417 Q6IHT7 10.67 1.712 to 0.0119 * ↓ 19.62 Uncharacteri 425 Q8IMJ0 13 4.045 to 0.0011 ** ↓ zed protein 21.95 isoform A Peroxiredoxi 427 Q960M4 9.333 0.3787 to 0.0372 * ↓ n 18.29 60S 446 Q9VTP4 15 6.045 to 0.0001 ** ↓ ribosomal 23.95 * protein L10a- 2 GEO07185p 449 X2JEM4 13.33 4.379 to 0.0008 ** ↓ 1 22.29 * Epoxide 465 Q7JRC3 12 3.045 to 0.0032 ** ↓ hydrolase 20.95 Paraplegin 473 Q9W4W8 10 1.045 to 0.0215 * ↓ bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

isoform A 18.95 GH26789p 476 Q9VVA4 14.67 5.712 to 0.0002 ** ↓ 23.62 * Uncharacteri 493 Q9VPJ9 12.33 3.379 to 0.0023 ** ↓ zed protein 21.29 isoform B Dihydroorotat 495 P32748 -15.67 -24.62 to - <0.000 ** ↑ e 6.712 1 ** dehydrogena se (quinone) mitochondrial

Supplemental table 3 – label-free MS identified 105 proteins with different abundance when comparing non-exercised Pink1- and WT flies

Protein Hea Databa Mea 95.00% Adjus Pink1- t- se n CI of diff. ted p- /- n.e ma identifi Diff. value relativ p er e to Ro WT w n.e NADPH--cytochrome P450 4 Q2759 11.3 2.379 to 0.0063 ** ↑ reductase 7 3 20.29 GH20802p 5 Q9W3L 14 5.045 to 0.0003 ** ↑ 4 22.95 * Transferrin 7 Q9VW 9.66 0.7120 to 0.0284 * ↑ V6 7 18.62 Fatty acyl-CoA reductase 10 Q9VLJ 16 7.045 to <0.00 ** ↑ 7 24.95 01 ** Heat shock protein 60A 11 O0264 9.33 0.3787 to 0.0372 * ↑ 9 3 18.29 FI01422p 12 A1Z6V 13 4.045 to 0.0011 ** ↑ 5 21.95 Chitinase-like protein Idgf4 15 Q9W30 9.66 0.7120 to 0.0284 * ↑ 3 7 18.62 Enoyl-CoA hydratase short chain 16 Q7JR5 9.66 0.7120 to 0.0284 * ↑ 1 isoform A 8 7 18.62 LD30155p 27 Q9VN2 10.6 1.712 to 0.0119 * ↑ 1 7 19.62 FI09602p 31 Q9VVU 11.3 2.379 to 0.0063 ** ↑ 1 3 20.29 Reticulon-like protein 32 Q9VM 14.3 5.379 to 0.0002 ** ↑ V9 3 23.29 * Sc2 34 Q9VZL 12.3 3.379 to 0.0023 ** ↑ 3 3 21.29 CG3835-RA 38 Q7K51 9.33 0.3787 to 0.0372 * ↑ 1 3 18.29 Ferritin 39 Q7KR 9 0.04537 0.0483 * ↑ U8 to 17.95 Sideroflexin-1-3 41 Q9VN1 10 1.045 to 0.0215 * ↑ 3 18.95 LP10861p 44 Q9W0 13 4.045 to 0.0011 ** ↑ M4 21.95 EG:BACR7A4.14 protein 48 Q9U1L 12.6 3.712 to 0.0016 ** ↑ 2 7 21.62 A-kinase anchor protein 200 50 Q9VLL 11 2.045 to 0.0087 ** ↑ 3 19.95 RE08669p 51 Q9V9 12 3.045 to 0.0032 ** ↑ W3 20.95 Cytochrome b5 52 Q9V4N 9.33 0.3787 to 0.0372 * ↑ 3 3 18.29 Cytochrome P450 4e2 53 Q2760 11.6 2.712 to 0.0046 ** ↑ 6 7 20.62 Sphingosine-1-phosphate lyase 61 Q9V7Y 12.6 3.712 to 0.0016 ** ↑ 2 7 21.62 Rm62 isoform H 64 E1JJ68 15 6.045 to 0.0001 ** ↑ bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

