Dual lysine and N‐terminal acetyltransferases reveal the complexity underpinning protein acetylation

Abstract Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N‐α‐acetylation (NTA) and ε‐lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5‐related N‐acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid‐localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.

(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. Depending on transfer agreements, referee reports obtained elsewhere may or may not be included in this compilation. Referee reports are anonymous unless the Referee chooses to sign their reports.) 1st Revision -Editorial Decision 24th Feb 2020 Manuscript Number: MSB-20-94 64 Tit le: Dual lysine and N-t erminal acet ylt ransferases reveal the complexit y underpinning prot ein acet ylat ion Thank you again for submit ting your work to Molecular Syst ems Biology. We have now heard back from the three referees who agreed to evaluat e your st udy. Overall, the reviewers think that the present ed findings are novel and relevant for the field. They raise however a series of concerns, which we would ask you to address in a revision.
Wit hout repeat ing all the point s list ed below, some of the more fundament al issues raised are the following: -Reviewer #1 refers to the need to provide further biochemical validations.
-Reviewer #2 points out that some follow up analysis on the biological significance of the dual specificity acetyltransferases would significantly enhance the impact of the study. Reviewer #3 expresses concerns along those lines, as they mention that the study is largely focused on in vitro data. I understand that this is a potentially challenging issue to address and I would be open to discussing in further detail potential suggestions you may have on how to address it.
All other issues raised by the reviewers would need to be convincingly addressed. Please let me know in case you would like to discuss any of the issues raised by the reviewers.
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Summary
In this st udy, Bienvenut . et at . ident ify some put at ive chloroplast acet ylt ransferases by searching for prot eins cont aining GNAT sequence and secondary st ruct ure homology and predict ed chloroplast localizat ion, using in silico tools like Prosit e, Target P and ot hers. They ident ify as many as 10 put at ive candidat es, which indeed share similar secondary st ruct ure feat ures and key residues wit h ot her GNATs, while most of them cont ain two predict ed Ac-CoA binding mot ifs, which is unusual. Expression of GFP fusion prot eins of all these GNATs in Arabidopsis prot oplast s confirms that 8 (7?) of 10 show chloroplast localizat ion. Furt her charact erizat ion of these enzymes is carried out by comparing the lysine acet ylome and N-t erminal acet ylome (GAP assay) of E.coli st rains overexpressing MBP-GNAT s or empt y vect or, mainly using mass spect romet ry. The result s are consist ent wit h these GNATs possessing bot h KAT and NAT act ivit y. Last ly, global NTA analysis comparison bet ween WT and GNAT2 knockout confirms that GNAT2 has NTA act ivit y in vivo.

General remarks
Previous underst anding about acet ylt ransferases in chloroplast is very limit ed so the novelt y of this st udy is high. Bienvenut . et al. ident ify new chloroplast GNATs, most of which have not been previously charact erized. Most surprisingly is the finding that of these chloroplast GNATs possess bot h KTA and NAT act ivit y, a dual act ivit y that does not appear to be conserved in higher eukaryot es. Overall, the cell based mass-spec st udies seem to largely support the conclusions of the aut hors, alt hough there is no biochemical validat ion, which would be required to demonst rat e direct effects (see below). Assuming that the results can be validated biochemically, and that the other relatively minor issues addressed, we believe that the study would be an exciting contribution to the field. Major points 1. It is unclear from the study as carried out, if the characterized GNATs carry out the KAT and NAT modifications directly or indirectly. This could be resolved if the authors prepared one or more of the plastid GNAT proteins as a recombinant protein and assayed it against an identified N-terminal or lysine sidechain peptides. This would not only address if the observed activities in cells are direct, but also if the catalytic subunits are necessary and sufficient for the two acetylation activities. The requirement for regulatory subunits could be evaluated by assessing the requirement of added cell extract to the recombinant enzyme for peptide acetyltransferase activity. Related to the issue above, there is concern that the E.coli GAP assays overexpressing GNATs reveal relaxed substrate specificity, while in vivo GNAT2 KO global NTA analysis displayed preferred small residues at downstream positions. In addition, if so many GNATs show such redundant and non-specific substrate specificity similar to archaeal NATs, why does a single GNAT2 KO show a significant decrease in only some types of substrates? These might be very complex questions to address in vivo but the vitro experiments would go a long way to demonstrating that these GNATs do indeed harbor NTA activity.
3. Line 120, The statement at the end of the introduction that states "has far reaching implications for the study of acetylation in eukaryotic organisms." Should be softened to "may have far reaching...." as this dual specificity may not be conserved in higher eukaryotes.
4. Line 189, change "two bases" to "two catalytic residues" 5. Line 201, the authors state "eight of the ten predicted... show plastid-associated localization", but in between Lines 205 -208, the data suggests "GNAT6 is probably not within chloroplast and GNAT8 and GNAT9 show cytosolic/nuclear localization." So only seven of them appear to have confirmed plastid localization. Figure EV2, the expression level of all 8 GNAT proteins appear to vary a lot. The authors should discuss how this might affect the data shown in Figure 3. 7. The authors should provide the identity and N terminal sequence of all substrates identified in the GAP assay in the supplementary information section.

