AtVPS13M1 is involved in lipid remodeling in low phosphate and is located at the mitochondria surface in plants

  1. Sébastien Leterme
  2. Catherine Albrieux
  3. Sabine Brugière
  4. Yohann Couté
  5. Julien Dellinger
  6. Benjamin Gillet
  7. Sandrine Hughes
  8. Julie Castet
  9. Amélie Bernard
  10. David Scheuring
  11. Marion Schilling
  12. Juliette Jouhet
  13. Morgane Michaud

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Abstract

VPS13 are conserved lipid transporters with multiple subcellular localizations playing key roles in many fundamental cellular processes. While the localization and function of VPS13 have been extensively investigated in yeast and animals, little is known about their counterparts in plants, particularly regarding their role in stress response. In this study, we characterized AtVPS13M1, one of the four VPS13 paralogs of the flowering plant Arabidopsis thaliana . We show that AtVPS13M1 binds and transports glycerolipids with a low specificity in vitro . AtVPS13M1 interferes with phospholipids degradation in response to phosphate starvation, a nutrient stress that triggers a massive remodeling of membrane lipids. AtVPS13M1 is mainly expressed in young dividing and vascular tissues. Finally, we show that AtVPS13M1 is mainly located at the surface of mitochondria in leaves. Overall, our work highlights the conserved role in lipid transport of VPS13 in plants, reveals their importance in nutrient stress response and opens important perspectives for the understanding of lipid remodeling mechanisms and for the characterization of this protein family in plants.

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    Reply to the reviewers

    We thank the reviewers for their general comment and for the critical evaluation of our analyses and results interpretation. Their comments greatly helped us to improve the manuscript.

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

    Summary: An analysis of an Arabidopsis VSP13 presumed lipid transport is provided. The analysis pretty much follows similar studies done on yeast and human homologs. Key findings are the identification of multiple products from the locus due to differential splicing, analysis of lipid binding and transport properties, subcellular location, tissue specific promoter activity, mutant analysis suggesting a role in lipid remodeling following phosphate deprivation, but no physiological or growth defects of the mutants. Major points: The paper is generally written and documented, the experiments are well conducted and follow established protocols. The following major points should be considered:

    There are complementary lipid binding assays that should be considered such as liposome binding assays, or lipid/western dot blots. All of these might give slightly different results and may inform a consensus. Of course, non-membrane lipids such as TAG cannot be tested in a liposome assay.

    Concerning lipid transfer proteins (LTPs), it is important to differentiate the lipid binding capacity related to the transport specificity (which lipids are transported by a LTP?) from the lipid binding capacity linked to the targeting of a LTP to a specific membrane (a LTP can bind a specific lipid via a domain distinct from the lipid transfer domain to be targeted in cells, but will not transport this lipid). Both aspects are of high interest to be determined. Our goal here was to focus on the identification of the lipids bound to AtVPS13M1 and to be likely transported, which is why we used a truncation (1-335) corresponding to the N-term part of the hydrophobic tunnel. Liposome binding assays and lipid dot blots are necessary to answer the question of the membrane binding capacity of the protein. We think that this aspect is out of the scope of the current article as it will require to express and purify other AtVPS13M1 domains that are known to bind lipids such as the two PH domains and the C2. This will be the scope of future investigations in our lab.

    Similarly, lipid transfer based only on fluorophore-labeled lipids may be misleading because the fluorophore could affect binding. It is mentioned that the protein in this assay is tethered by 3xHis to the liposomes. Un less I ma missing something, I do not understand how that should work. This needs to be better explained.

    We truly agree with Reviewer 1 that the presence of a fluorophore could affect lipid binding to the protein. In this assay, lipids are labeled on their polar head and it is therefore difficult to conclude about the specificity of our protein in term of transport. This assay is used as a qualitative assay to show that AtVPS13M1(1-335) is able to transfer lipids in vitro, and in the manuscript, we did not make any conclusion about its transport specificity based on this assay, but rather used the binding assay to assess the binding, and likely transport, specificity of AtVPS13M1. FRET-based assay is a well-accepted assay in the lipid transfer community to easily probe lipid transport in vitro and has been used in the past to assess transfer capacity of different proteins, including for VPS13 proteins (for examples, see (Kumar et al., 2018; Hanna et al., 2022; Valverde et al., 2019)).