23.95 * 40S ribosomal protein S14b 65 C0HKA 13 4.045 to 0.0011 ** ↑ 1 21.95 LD12946p 66 Q9VX 10.6 1.712 to 0.0119 * ↑ M4 7 19.62 60S ribosomal protein L5 67 Q9W5 9.66 0.7120 to 0.0284 * ↑ R8 7 18.62 Probable cytochrome P450 9f2 68 Q9VG8 10.3 1.379 to 0.0161 * ↑ 2 3 19.29 HDC00331 70 Q6IHY - -19.29 to 0.0161 * ↓ 5 10.3 -1.379 3 Sodium/potassium-transporting 72 Q2404 -9 -17.95 to 0.0483 * ↓ ATPase subunit beta-1 6 -0.04537 Receptor of activated protein 73 M9PC 10.6 1.712 to 0.0119 * ↑ kinase C 1 isoform C C1 7 19.62 GH22187p 74 Q7YTZ 9 0.04537 0.0483 * ↑ 4 to 17.95 Probable cytochrome P450 28a5 75 Q9V41 25.6 16.71 to <0.00 ** ↑ 9 7 34.62 01 ** RE29555p 79 Q9VC1 9.33 0.3787 to 0.0372 * ↑ 8 3 18.29 GH11762p 80 Q9VY0 9 0.04537 0.0483 * ↑ 5 to 17.95 Polyadenylate-binding protein 81 P2118 13 4.045 to 0.0011 ** ↑ 7 21.95 Cytochrome P450 9b2 82 Q9V4I1 14.6 5.712 to 0.0002 ** ↑ 7 23.62 * Dolichyl- 83 A0A0B 11.6 2.712 to 0.0046 ** ↑ diphosphooligosaccharide-- 4LFR4 7 20.62 protein glycosyltransferase subunit 2 Fatty acyl-CoA reductase 84 Q9VG8 11.3 2.379 to 0.0063 ** ↑ 7 3 20.29 LD30122p 86 Q9W4 14.3 5.379 to 0.0002 ** ↑ N8 3 23.29 * 40S ribosomal protein S23 88 Q8T3U 9.66 0.7120 to 0.0284 * ↑ 2 7 18.62 Uncharacterized protein isoform 89 Q86BQ 12.6 3.712 to 0.0016 ** ↑ A 3 7 21.62 Poly(U)-specific 90 Q9VZ4 10 1.045 to 0.0215 * ↑ endoribonuclease homolog 9 18.95 Zipper isoform H 91 A0A0B 15.6 6.712 to <0.00 ** ↑ 4JD95 7 24.62 01 ** Glutamine synthetase 93 E1JHQ 9 0.04537 0.0483 * ↑ 1 to 17.95 RH34413p 95 Q9VU3 11 2.045 to 0.0087 ** ↑ 5 19.95 Ribosomal protein L11 isoform B 97 A0A0B 9.33 0.3787 to 0.0372 * ↑ 4LGZ5 3 18.29 ATP binding cassette subfamily B 98 Q7KVB 17.6 8.712 to <0.00 ** ↑ member 7 isoform B 1 7 26.62 01 ** GM05240p 99 Q9VC 11.6 2.712 to 0.0046 ** ↑ bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