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8. GNAT2 AT1G32070.2 in Figure1A is indicated by the authors to be reported in the literature to possess both NAT and KAT activity. In light of this previous report, the novelty of this study would be increased if it focused on another member to demonstrate this dual activity. 9. Based on the GAP assay, the plastid GNATs appear to have a significant overlap in NAT specificity ( Figure 3B) and GNAT2 is not the most active GNAT ( Figure 3A). In light of this, the authors should discuss this result in the context of the results for the GNAT2 KO. Why do the other NATs not compensate when GNAT2 is knocked out? 10. Line 235, the authors state "GNATs acetylate target proteins in their vicinity". It is still unknow if auto lysine acetylation of MBP-GNATs is inter-or intra-molecular. It is safer to say that highly acetylated MBP peptide might be due to the unusually high abundance of MBP present in the overexpressed cells.
11. Line 298, change "and could quantified half of them" to "and could quantify half of them" 12. The data in Figure 4G requires more explanation. Why is there sequence preferences for positions 7-10. This is quite unusual for NTA's. What does the green annotation of threonine residues meant to symbolize? 13. Figure EV4, The authros should add numbers for each peptide substrate identified for GNAT6,4,7,5,10.
14. Line 338 and 339, the authors state that the GNATs have only partial redundancy on NAT activity, which is contradictory to Figure 3B . To evaluate if GNAT2 has its own plastic "unique" or "relaxed" NTA, the authors could take the top hit substrates and test them as substrates for other GNATs in-vitro. 15. Line 348, The authors should add references for the NAT complex assemblies. 16. line 348, "(8 as to 2019, named NatA/ B/ C/ D/ E/ F/ NAA80)", the authors state 8, but only list 7.
17. Line 351, authors should define NME and the requirement for it. 18. Line 354, change "narrower of proteins" to "narrower set of proteins" 19. The first and second paragraph (line 341-372) of the "discussion" sounds like it could fit in the introduction section. 20. Dinh et al 2015 identified NAA70 as AT2G39000, which the authora named NSI/GNAT2, in line 373. However, in Figure 1A, GNAT2 is listed as AT1G32070.2, while AT2G39000.1 is indicated as GNAT4, which is confusing to readers in terms of naming. 21. Line 402, The sentence that reads "Surprisingly, all identified GNATs display a clear dual KAT and NAT activity, closing the debate of the possibility that the same enzyme can have both activities." is misleading. As far as we are aware there is no debate about whether an enzyme can catalyze both NAT and KAT reaction. The debate centers around whether Naa10 can carry out both activities. In addition, as the authors point out, the structures of the yeast and metazoan NAT substrate binding sites are not appropriately configured to accommodate an internal lysine side chain. The plastid GNAT may indeed have a different type of substrate binding site that may allow it to accommodate both N-terminal and internal amino group substrates. 22. The latter part of the sentence that begins on line 430, which reads "Here we show, that NSI/GNAT2 actually displays an additional NTA activity in vivo next to its KA activity, but GNAT2 inactivation differently affects its KA and NTA targets, suggesting a different acetylation recognition mode." is confusing. It is clear that GNAT2 must have different sequence specificity modes since it acetylates one substrate that has no N-terminal residues and another that does, and it does these in different substrates so it will clearly have different effects. Are the authors trying to say something else?
Reviewer #2: The manuscript titled "Dual lysine and N-terminal 1 acetyltransferases reveal the complexity underpinning protein acetylation" by Bienvenut et al. show enzymatic evidence in Arabidopsis plastids for a new family of GCN5-related N-acetyltransferases, that possess dual substrate specificity for both N-and -lysine. The authors' study is significant for several reasons: (1) it is well-documented that distinct acetyltransferases exist for both these substrates, but there is controversy of whether a single enzyme can have dual specificity, (2) the cellular and biological context of lysine acetylation and N-terminal acetylation have unique features, and (3) the functional consequence of acetylation on protein function is less well understood compared to the number of documented sites of modification. Improved characterization of acetyltransferase enzymes and their specificities should support these efforts. In the authors study, ten putative GNAT proteins were identified by computational predictions, of which 8 were plastid-localized. Using western blotting and global acetylome profiling with quantitative mass spectrometry, the authors documented that six of the eight GNATs display dual acetyltransferase activities. The KA and NAT profiling experiments of E. coli lysates derived from GNAT-expressing strains clearly indicated unique substrate specificities (at least among E. coli proteins) and relative GNAT activities, but of course these results should be translated to plastids with some caution. Importantly, the authors did directly support these experiments with targeted knockout of GNAT2 in planta, showing reduced levels of both NTA and KA on plastid proteins.
Overall, the authors two-step in silico and experimental validation approaches efficiently identified and evaluated the top candidate enzymes in Arabidposis. The in-silico sequence and structural conservation analysis was extremely thorough, highlighting shared and unique features of the putative plastid NATs compared to known cytoplasmic counterparts. Moreover, the experimental design was performed with replicate numbers that were fit-for-purpose and with the appropriate controls. The experimental results were clearly explained, and the figures were impactful. Overall, the study was well conducted and definitively addresses the debate of acetyltransferase dual specificity. The only point lacking, as described in detail below, is the lack of biological context for this dual specificity enzymes, for instance, in chloroplasts. If the authors could provide additional insight here, this study would be significantly elevated and should be considered for publication.