    To be able to transfer lipids from one liposome to another, both liposomes have to be in close proximity. Therefore, we attached our protein on donor acceptors, to favor the transport of the fluorescent lipids from the donor to the acceptor liposomes. Then, we progressively increased acceptor liposomes concentration to favor liposome proximity and the chance to have lipid transfer. We added a scheme on Figure 3B of the revised version of the manuscript to clarify the principle of the assay. In addition, we provided further control experiments suggested by Reviewers 2 and 3 showing that the fluorescence signal intensity depend on AtVPS13M1(1-335) protein concentration and that no fluorescence increase is measured with a control protein (Tom20.3) (see Figure 3C-D of the revised manuscript).

    The in vivo lipid binding assay could be obscured by the fact that the protein was produced in insect cells and lipid binding occurs during the producing. What is the evidence that added plants calli lipids can replace lipids already present during isolation.

    Actually we don’t really know whether the insect cells lipids initially bound to AtVPS13M1(1-335) are replaced by calli lipids or whether they bound to still available lipid binding sites on the protein. But we have two main lines of evidence showing that our purified protein can bind plant lipids even in the presence of insect cells lipids: 1) our protein can bind SQDG and MGDG, two plants specific lipids, and 2) as explained p.8 (lines 243-254), lipids coming from both organisms have a specific acyl-chain composition, with insect cells fatty acids mainly composed of C16 and C18 with 0 or 1 unsaturation whereas plant lipids can have up to 3 unsaturations. By analyzing and presenting on the histograms lipid species from insect cells, calli and those bound to AtVPS13M1(1-335), we were able to conclude that for all the lipid classes besides PS, a wide range of lipid species deriving from both organisms was bound to our protein. The data about the lipid species bound to AtVPS13M1(1-335) are presented in Figure 2E and S2.

    The effects on lipid composition of the mutants are not very drastic from what I can tell. Furthermore, how does this fit with the lipid composition of mitochondria where the protein appears to be mostly located?

    It is true that lipid composition variations in the mutants are not drastic but still statistically significant. As a general point in the field of lipid transfer, it is not very common to have major changes in total lipidome on single mutants of lipid transfer proteins because of a high redundancy of lipid transport pathway in cells. This is particularly true for VPS13 proteins, as exemplified by multiple studies. Major lipid phenotypes can be revealed in specific conditions, such as phosphate starvation in our case, or when looking at specific organelles or specific tissues and/or developmental stages. This is explained and illustrated by examples in the discussion part p. 16 (line 526-532). In addition, as suggested by Reviewer 3, we performed further lipid analysis on calli and also on rosettes under Pi starvation and found a similar trend (Figure 4 and S4 of the revised version of the manuscript). Thus, we believe that, even if not drastic, these variations during Pi starvation are a real phenotype of our mutants.

    As we found that our protein is located at the mitochondrial surface, we agree that Reviewer 1’s suggestion to perform lipidomic analyses on isolated mitochondria will be of high interest but this will be the scope of future studies that we will performed in our lab. First, we would like to identify all the organelles at which AtVPS13M1 is localized before performing subfractionations of these different organelles from the same pool of cell cultures grown in presence or absence of phosphate.

    For the localization of the fusion protein, has it been tested whether the furoin is functional? This should be tested (e.g. by reversion of lipid composition).

    As we did not observe major developmental phenotypes in our mutants, complementation should be indeed tested by performing lipidomic analyses in calli or plants grown in presence or absence of Pi, which is a time-consuming and expensive experiment. Because we used the fusions mainly for tissue expression study and subcellular localization and not for functional analyses, we believe that this is not an essential control to be performed for this work.

    It is speculated that different splice forms are located to different compartments. Can that be tested and used to explain the observed subcellular location patterns?

