Acetyl-CoA dependent lysine acetylation of mitochondrial proteins

Acetyl-CoA is another metabolite in mitochondria which can impact mitochondrial functions due to lysine acetylation of proteins. This might occur under oxidizing conditions when CS activity is impaired and acetyl-CoA levels might rise and would be available for lysine

2. Summarizing Discussion acetylation. In the previous chapter MRR was discussed in the context of mitochondrial impairment and the influence of increased citrate levels on MRR. In the following section the impact of lysine acetylation on plant mitochondrial function will be presented.

Since a lack of knowledge on the extent of lysine acetylated proteins in plant mitochondria existed, it was our goal to shed light on the existing plant mitochondrial acetylome and to reveal putative regulatory functions.

By mass spectrometry (MS) analysis, 120 lysine-acetylated mitochondria proteins of Arabidopsis including 243 lysine-acetylated sites were discovered ([6] Tab.1, method see publication [7]). We categorized the identified lysine-acetylated proteins into functional groups and discovered that 17% of the identified proteins belong to the TCA cycle and 13%

to the respiratory chain ([6] Fig. 1). Relative to the total amount of proteins participating in the TCA cycle on the basis of MapMan bin annotations, 38% of them are lysine-acetylated.

Our results are consistent with other studies in bacteria and animals showing that actually all TCA cycle enzymes were identified as lysine-acetylated (Wang et al., 2010; Zhao et al., 2010). Compared to the total number of proteins identified from the TCA-cycle, only 12% of the proteins participating in the ETC were identified although the ETC consists of many more proteins than the TCA cycle. The most likely explanation for this is that the TCA cycle enzymes are situated in the matrix of mitochondria and usually do not appear in SC, thus they are probably more accessible for lysine acetylation. Proteins of the ETC are partially hidden in the inner mitochondrial membrane and they likely form SC which would prevent the acetylation of lysine residues. To verify the MS analysis we performed two dimensional blue native (2D-BN-PAGE) as second approach to detect mitochondrial lysine acetylated proteins followed by Western blot analysis using the anti-acetyllysine antibody ([6] Fig. 4, method see publication [7]). We confirmed the very low abundance of lysine-acetylated proteins in SC I/III2. In line with the MS analysis, CV exhibited the highest number of lysine acetylation sites within the ETC, and CIII carried no obvious lysine acetylated proteins. In Fig. 10, lysine-acetylated proteins are depicted within the TCA cycle and the ETC. The following chapter provides some more insights into the possible regulatory functions of lysine acetylation on important enzymes.

Fig. 10: Overview of identified Arabidopsis lysine-acetylated proteins from mitochon- drial energy metabolism. Proteins functional categories were annotated using MapMan (Thimm et al., 2003). Boxes in blue indicate identified non-acetylated proteins before enrichment, red boxes show identified lysine-acetylated proteins and white boxes indicate TAIR10 proteins not identified in the LC-MS/MS analysis. AOX, alternative oxidase; TCA, tricarboxylic acid; TP, transporter; I, complex I; II, complex II; III, complex III; IV, complex IV (publication [6]).

Comparison of lysine acetylation of mitochondrial proteins in different species

The mitochondrial pyruvate dehydrogenase complex (PDC) acts as a link between glycolysis and the TCA cycle. It catalyzes the rate limiting step of the TCA cycle which is the oxidative decarboxylation of pyruvate by the release of acetyl-CoA and the reduction of NAD⁺ to NADH. Acetyl-coA is the substrate of protein acetylation, and it is the first substrate of the TCA cycle and plays an important role for fatty acid synthesis, isoprenoids and a number of secondary metabolites. Hence, regulation of the PDC activity serves as an important switch

2. Summarizing Discussion for many metabolic pathways. Feedback activity regulation of the PDC by lysine acetylation seems likely since we have shown in publication [6] that acetylation is dependent on the amount of acetyl-CoA. However, such a mechanism for PDC is not proven yet, whereas in yeast it was demonstrated that the deletion of PDC results in a general decrease in lysine acetylation, the study also indicating that there is a direct link between intracellular acetyl-CoA concentrations and the acetylation state of proteins (Weinert et al., 2014). Recently, it has been discovered in mammalian cells that lysine acetylation and phosphorylation act hierarchically in concert to control molecular composition as well as activity of PDC (Fan et al., 2014). In future research the overlap in regulatory pathways concerning lysine acetylation and phosphorylation is a promising path for research in plants.

For CS we were able to demonstrate that the activity was regulated in a redox dependent manner by the action of TRX (publication [3]). Nothing is known so far as to how lysine acetylation affects CS activity. As an acetyl-CoA consuming enzyme, CS also controls mitochondrial acetyl-CoA levels. Weinert et al., 2014 demonstrated that the lack of CS in yeast leads to increased acetyl-CoA levels in the mitochondria which correlate with an increase in lysine acetylation. Additionally, CS indirectly affects acetyl-CoA levels in the cytosol because citrate can be exported from mitochondria and converted by ATP-dependent citrate-lyase into acetyl-CoA (Fatland et al., 2002; Fatland et al., 2005). It would be interesting to investigate the effect of lysine acetylation on CS also in correlation with the knowledge that citrate possibly leads to MRS [2].