Primary comment
While the authors' demonstration of dual specificity KAT/NATs was convincing and significant, the authors demonstrated in a previous study (Koskela et al, 2018) that GNAT2 KO led to reduced KA levels and defective state transitions of the chloroplasts. While the current study now extends these previous findings by determining that GNAT2 KO also leads to reduced NTA levels in chloroplasts and reveals partial GNAT redundancy, the study lacks a defining experimental result that demonstrates the biological significance of dual specificity. I recognize that without direct evidence of substrate binding characteristics, it is difficult to design experiments that distinguish these activities. Yet, based on the authors structural analysis (Fig 5), perhaps they have considered more subtle genetic ablations of GNAT2 in Arabidopsis?
Minor comment Pg 16, line 54, a missing word or incorrect sentence structure?
Reviewer #3: The manuscript 'Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation' by Bienvenut et al. is a comprehensive investigation into a novel group of enzymes found in plastids. This study represents an important step in defining enzymes responsible for one of the most common protein modifications, namely acetylation. The authors find that among 8 identified plastid acetyltransferases, most of them display a quite convincing dual activity targeting both protein N-termini (alpha amino groups) and lysine side chains (epsilon amino groups). The general opinion is that protein acetyltransferases are either NATs catalyzing Nterminal acetylation or KATs catalyzing lysine acetylation. In many cases this is probably also true, but this study leans support to an more open model where the same enzyme may have both activities. Candidate proteins were expressed in E. coli and the resulting acetylomes were analyzed with respect to both lysine and N-terminal acetylation events. Further sequence analysis defined motif signatures resembling known AcCoA binding motifs etc. One enzyme, GNAT2, was more thoroughly studied by including important in vivo experiments truly defining this enzyme as both a NAT and a KAT. The weakness of the study is the overrepresentation of in vitro data that may be over-interpreted, but the GNAT2 in vivo data are convincing and makes the overall presentation of this group of enzymes valid.
Minor issues: 1) The language is mostly good, but some typos and artistic twists here and there might be adjusted for a clearer presentation.  Figure S1. GNAT4 and GNAT7 is duplicated while GNAT3 and GNAT10 are missing. 6) Line 129-130: Please include include info on new/old names correlating the GNAT1-10 nomenclature with NAA70 and NSI enzymes. In Table S1 there is no mention of NAA70 or NSI while the text does not mention GNAT names when listing NAA70 and NSI. 7) According to Figure 1B, only GNAT9 is missing a chloroplastic transit peptide while according to Table S1 GNAT9, GNAT6 and GNAT3 all miss a chloroplastic transit peptide (while having a mitochondrial targeting signal). Table S1 text says: 'Subcellular 52 localization was predicted with Target P. However, only eight of them possess a clear TP..'. Please clarity and unify to avoid confusion. 8) Figure 2A: Please combine this figure with the Coomassie stained gels in Fig EV2 in order to allow the reader to compare the bands. Also, GNAT4 anti-Ac-Lys should be repeated with less loading/less signal to allow for better interpretation.