    Indeed some splice forms can modify the sequence of domains putatively involved in protein localization. This could be tested by producing synthetic constructs with one specific exon organization, which is challenging according to the size of AtVPS13M1 cDNA (around 12kb). In addition, our long-read sequencing experiment and PCR analyses revealed the existence of six transcripts, a major one representing around 92% and the five others representing less than 2.5% (Figure 1D). Among the five less abundant transcripts, four produce proteins with a premature stop codon and are likely to arise from splicing defects as explained in the discussion part p. 15 (lines 488-496). One produces a full-length protein with an additional loop in the VAB domain but because of the low abundance of this alternative transcript (1.4%), we believe it does not contribute significantly to the major localization we observed in plants and did not attend to analyze its localization.

    GUS fusion data only probe promoter activity but not all levels of gene expression. That caveat should be discussed.

    We are aware of this drawback and that is the reason why we fused the GUS enzyme directly to our protein expressed under its native locus (i.e. with endogenous promoter and exons/introns) as depicted in Figure 5A. Therefore, our construction allows to assess directly AtVPS13M1 protein level in plant tissues.

    Minor points:

    1. Extraplastidic DGDG and export from chloroplasts following phosphate derivation was first reported in PMID: 10973486.

    We added this reference in the text.

    Check throughout the correct usage of gene expression as genes are expressed and proteins produced.

    Many thanks for this remark, we modified the text accordingly

    In general, the paper is too long. Redundancies between introduction, results and discussion should be removed to streamline.

    We reduced the text to avoid redundancy.

    I suggest to redraw the excel graphs to increase line thickness and enlarge font size to increase presentation and readability.

    We tried as much as we can to enlarge graphs and font size increasing readability.

    Reviewer #1 (Significance (Required)):

    Significance: Interorganellar lipid trafficking is an important topic and especially under studied in plants. Identifying components involved represents significant progress in the field. Similarly, lipid remodeling following phosphate derivation is an important phenomenon and the current advances our understanding.

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

    Summary: The manuscript "AtVPS13M1 is involved in lipid remodelling in low phosphate and is located at the mitochondria surface in plants" by Leterme et al. identifies the protein VPS13M1 as a lipid transporter in Arabidopsis thaliana with important functions during phosphate starvation. The researchers were able to localise this protein to mitochondria via GFP-targeting in Arabidopsis. Although VPS13 proteins are well described in yeast and mammals, highlighting their importance in many vital cellular processes, there is very little information on them in plants. This manuscript provides new insights into plant VPS13 proteins and contributes to a better understanding of these proteins and their role in abiotic stress responses, such as phosphate starvation.

    Major points:

    • Please describe and define the domains of the VPS13M1 protein in detail, providing also a figure for that. Figure 1 is mainly describing possible splice variants, whereas the characteristics of the protein are missing.

    We have added information on AtVPS13M1 domain organization in the introduction (p.4, lines 103-109) and referred to Figure 1A that described protein domain organization. We did not added too much details as plant VPS13 protein domains organization was extensively described in two previous studies cited several times in the manuscript (Leterme et al., 2023; Levine, 2022).

    • Please compare the expression level of VPS13M1 in the presence and in the absence of phosphate.

    Many thanks for this suggestion. We performed qRT-PCR analyses of AtVPS13M1 from mRNA extracted from calli grown six days in presence and absence of phosphate. The results obtained did not reveal variations in mRNA level. The results were added in Figure S1A of the revised version of the manuscript and discussed in p.5 (lines 154-156).

    • Page 9, second paragraph: Here, the lipid transport capability of AtVPS13M1 is described. Varying concentrations of this recombinant protein should be used in this test. Further, it is not highlighted, that a truncated version of VSP13M1 is able to transport lipids. This is surprising, since this truncated version is less than 10% of the total protein (only aa 1-335).

    We agree with reviewer 2 that increasing protein concentration is an important control to perform. We included an experiment with an increasing quantity of protein (2X and 4X) in the revised version of the manuscript and showed that the signal intensity increased faster when protein concentration is higher (Figure 3D of the revised manuscript). As requested by Reviewer 3, we also included a negative control with Tom20.3 to show that the signal increase after the addition of AtVPS13M1(1-335) is specific to this protein (Figure 3C of the revised manuscript).