Concerning the remaining enzymes in the TCA cycle, it is known for mammalian cells that the activity of malate dehydrogenase is stimulated by lysine acetylation while isocitrate dehydrogenase as well as succinate dehydrogenase activities are repressed (Guan and Xiong, 2011). Similar observations were made for the cytosolic NAD⁺-dependent malate dehydrogenase from Arabidopsis (Finkemeier et al., 2011). These opposing effects are not yet understood in detail and might reflect the complex network of specific KATs and KDACs as regulators of these enzymes.

Taking a closer look at the ETC, the only subunit of CI that was identified by MS analysis as lysine-acetylated in Arabidopsis was the gamma carbonic anhydrase 2 (CA2) which is involved in the assembly of CI as well as in SC formation (Perales et al., 2005). It will be of great relevance to find out if acetylation of CA2 is involved in CI stabilization. As previously, explained the loss of CI in the ndufa1 mutant has an impact on transcription levels and therefore it would be interesting to investigate if acetylation of CA2 contributes into MRR by stabilizing or destabilizing CI.

The OXPHOS protein complex with the highest number of lysine-acetylated proteins is CV.

Also in other species, CV has an outstanding number of lysine acetylation sites but actually very little is known about their function. In mice, it was already demonstrated that the number of acetylation sites on CV varies during fasting or feeding, suggesting that lysine acetylation is a dynamic PTM which depends on cellular energy status (Kane and Van Eyk, 2009). Only for the oligomycin sensitivity conferral protein (OSCP) subunit in mammalian cells it has been reported that an increase in lysine acetylation leads to decreased ATP levels. The regulation of the OSCP acetylation status is dependent on the action of Sirt3 (Wu et al., 2013).

In our studies the OSCP subunit was not identified as a lysine-acetylated protein. This might suggest that the OSCP subunit of Arabidopsis might not be regulated by lysine acetylation or that an unknown KDAC regulates the deacetylation of OSCP in Arabidopsis.

Beside proteins of the TCA cycle and the ETC, mitochondrial transporter proteins were also discovered as lysine-acetylated. Strikingly, all mitochondrial voltage dependent anion channels 1-3 (VDAC1-3) were found to be acetylated which was also observed in other species. VDACs play an essential role in regulating metabolite transport between mitochondria and the cytoplasm (Colombini, 2004; Homble et al., 2012). In mouse and human liver mitochondria for example, VDACs contain several lysine acetylated sites (Kerner et al., 2012; Kim et al., 2006; Schwer et al., 2009; Yang et al., 2011; Zhao et al., 2010).

Because VDACs are the most abundant mitochondrial outer membrane proteins it is of high interest to better understand their regulation and the involvement of lysine acetylation.

Besides VDACs, we also identified a dicarboxylate/tricarboxylate carrier as lysine-acetylated which possibly transports citrate. As described earlier citrate plays a pivotal role in MRR as well as in the control of acetyl-CoA levels in the cytosol. By controlling the transport of citrate into the cytosol an additional layer of regulating acetyl-CoA levels would be provided.

The ATP/ADP carriers 1-3 (AAC1-3) were also found to be acetylated which was investigated in more detail in publication [5] and will be discussed below (subchapter 2.7).

Mechanisms that regulate lysine acetylation levels

As mitochondria show a high number of lysine-acetylated proteins the question arose how this PTM is transferred onto the protein. In the nucleus the acetylation of histone proteins is catalyzed by HATs. Interestingly, no HAT/KAT has been found in plant organelles until now and also for other species just one single KAT, the GCN5-related KAT, GCNAL1 was found to be localized in human mitochondria (Scott et al., 2012). However, lysine acetylation was also found on mitochondrial encoded proteins which implies that the acetylation reaction can

2. Summarizing Discussion occur within the organelle (Hirschey et al., 2011). In contrast, the knowledge about orgarnellar KDACS is increasing rapidly with sirtuins as predominant non-nuclear localized deacetylases. The action of sirtuin homologues in Arabidopsis will be discussed in the next chapter and publication [5]. In plants, no mitochondrial KAT has been identified so far but we were able to demonstrate that lysine acetylation can also occur non-enzymatically similar to what was observed for mice mitochondria. Wagner and Payne, 2013 demonstrated that lysine acetylation can occur non-enzymatically on isolated mice mitochondria in in vitro experiments. Non-enzymatic lysine acetylation requires a high pH which is part of the physiology of mitochondria. In contrast to cytosolic pH of 7, the mitochondrial matrix has a pH of 7.9-8.0. Additionally, acetyl-CoA levels can reach concentrations of around 1.5 mM.

To mimic these conditions we isolated plant mitochondria and treated them with different pH and with or without addition of acetyl-CoA ([6] Fig. 5). At pH 8 and an acetyl-CoA concentration of 5 mM a maximum level of acetylation was reached, which could be even increased by denaturing mitochondria before treatment. The presence of non-enzymatic acetylation may explain the absence of KATs in organelles. However, it does not rule out the presence of KATs, also because the pH in the intermembrane space (IMS) is even lower than in the cytosol (Porcelli et al., 2005) and acetylated lysines were identified on IMS proteins.

Therefore, it would be interesting to discover which of the acetylated proteins we found in our study are localized to the IMS. But if the control is not on the level of KATs, it is likely that acetylation control is based on KDACs which is already proposed for SIRT3 in mice mitochondria. It protects mitochondrial enzymes and proteins from chemical acetylation-induced impairment (Hirschey et al., 2011). In Arabidopsis, two sirtuin-type like KDACs exist, whereas one of the two is localized in mitochondria [5] and which will be discussed in the next section.