Summary
In this study, Bienvenut. et at. identify some putative chloroplast acetyltransferases by searching for proteins containing GNAT sequence and secondary structure homology and predicted chloroplast localization, using in silico tools like Prosite, TargetP and others. They identify as many as 10 putative candidates, which indeed share similar secondary structure features and key residues with other GNATs, while most of them contain two predicted Ac-CoA binding motifs, which is unusual. Expression of GFP fusion proteins of all these GNATs in Arabidopsis protoplasts confirms that 8 (7? We would like to thank the reviewer for their comments and suggestions. We selected two GNATs, representing two subtypes from the different branches of the phylogenetic tree (Fig. EV1), for protein purification and in vitro activity tests. For the activity assay, we used a HPLC-based method, which allows the detection of the appearance of the acetylated peptide substrate over time. We used standard peptides, which were previously developed for lysine deacetylase as well as acetyltransferase enzyme assays by our collaborators (laboratory of Prof. Dirk Schwarzer, University of Tübingen), who are now included as co-authors on the manuscript. For this study, they additionally synthetized the same peptide with different alpha amino acids possessing either a free or acetylated N-terminus. Hence, these peptides allowed an unambiguous determination of both N-alpha and -lysine activities on the same peptide sequence. Interestingly, both enzymes were active without any auxiliary subunits. Furthermore, the assays revealed that both enzymes have relaxed substrate specificities, with some preferences for particular N-terminal amino acids. This nicely correlates the data obtained from the GAP assay and the study of the N-terminomics analysis of the GNAT knockout line. These new data are now displayed in a new Concerning the use and the interpretation of the data from the GAP assay, we would like to point out, as we reported in the result section, that this test has been previously validated with cytosolic AtNAA10 ( ). This specificity also tightly fits that observed in other organisms. Additionally, we recently characterized AtNAA60 and AtNAA50; both studies are now under minor revision in two different journals and the two manuscripts include a GAP assay for both enzymes. The data reveal similar substrate specificities with known orthologous NAAs. We assume that the GAP assay as a result is very robust, relevant and reflecting the biological observations. Its unique advantage is to sample a large array of sequences at once without any of the a priori and limitations which biochemical studies usually direct (choice of peptide sequence, limited sequence corpus, additional chemical groups for detection…).
Concerning the issue of relaxed substrate specificity of plastid GNATs observed with the GAP assay, there is kind of a misunderstanding and our presentation was likely misleading. We now better address this issue in the new result section. In brief, the GAP assay revealed that the NTA substrate specificity of any of the plastid GNATs gathered NatA/B/C/D/E specificities. Such specificities were classified as mainly depending on the nature of aminoacids at positions 1>2>3. For instance, all of six most active plastid AtGNATs were very efficient for N-termini starting with an initiator Met, but with clear differences induced by the amino acid at position 2. To better illustrate this issue, we decided to increase the threshold criteria from 2% to 5% to display them in the new Figure 3A for each GNAT.
In addition, we now include a new dashed line showing how many substrates are N-acetylated with a value above 30%, which is very significant. The complete dataset is now available in Dataset EV3. Both the new Figure 4A and Dataset EV3 now nicely illustrate that some sequences are only recognized by one GNAT and not by the others, while some others are recognized by several GNATs. A few are even N-α-acetylated by all of them. Though our GAP data cover almost 400 sequences, we have too little information to conclude fully on the specificity of each GNAT. An attempt towards this end is given in Figure 4B where we built a logo illustrating AtGNAT2 specificity with respect to the seven others AtGNATs; this reveals unique features which are now discussed. Altogether, the data reveal that plastid GNATs have only partial overlapping relaxed substrate specificity. Because other plastid GNATs are most likely able to still acetylate plastid proteins in the context of the AtGNAT2 mutant (nsi-1), the characterization of this mutant reflects mostly the most sensitive AtGNAT2 NTA-and KA-substrates rather than its substrate specificity. Furthermore, the NTA of different N-termini of the same proteins, such as F16P1, were affected in AtGNAT2-defective plant lines, suggesting long distance contacts between AtGNAT2 and their substrates. This can explain the selectivity of AtGNAT2 for specific substrates. The new Dataset EV3 shows all GAP data while Figure 4 illustrates some examples addressing the issue related to "overlapping substrate specificity". We also discuss and compare Figure 4B and Figure 5G and reveal similar features.