    The transport ability of the N-terminal part of VPS13 was demonstrated in yeast and mammals VPS13D (Kumar et al., 2018; Wang et al., 2021). We highlighted this p. 7 (lines 213-218) of the revised version of the manuscript. This is explained by the inherent structure of VPS13 proteins that are composed of several repeats of the same domain type called RBG (for repeating β-groove), each forming a β-sheet with a hydrophobic surface. The higher the number of RBG repeats, the longer the hydrophobic tunnel is. The (1-335) N-terminal region corresponds to two RBG unit repeats forming a “small” tunnel able to bind and transfer lipids. The number of RBG repeats has influence on the quantity of lipids bound per protein in vitro, the longest the protein is, the highest the number of lipid molecules bound is (Kumar et al., 2018), but the effect on protein length on in vitro lipid transfer capacity has not been investigated yet to the best of our knowledge.

    • Also, for phenotype analysis, T-DNA insertion mutants are used that still contain VPS13M1 transcripts. Although protein fragments where not detected by proteomic analysis, this might be due to low sensitivity of the proteomic assay. Further the lipid transport domain of VPS13M1 (aa 1-335) might not be affected by the T-DNA insertions at all. Here more detailed analysis needs to be done to prove that indeed loss-of protein function occurs in the mutants.

    We do not have other methods than proteomic to test whether our mutants are KO or not. We tried unsuccessfully to produce antibodies. Mass spectrometry is the most sensitive method but the absence of detection indeed does not mean the absence of the protein. From proteomic data, we can conclude that at least, our mutants present a decrease in AtVPS13M1 protein level, thus we called them “knock down” in the revised version of the manuscript and added the following sentence p. 9 (lines 297-300): “As the absence of detection of a protein by mass spectrometry-based proteomics does not allow us to strictly claim the absence of this protein in the sample, we concluded that AtVPS13M1 expression in both atvps13m1-1 and atvps13m1-4 was below the detection limit and consider them as knock down (KD) for AtVPS13M1.”

    • Localisation in mitochondria: As the Yepet signal is very weak, a control image of not transfected plant tissue needs to be included. Otherwise, it might be hard to distinguish the Yepet signal from background signal. The localisation data presented in Figure 5 does not allow the conclusion that VPS13M1 is localized at the surface of mitochondria as stated in the title. It only indicates (provided respective controls see above) that VPS13M1 is in mitochondria. Please provide more detailed analysis such as targeting to tobacco protoplasts, immunoblots or in vitro protein import assays. Also test +Pi vs. -Pi to see if VPS13M1 localisation is altered in dependence of Pi.

    Indeed our Yepet signal is not very strong but on the experiments we performed on Col0 non-transformed plants, we did not very often see fluorescence background in the leaves’ vascular tissue, that is why we focused our study on this tissue. We sometimes observed some background signals in some cells that are clearly different from AtVPS13M1-3xYepet signals and never co-localized with mitochondria. Examples of these aspecific signals are presented in Figure S6E of the revised version of the manuscript.

    We agree with reviewer 2 that our confocal images suggested, but not demonstrated, a localization at the surface of mitochondria. To confirm the localization, we generated calli cell cultures from AtVPS13M1-3xYepet lines and performed subcellular fractionations and western blot analyses confirming that AtVPS13M1 was indeed enriched in mitochondria and also in microsomal fractions (Figure 6G of the revised version). Then we performed mild proteolytic digestion of the isolated mitochondria with thermolysin and show that AtVPS13M1 was degraded, as the outer membrane protein Tom20.3, but not the inner membrane protein AtMic60, showing that AtVPS13M1 is indeed at the surface of mitochondria (Figure 5H of the revised manuscript). We believe that this experiment, in addition to the confocal images showing a signal around mitochondria, convincingly demonstrates that AtVPS13M1 is located at the surface of mitochondria.

    The localization of AtVPS13M1 under Pi starvation is a very important question that we tried to investigate without success. Indeed, we intended to perform confocal imaging on seedlings grown in liquid media to easily perform Pi starvation as described for the analysis of AtVPS13M1 tissue expression with β-glucuronidase constructs. However, the level of fluorescence background was very high in seedlings and no clear differences between non-transformed and AtVPS13M1-3xYepet lines were observed, even in root tips where the protein is supposed to be the most highly expressed according to β-glucuronidase assays. Example of images obtained are presented in Figure R1. We concluded that the level of expression of our construct was too low in seedlings. The constructions of lines with a higher AtVPS13M1 expression level, by changing the promotor, to better analyze AtVPS13M1 in different tissues or in response to Pi starvation will be the scope of future work in our laboratory in order to investigate AtVPS13M1 localization under low Pi.