C6 7 20.62 BcDNA.GH10229 104 Q9Y11 15.6 6.712 to <0.00 ** ↑ 4 7 24.62 01 ** Probable transaldolase 106 Q9W1 12 3.045 to 0.0032 ** ↑ G0 20.95 Heat shock protein 22 113 P0251 19.3 10.38 to <0.00 ** ↑ 5 3 28.29 01 ** CG6459 protein 114 Q7JXC 13.6 4.712 to 0.0005 ** ↑ 4 7 22.62 * Phosphatidate 116 X2JC5 12 3.045 to 0.0032 ** ↑ cytidylyltransferase 5 20.95 60S ribosomal protein L9 118 P5088 10.6 1.712 to 0.0119 * ↑ 2 7 19.62 NADH dehydrogenase 119 A4V44 - -18.29 to 0.0372 * ↓ (Ubiquinone) 75 kDa subunit 9 9.33 -0.3787 isoform B 3 AT07710p 120 Q9VH 9.66 0.7120 to 0.0284 * ↑ W0 7 18.62 40S ribosomal protein S7 123 Q8IMI7 9.33 0.3787 to 0.0372 * ↑ 3 18.29 LD24105p 127 Q9W3 12 3.045 to 0.0032 ** ↑ N9 20.95 Fatty acid synthase 3 130 Q7PLB 10 1.045 to 0.0215 * ↑ 8 18.95 Putative fatty acyl-CoA reductase 133 Q960W 9 0.04537 0.0483 * ↑ CG8306 6 to 17.95 Fatty acid synthase 2 isoform A 135 Q9VQL 10 1.045 to 0.0215 * ↑ 6 18.95 Microsomal glutathione S- 137 Q8SY1 9.33 0.3787 to 0.0372 * ↑ transferase-like isoform A 9 3 18.29 Annexin 140 A0A0B 11.3 2.379 to 0.0063 ** ↑ 4KH34 3 20.29 Fatty acyl-CoA reductase 145 Q9VES 17.3 8.379 to <0.00 ** ↑ 7 3 26.29 01 ** Probable cytochrome P450 6a23 147 Q9V77 -11 -19.95 to 0.0087 ** ↓ 1 -2.045 Imaginal disk growth factor 6 149 Q2399 9.66 0.7120 to 0.0284 * ↑ 7 7 18.62 Calnexin 99A isoform A 150 Q9VAL 9 0.04537 0.0483 * ↑ 7 to 17.95 C-1-tetrahydrofolate synthase 151 O9655 10.6 1.712 to 0.0119 * ↑ cytoplasmic 3 7 19.62 Peptidyl-prolyl cis-trans 157 Q9W22 9.33 0.3787 to 0.0372 * ↑ isomerase 7 3 18.29 GH01077p 161 Q9VFF - -18.29 to 0.0372 * ↓ 0 9.33 -0.3787 3 Epoxide hydrolase 162 Q7KB1 9.33 0.3787 to 0.0372 * ↑ 8 3 18.29 Catalase 167 P1733 9 0.04537 0.0483 * ↑ 6 to 17.95 HL01062p 168 Q9VU1 10 1.045 to 0.0215 * ↑ 7 18.95 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Tropomyosin 2 isoform E 169 A0A0B - -19.62 to 0.0119 * ↓ 4KHJ9 10.6 -1.712 7 Maltase A1 179 P0719 9.66 0.7120 to 0.0284 * ↑ 0 7 18.62 GH02075p 219 Q9VPR 16.6 7.712 to <0.00 ** ↑ 1 7 25.62 01 ** Eukaryotic translation initiation 226 A0A0B 17 8.045 to <0.00 ** ↑ factor 3 subunit C 4LFL2 25.95 01 ** 60S acidic ribosomal protein P0 230 M9PG7 9.33 0.3787 to 0.0372 * ↑ 6 3 18.29 RE74312p 232 Q9VD2 10 1.045 to 0.0215 * ↑ 9 18.95 Uncharacterized protein isoform 240 M9MSJ 10.6 1.712 to 0.0119 * ↑ B 3 7 19.62 UDP-glucuronosyltransferase 292 Q9W2J - -22.29 to 0.0008 ** ↓ 4 13.3 -4.379 * 3 Actin indirect flight muscle 315 P8396 - -29.62 to <0.00 ** ↓ 7 20.6 -11.71 01 ** 7 UDP-glucose:glycoprotein 329 Q0933 13.6 4.712 to 0.0005 ** ↑ glucosyltransferase 2 7 22.62 * Translocon-associated protein 334 Q9VUZ 10.3 1.379 to 0.0161 * ↑ subunit beta 0 3 19.29 GM14617p 352 Q9V9T 17 8.045 to <0.00 ** ↑ 5 25.95 01 ** Levy isoform A 353 Q9W1 - -27.29 to <0.00 ** ↓ N3 18.3 -9.379 01 ** 3 Extended synaptotagmin-like 357 A0A0B 21.3 12.38 to <0.00 ** ↑ protein 2 isoform D 4KGU9 3 30.29 01 ** RH59310p 381 Q9W3 3.33 -5.621 to 0.774 n ↑ N1 3 12.29 s 40S ribosomal protein S18 383 P4109 9.66 0.7120 to 0.0284 * ↑ 4 7 18.62 GEO07462p1 387 M9MR 15 6.045 to 0.0001 ** ↑ C9 23.95 * Uncharacterized protein isoform 390 X2JEA 12.3 3.379 to 0.0023 ** ↑ B 2 3 21.29 Aminomethyltransferase 392 Q9VKR 17.6 8.712 to <0.00 ** ↑ 4 7 26.62 01 ** Uncharacterized protein 395 Q8IPP 10 1.045 to 0.0215 * ↑ 8 18.95 HDC01001 417 Q6IHT 14.6 5.712 to 0.0002 ** ↑ 7 7 23.62 * Fatty acyl-CoA reductase 423 Q9VES 9.33 0.3787 to 0.0372 * ↑ 6 3 18.29 FI03659p 426 C8VV6 15 6.045 to 0.0001 ** ↑ 0 23.95 * Heavy metal tolerance factor 1 428 Q9VF2 9.66 0.7120 to 0.0284 * ↑ 0 7 18.62 60S ribosomal protein L10a-2 446 Q9VTP 9.33 0.3787 to 0.0372 * ↑ 4 3 18.29 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.06.425659; this version posted January 7, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

NADH-cytochrome b5 reductase 448 Q9I7R 11.6 2.712 to 0.0046 ** ↑ 1 7 20.62 GEO07185p1 449 X2JEM 9 0.04537 0.0483 * ↑ 4 to 17.95 GH07506p 454 Q9W1I 10.3 1.379 to 0.0161 * ↑ 7 3 19.29 Mitochondrial proton/calcium 458 P9192 9.66 0.7120 to 0.0284 * ↑ exchanger protein 7 7 18.62 Sulfhydryl oxidase 470 Q9VD6 - -23.62 to 0.0002 ** ↓ 1 14.6 -5.712 * 7 Paraplegin isoform A 473 Q9W4 9.66 0.7120 to 0.0284 * ↑ W8 7 18.62 GH26789p 476 Q9VVA 13 4.045 to 0.0011 ** ↑ 4 21.95