Minor points
2. line 55-57, the authros should also mention that some NATs functional predominantly posttranslationally, like Naa60 (NatF) and Naa80 (NatH). 3. Line 120, The statement at the end of the introduction that states "has far reaching implications for the study of acetylation in eukaryotic organisms." Should be softened to "may have far reaching...." as this dual specificity may not be conserved in higher eukaryotes.
This sentence has been softened as suggested.

Line 189, change "two bases" to "two catalytic residues"
This has been changed accordingly.
5. Line 201, the authors state "eight of the ten predicted... show plastid-associated localization", but in between Lines 205 -208, the data suggests "GNAT6 is probably not within chloroplast and GNAT8 and GNAT9 show cytosolic/nuclear localization." So only seven of them appear to have confirmed plastid localization.
This is correct, only seven of them are localized within plastids. However, the term plastid-associated localization is not dedicated only for proteins that are localized inside plastids but applies to all that are connected to the organelle; the same is valid for other compartments. GNAT6 was predicted as an organellar-localized protein and a transit peptide was also predicted for this protein. In our GFPimages, GNAT6 was observed as associated to chloroplast. Thus, eight plastid-associated GNATs is the correct term to use. Additionally, we have repeated the localization analysis for GNAT6 with some subcellular localization markers. Importantly, the GNAT6-GFP signal does not overlap with mitochondria. In addition to the chloroplast-associated localization we now confirmed some additional nuclear localization for this protein, which is shown by co-expression with a nuclear envelope marker protein. Figure EV2, the expression level of all 8 GNAT proteins appear to vary a lot. The authors should discuss how this might affect the data shown in Figure 3.

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The expression level of GNATs does not seem to influence the NTA and KA activity. For instance, a low level of both NTA and KA activity was observed with GNAT1 even if this protein is the most highly expressed in bacteria. In contrast, GNAT4 was among the less expressed GNATs but its NTA and KA activity was among the highest one. We have now indicated in Figure 3A the relative level of expression of each GNAT just under the panel that displays the number of substrates. GNAT1 is the best expressed but instead it modifies the lowest number of proteins with high efficiency (see dashed line); this is in contrast to GNAT5. This is discussed now lines 335-338.

The authors should provide the identity and N terminal sequence of all substrates identified in the GAP assay in the supplementary information section.
A new dataset of the identified E.coli N-termini in the GAP assay is now provided as Dataset EV3. This is also mentioned in the manuscript line 321.