    Phenotype analysis needs to be done under Pi stress and not under cold stress! Further, root architecture and root growth should also be done under Pi depletion. Here the title is also misleading, it is not at all clear why the authors switch from phosphate starvation to cold stress.

    In the revised version of the manuscript, we analyzed the seedlings root growth of two mutants (atvps13m1-3 and m1-4) under low Pi and did not notice significant differences (Figure 7E, S7D of the revised version). We analyzed growth under cold stress because this stress also promotes remodeling of lipids, but we agree that it goes beyond the scope of this article that is focused on Pi starvation and we removed this part from the revised manuscript.

    Minor points: Page 3, line 1: what does the abbreviation VPS stand for?

    The definition of VPS (Vacuolar Protein Sorting) was added.

    Page 3, line 1: change "amino acids residues" to "amino acid residues"

    This was done.

    Page 3, line 8 - 12: please rewrite this sentence. You write, that because of their distribution VPS13 proteins do exhibit many important physiological roles. The opposite is true: They are widely distributed in the cell because of their involvement in many physiological processes.

    We changed the sentence to “ VPS13 proteins localize to a wide variety of membranes and membrane contact sites (MCSs) in yeast and human (Dziurdzik and Conibear, 2021). This broad distribution on different organelles and MCSs is important to sustain their important roles in numerous cellular and organellar processes such as meiosis and sporulation, maintenance of actin skeleton and cell morphology, mitochondrial function, regulation of cellular phosphatidylinositol phosphates level and biogenesis of autophagosome and acrosome (Dziurdzik and Conibear, 2021; Hanna et al., 2023; Leonzino et al., 2021).”

    Page 6, line6: change "cDNA obtained from A. thaliana" to "cDNA generated from A. thaliana.

    This was done.

    Page 6, line 10: change" 7.6kb" to "7.6 kb"

    This was done.

    Page 7: address this question: can the isoforms form functional VPS13 proteins? This might help to postulate whether these isoforms are a result of defective splicing events.

    We addressed this aspect in the discussion p.15 at lines 486-502.

    Figure 2 B: Change "AtVPS13M1"to "AtVPS13M1(1-335)"

    This was done.

    Figure 2, legend: -put a blank before µM in each case.

    This was done.

    -Change 0,125µM to 0.125 µM

    This was done.

    -what does "in absence (A-0µM)" mean?

    This means that the Acceptor liposomes are at 0 µM. To clarify, we changed it to “Acceptor 0 µM” in the revised version of the manuscript (Figure 3C).

    -Which statistical analysis was employed?

    We performed a non-parametric Mann-Whitney test in the revised version of the manuscript. This was indicated in the legend.

    -Further, rewrite the sentence "Mass spectrometry (MS) analysis of lipids bound to AtVPS13M1(1-335) or Tom20 (negative control) after incubation with calli total lipids. Results are expresses in nmol of lipids per nmol of proteins (C) or in mol% (D)". -"C" and "D" are not directly comparable, as in "C" no Tom20 was used and in "C" no insect cells were used.

    -Further, in "D" the experimental setup is not clear. AtVPS13(1-335) is supposed to be purified protein after incubation with calli lipids (figure 2, A). Further, in the same figure, lipid composition of "insect cells" and "calli-Pi" are compared àwhy? Please clarify this.