GNAT2 AT1G32070.2 in Figure1A is indicated by the authors to be reported in the literature to possess both NAT and KAT activity. In light of this previous report, the novelty of this study would be increased if it focused on another member to demonstrate this dual activity.
Except GNAT4, none of the identified plastid GNATs was known to have both NTA and auto-KA activities; GNAT2 was previously shown by us to display significant KA activity (Koskela 2018 TPC) in addition to low serotonin acetyltransferase activity (Lee et al. 2014, PMID: 25250906) as now cited in the legend to the figure (line 1307). The NTA activity of GNAT2 has never been reported either in vitro or in vivo. 9. Based on the GAP assay, the plastid GNATs appear to have a significant overlap in NAT specificity ( Figure 3B) and GNAT2 is not the most active GNAT ( Figure 3A).

In light of this, the authors should discuss this result in the context of the results for the GNAT2 KO. Why do the other NATs not compensate when GNAT2 is knocked out?
Please see answer to major point 1.

Line 235, the authors state "GNATs acetylate target proteins in their vicinity". It is still unknow if auto lysine acetylation of MBP-GNATs is inter-or intra-molecular. It is safer to say that highly acetylated MBP peptide might be de to the unusually high abundance of MBP present in the overexpressed cells.
The sentence has been changed accordingly.
11. Line 298, change "and could quantified half of them" to "and could quantify half of them" The sentence has been changed accordingly. Figure 4G requires more explanation. Why is there sequence preferences for positions 7-10. This is quite unusual for NTA's.

The data in
As discussed before, GNAT2 displays unambiguous specificity but it is still unclear where it comes from and it is unrelated to positions 1-3. We also know that there is specificity beyond the first residues as the same protein with two different N-termini is a substrate of GNAT2. We thus have considered looking at the first 10 residues to reveal whether there is something appearing downstream. The sequence logo in Figure 4G (now Figure 5G) shows no strong specificity appearing clearly as only Thr or Leu are retrieved in less than 1/3 of the 10 affected proteins. A comparison with the data discussed with the new Figure 4B is now available, showing similar features relative to GNAT2 specificity.

What does the green annotation of threonine residues meant to symbolize?
The color symbol is associated to the default choice, which the software Icelogo proposes for each class of amino acid. Green is for the class of small hydrophilic uncharged residues including Ser, Thr, Gly… Asp or Glu would be red. Lys, Arg, His would be blue. Asn or Gln, purple. Hydrophobic (Ala, Ile, Val, Leu, Trp, Tyr, Phe, Pro) residues are black. This is now indicated in the legend to the new Figure 4B where all colors are retrieved and reminded in the legend of this figure (now Figure 5).
Numbers of each peptide substrate identified for the different GNAT are now reported in the new version (now Figure EV6).
14. Line 338 and 339, the authors state that the GNATs have only partial redundancy on NAT activity, which is contradictory to Figure 3B . To evaluate if GNAT2 has its own plastic "unique" or "relaxed" NTA, the authors could take the top hit substrates and test them as substrates for other GNATs invitro.
A new Figure 4 and Dataset EV3 have been prepared according to the reviewer's suggestion, which better highlight partial overlapping substrate specificity. See also answer to major points.

Line 348, The authors should add references for the NAT complex assemblies.
We Number has been corrected.

Line 351, authors should define NME and the requirement for it.
NME and its requirement have been added in addition to two references.

Line 354, change "narrower of proteins" to "narrower set of proteins"
Done 19. The first and second paragraph (line 341-372) of the "discussion" sounds like it could fit in the introduction section.
We preferred to have a more balanced introduction pointing on the GNAT family to which KAT and NAT enzymes belong. Figure 1A, GNAT2 is listed as AT1G32070.2, while AT2G39000.1 is indicated as GNAT4, which is confusing to readers in terms of naming.

Dinh et al 2015 identified NAA70 as AT2G39000, which the authors named NSI/GNAT2, in line 373. However, in
AT2G39000 corresponds to NAA70 and now GNAT4, whereas we clearly stated that AT1G32070.2 was previously named NSI and now GNAT2. Figure and text  Since the most affected KA and NTA proteins in the GNAT2 mutant context are different, it is most likely that the recognition mode of GNAT2 for them is different. We changed the sentence to avoid confusion with the activity (lines 522-524).