    C and D are two different representations of the same results providing different types of information. In C., the results are expressed in nmol of lipids / nmol of proteins to assess 1) that the level of lipids found in AtVPS13M1(1-335) purifications is significantly higher than what we can expect from the background (assessed using Tom20) and 2) what are the classes of lipids that associate or not to AtVPS13M1(1-335). In D. the lipid distribution in mol% is presented for AtVPS13M1(1-335) as well as for total extracts from calli and insect cells to be able to compare if one lipid class is particularly enriched or not in AtVPS13M1(1-335) purifications compared to the initial extracts with which the protein was incubated. As an example, it allows to deduce that the absence of DGDG detected in the AtVPS13M1(1-335) purifications is not linked to a low level of DGDG in the calli extract, because it represented around 15 mol%, but likely to a weak affinity of the protein for this lipid. We did not represent the Tom20 lipid distribution on this graph because it represents background of lipid binding to the purification column and might suggest that Tom20 binds lipids. We changed the legend in this way and hope that it is clearer now: “C-D. Mass spectrometry (MS) analysis of lipids bound to AtVPS13M1(1-335) or Tom20 (negative control) after incubation with calli total lipids and repurification. Results are expresses in nmol of lipids per nmol of proteins in order to analyze the absolute quantity of the different lipid classes bound to AtVPS13M1(1-335) compared to Tom20 negative control (C), and in mol% to assess the global distribution of lipid classes in AtVPS13M1(1-335) purifications compared to the total lipid extract of insect cells and calli (D).”

    Figure 3: -t-test requires a normal distribution of the data. This is not possible for an n=3. Please use an adequate analysis.

    We performed more replicates and used non-parametric Mann-Whitney analyses in the revised version of the manuscript.

    -Please clarify the meaning of the letters on the top of the bars in the legend.

    This corresponded to the significance of t-tests performed in the first version of the manuscript that were reported in Table S3. As in the new version we performed Mann-Whitney tests, we highlighted the significance by stars and in the figure legends.

    Please, make it clear that two figures belong to C.

    This was clarified in the legend.

    -Reorganise the order of figure 3 (AàBàCàD)

    Because of the configuration of the different histograms presented in the figure, we were not able to change the order but we believed that the graphs can be easily red this way.

    Page 10, 3. Paragraph: since the finding, that no peptides were found in the VSP13M1 ko lines, although transcription was not altered, is surprising, please include the proteomic data in the supplement

    Proteomic data were deposited on PRIDE with the identifier PXD052019. They will remain not publicly accessible until the acceptance of the manuscript.

    Page 11, line 17: The in vitro experiments showed a low affinity of VSP13M1 towards galactolipids. It is further claimed that this is consistent with the finding of the AtVSP13M1 Ko line in vivo, that in absence of PI, no change in DGDG content could be observed. However, the "absence" of VSP13M1 in vivo might still result in a bigger VSP13M1 protein, than the truncated form (1-335) used for the in vitro experiments

    It is true that our in vitro experiments were performed only with a portion of AtVPS13M1 and that the length of the protein could influence protein binding specificity. We removed this assessment from the manuscript.

    Page 13, lane 8: you should reconsider the use of a triple Yepet tag: If two or more identical fluorescent molecules are in close proximity, their fluorescence emission is quenched, which results in a weak signal (as the one that you obtained). See: Zhuang et al. 2000 (PNAS) Fluorescence quenching: A tool for single-molecule protein-folding study

    Many thanks to point this paper. We use a triple Yepet because AtVPS13M1 has a very low level of expression and because this strategy was used successfully to visualize proteins for which the signal was below the detection level with a single GFP (Zhou et al., 2011). The quenching of the 3xYepet might also depend on the conformation they adopt on the targeting protein.

    Page 13, line 14: change 1µm to 1 µm

    This was done.

    Page 13, line 29: please reduce the sentence to the first part: if A does not colocalize with B, it is not necessary to mention that B does not colocalise with A.

    The sentence was modified accordingly.

    Page 14, 2. Paragraph: it is not conclusive that phenotype analysis is suddenly conducted with plants under cold stress, since everything was about Pi-starvation and the role of VSP13M1. Lipid remodelling under Pi stress completely differs from the lipid remodelling under cold stress.

    We eliminated this part in the revised version of the manuscript.

    Page 14, line 20: change figure to Figure

    This was done.

    Page 07, line 17: change artifact to artefact

    This was done.

    Reviewer #2 (Significance (Required)):

    General assessment: The paper is well written and technically sound. However, some points could be identified, that definitely need a revision. Overall, we got the impression that so far, the data gathered are still quite preliminary and need some more detailed investigations prior to publication (see major points).

    Advance: The study definitely fills a gap of knowledge since not much is known on the function of plant VPS13 proteins so far.

    Audience: The study is of very high interest to the plant lipid community but as well of general interest for Plant Molecular Biology and intracellular transport.

    Our expertise: Plant membrane transport and lipid homeostasis.

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

    The manuscript by Leterme et al. (2024) describes the characterization of VPS13M1 from Arabidopsis. VPS13 proteins have been analyzed in yeast and animals, where they establish lipid transfer connections between organelles, but not much is known about VPS13 proteins in plants. First, different splicing forms were characterized, and the form A was identified as the most relevant one with 92% of the transcripts. The protein (just N-terminal 335 amino acids out of ca. 3000 amino acids) was expressed in insect cells and purified. Next, the protein was used for lipid binding assays with NBD-labeled lipids followed by analysis in polyacrylamide gel electrophoresis. VPS13M1 bound to PC, PE, PS and PA. Then, the protein from insect cells was incubated with Arabidopsis callus lipids, and lipids bound to VPS13M1 analyzed by LC-MS/MS. Lipid transfer between liposomes was measured by the change in fluorescence in donor liposomes derived from two labeled lipids after addition of the protein caused by lipid transfer and dilution to acceptor liposomes. T-DNA insertion mutants were isolated and the lipids measured in callus derived from these mutants. Protein localization in different plant organs was recorded with a GUS fusion construct transferred into transgenic plants. The protein was localized to mitochondria using a VPS13M1-Yepet fusion construct transferred into mutant plants. The mutant plants show no visible difference to wild type, even when the plants were grown under stress conditions like low temperature. The main message of the title is that VPS13M1 localizes to the mitochondria which is well documented, and it is involved in lipid remodeling under low phosphate conditions.

    The lipid transfer assay shown in Figure 2F lacks a negative control. This would be the experiment with donor and acceptor liposomes in the presence of another protein like Tom20.

    Many thanks for this suggestion. In the revised version of the manuscript, we performed a fluorescent lipid transport assay with Tom20.3 in the presence of 25 µM of donor liposomes and 1.5 mM of acceptor liposomes, the condition for which we observed a maximum of transport for AtVPS13M1(1-335). As expected, no fluorescence increase was observed. The results are presented in the Figure 3C of the revised manuscript.

    The lipid data (Fig. 3 and Fig. S4) do not sufficiently support the second claim, i.e. that the protein is involved in lipid remodeling under low P. Data in Fig. 3C are derived from only 3 replicates and in Fig. S4 from only 2 replicas with considerable error bars. Having only 2 replicates is definitely not sufficient. Fig. 3C shows a suppression in the decrease in PE and PC at 4 d of P deprivation (significant for two mutants for PE, for only one for PC). Fig. S4A shows suppression of the decrease in PC at 6 d after P deprivation (significant for both mutants), but no significant effect on PE. Fig. 4SB shows no significant change in PE or PC at -P after 8 d of P deprivation. The data are not consistent. There are also problems with the statistics in Fig. 3 and Fig. S4. The authors used T-test, but place letters a, b, c on top of the bars. Usually, asterisks should be used to indicate significant differences. Data indicate medians and ranges, not mean and SD. In Fig. S4, how can you indicate median and range if you have only 2 replicates? Why did the authors use callus for lipid measurements? Why not use leaves and root tissues? What does adjusted nmol mean? What does the dashed line at 1.05 on the y axis mean? Taken together, I suggest to repeat lipid measurements with leaves and roots from plantets grown under +P and -P conditions in tissue culture with 5 replcates. Significant differences can be analyzed on the level of absolute (nmol per mg FW/DW) or relative (%) amounts.