Reviewer #2:
The manuscript titled "Dual lysine and Yet, based on the authors structural analysis (Fig 5), perhaps they have considered more subtle genetic ablations of GNAT2 in Arabidopsis?
We do appreciate the reviewer's comment, however, to answer this most interesting and intriguing issue this will require a dedicated study of probably several years due to the time it takes for plant complementation and mutation analyses. This goes well beyond this study. For instance, the nsi knockout induces a photosynthetic phenotype. In Koskela et al (2018), we showed that the KA status of protein components belonging to PSI, PSII and LHC were modified in the KO compared to the WT. In the Koskela (2018) paper, we additionally demonstrated that deletion of the enzyme GNAT2 (NSI) resulted in a complete loss of the ability of Arabidopsis plants to perform state transitions and more recently that the loss of NSI/GNAT2 had no effect on plant growth under control growth conditions, but it was severely affected when plants were subjected to fluctuating light conditions (Koskela et al, 2020). Deletion of the enzyme GNAT2 (NSI) was shown to induce a decrease on KA, but not phosphorylation of the LHCII proteins (Koskela et al, 2018). Among the affected proteins in the NTA acetylome of the present study, we did not retrieve any protein from the light reactions such as LHCII, which was found as a target protein for Lys-acetylation of NSI. Hence, the identified NTA substrates of NSI do not support the role for NTA acetylation on the photosynthesis phenotype. Altogether, these studies suggest that KA is a major modification influencing state transition plastid. Of course, these studies are not exhaustive and a further deeper characterization of isolated PSII-LCHII complexes under different light conditions will be needed to fully clarify the impact of KA and NTA activity of GNAT2 and even other plastid GNATs in photosynthesis. Concerning more subtle genetic ablations, we have not in hands yet the 3D structure of GNAT2 or any other plastid GNAT, which we would need for a targeted analysis. Indeed, plastid GNATs belong to the NAA family but it remains impossibleeven using 3D models derived from known structures -to predict the various substitutions which would make "subtle" alterations such as blocking Lys with respect to N-terminal acetylation or vice versa. Our data indicate that plastid GNATs are part of a new NAA family which covers at least two subtypes, GNAT1/2/3 and GNAT4/5/6/7 and 10 (now displayed in Figure EV1). Hence, deeper investigations will keep us and others busy for the coming years.

Minor comment
Pg 16, line 54, a missing word or incorrect sentence structure?
Unfortunately, on page 16 there is no line 54, hence we do not know which sentence you are referring to. The manuscript was checked again for any grammar or spelling mistakes. We hope we detected all of them and removed them now.

Reviewer #3:
The Two sentences are now added to indicate first that NAA40, 50 and 60 have been suggested to have low KA activity, in addition to their high NTA activity (lines 115-117). The second sentence focuses on NAA10. Figure S1. GNAT4 and GNAT7 is duplicated while GNAT3 and GNAT10 are missing.

5)
We thank the reviewer for pointing out this issue. We do apologize and we have now corrected all labels, dealing with GNATs. This confusion is mainly due to the new nomenclature, which we have adopted for the purpose of the publication and we mixed up new and old for some reason. We also corrected the legend to fit with the proper Arabidopsis entries.
6) Line 129-130: Please include info on new/old names correlating the GNAT1-10 nomenclature with NAA70 and NSI enzymes. In Table S1 there is no mention of NAA70 or NSI while the text does not mention GNAT names when listing NAA70 and NSI.
Old and new nomenclatures are now reported in the legend to Figure EV1. Figure 1B, only GNAT9 is missing a chloroplastic transit peptide while according to As explained in M&M, when we searched for plastid NAT/KAT, we used parallel in silico strategies, including subcellular prediction tools. The prediction tools are very powerful but far to be robust particularly with plants. For instance, Target P encounters frequent erroneous predictions between mitochondrial (Mt) and chloroplast (Cp) localisations. Thus, we considered both Cp and Mt predicted candidates. In the independent search of KAT, we retrieved a list of 35 Arabidopsis candidates but only 10 (the same retrieved as in the NAT search), which contained a putative organellar targeting peptide according to Target P-1.1 predictor. In Table EV1 (even if a Mt localization was predicted for GNAT3, GNAT6 and GNAT9) only GNAT 9 missed a TP accordingly to a more in-depth analysis using N-TerPred, ChloroP, and multiple sequence alignment of plant orthologues. This is more clearly indicated in the table now. 8) Figure 2A: Please combine this figure with the Coomassie stained gels in Fig EV2 in order to allow the reader to compare the bands. Also, GNAT4 anti-Ac-Lys should be repeated with less loading/less signal to allow for better interpretation.

7) According to
We now have combined both figures and included a less exposed blot for GNAT4, in addition to the overexposed blot. In comparison to the other GNATs, the luminescence signal of the acetylated proteins was already overexposed after a few seconds, indicating the strong activity of this enzyme, which was confirmed in the quantitative mass spectrometry analysis. This has been corrected.
10) Line 349: remove 'narrow' since NatA for instance displays a broad substrate specificity.
"Narrow" has been changed with "well-defined". 11) For Discussion: Please revisit the interpretation of findings in Koskela, Plant Cell, 2018 in light of these new findings on the NAT-activity of GNAT2. (activities and mechanisms leading to phenotype etc) We now have extended the discussion regarding the photosynthetic phenotype of GNAT2. However, in contrast to the detected substrates for K-acetylation, none of the detected NTA substrates of GNAT2 allows a direct explanation for the observed state transition phenotype.
13th May 2020 2nd Editorial Decision 13th May 2020 Manuscript Number: MSB-20-9464R Title: Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acet ylat ion Thank you for sending us your revised manuscript . We have now heard back from reviewer #1 who was asked to evaluat e your revised st udy. As you will see below, the reviewer is sat isfied wit h the modificat ions made and thinks that the st udy is now suit able for publicat ion. As such, I am glad to inform you that we can soon accept your manuscript for publicat ion, pending some minor edit orial issues list ed below.
-Our dat a edit ors have not iced some unclear or missing informat ion in the figure legends, please see the at tached .doc file. Please make all request ed text changes using the at tached file and *keeping the "t rack changes" mode* so that we can easily access the edit s made.
-There is a callout to Dataset EV6, but no Dataset EV6 exists. Could you please edit the callout accordingly?
-The EV Dataset and EV Table legends can be removed from the main text. Please provide them only in the respective .xls files.
-Please provide high-resolution individual files for each of the main and EV figures.
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REFEREE REPORTS
Reviewer #1: The aut hors have sat isfact orily addressed our major and minor concerns in the revised manuscript . They have performed in vit ro assays to confirm that GNAT2 and GNAT10 each independent ly can have acet ylat ion act ivit y toward bot h N termini and int ernal lysine subst rat es. The aut hors also made new figures to demonst rat e more clearly the subst rat e overlap of these GNATs. In addit ion, minor issues have been addressed. We feel that the st udy is now suit able for publicat ion. Thank you for sending us your revised manuscript and for performing the final request ed minor changes. We are now sat isfied wit h the modificat ions made and I am pleased to inform you that your paper has been accept ed for publicat ion.

B-Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner. a statement of how many times the experiment shown was independently replicated in the laboratory.
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As it is commonly accepted that 3 biological replicates are a minimum requirement for statistical analysis, we chose to design the experiments with 4 biological replicates in each condition to ensure meeting this requirement while remaining easily managable regarding the experimental procedures. The assumption of the test is that most proteins acetylation levels should not vary when comparing the two groups. To verify this assumption, we calculated, using built-in statistics tools from Microsoft Excel, the average of ratio, the median as well as the mode, giving respectively 1.024, 1.000 and 1.000. This seems to indicate that we have indeed a normal distribution of the KO/WT protein N-terminal acetylation levels, centered around 1 as most proteins remain unchanged.
-na na 1. Data the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.

E-Human Subjects na
The variances of the two groups were calculated using built-in statistics tools available in Microsoft Excel software, based on the protein N-terminal acetylation levels observed, yielding a value of 2138.8 for the wild type group and 2108.5 for the ko group. These values are similar enough for the two groups to be considered with the same variance.
The antibody used in the study was the ant-acetyllysine antibody from Immunechem (ICP0380), which was used in more than 75 publications and specific detecteion of acetylated proteins was validated using blocking solution, which included acetylated BSA (see Finkemeier et