    Here are our answers to concerns about the design of our lipidomics experiments:

    We used calli for lipid measurement because it is very easy to control growth conditions and to performed phosphate starvation from this cell cultures. The second reason is that it is a non-photosynthetic tissue with a high level of phospholipids and a low level of galactoglycerolipids and it is easier to monitor the modification of the balance phospholipids/galactoglycerolipids in this system. The lipid analysis on calli at 4 days of growth in presence or absence of Pi were performed on 3 biological replicates but on two different mutants (atvps13m-1 and m1-3) and we drew our conclusions based on variations that were significant for both mutants. In the revised version of the manuscript, we performed further lipidomic analyses on calli from Col0 and another mutant (atvps13m1-2) after 6 days of growth in presence or absence of Pi (Figure 4E, S4A-C, n=4-5) and added new data on a photosynthetic tissue (rosettes) from Col0 and atvps13m1-3 mutant. For rosettes analysis, seeds were germinated 4 days in plates with 1 mM Pi and then transferred on plates with 1 mM or 5 µM of Pi. Rosettes were harvested and lipids analyzed after 6 days (Figure 4F-G, S4D, n=4-5). All the data were represented with medians and ranges because we believe that median is less sensitive to extreme values than mean and might better represent what is occurring. Ranges highlight the minimal and maximal value of the data analyzed and we believe it is a representative view of the variability we obtained between biological samples.

    Lipid measurement are done by mass spectrometry. As it was already reported, mass spectrometry quantification is not trivial as the intensity of the response depends on the nature of the molecule (for a review, see (Jouhet et al., 2024)). To counteract this ionisation problem, we developed a method with an external standard that we called Quantified Control (QC) corresponding to an A. thaliana callus lipid extract for which the precised lipid composition was determined by TLC and GC-FID. All our MS signals were “adjusted” to the signal of this QC as described in (Jouhet et al., 2017). Therefore our lipid measurement are in adjusted nmol. In material and method we modified the sentence accordingly p22 lines 720-723: “Lipid amounts (pmol) were adjusted for response differences between internal standards and endogenous lipids and by comparison with a quality control (QC).” This allows to represent all the lipid classes on a same graph and to have an estimation of the lipid classes distribution. To assess the significance of our results, we used in the revised version of the manuscript non-parametric Mann-Whitney tests and added stars representing the p-value on charts. This was indicated in the figure legends.

    Here are our answers to concerns about the interpretation of our lipidomics experiments:

    To summarize, in the revised version of the manuscript, lipid analyses were performed in calli from 3 different mutants (two at day 4, one at day 6) and in the rosettes from one of these mutants. All the results are presented in Figure 4 and S4. In all the experiments, we found that in +Pi, there is no major modifications in the lipid content or composition. In –Pi, we found that the total glycerolipid content is always higher in the mutant compared to the Col0, whatever the tissue or mutant considered (Figure 4A and S4A, D). In calli, this higher increase in lipid content is mainly due to an accumulation of phospholipids and in rosettes, of galactolipids. Because of high variability between our biological replicates, we did not always found significant differences in the absolute amount of lipids in –Pi. However, the analysis of the fold change in lipid content in –Pi vs +Pi always pointed toward a reduced extent of phospholipid degradation. We also added in these graphs the fold change for the total phospholipids and total galactolipids contents in the revised version of the manuscript. We believe that the new analyses we performed strengthen our conclusion about the role of AtVPS13M1 in phospholipid degradation and not on the recycling of precursors backbone to feed galactoglycerolipids synthesis at the chloroplast envelope.

    Page 9, line 15: Please use the standard form of abbreviations of lipid molecular species with colon, e.g. PC32:0, not PC32-0

    The lipid species nomenclature has been changed accordingly.

    Page 11, line 4, (atvps13m1.1 and m1.3: please indicate the existence of mutant alleles with dashes, i.e. (atvps13m1-1 and atvps13m1-3

    Names of the mutants have been changed accordingly.

    Page 14, line 21: which line is indicated by atvps13m1.2-4? What does -4 indicate here?

    This indicates that mutants m1-2 to m1-4 were analyzed.

    Page 16, line 25: many abbreviations used here are very specific and not well known to the general audience e.g. ONT, IR, PTC, NMD etc. I think it is OK to mention them here, but still use the full terms, given that they are not used very frequently in the manuscript.

    We kept ONT abbreviation because it was cited many times in both the results and discussion part. IR, PTC and NMD were cited only in the discussion and were eliminated.

    Page 19, line 11. The authors cite Hsueh et al and Yang et al for LPTD1 playing a role in lipid homeostasis during P deficiency. But Yang et al. described the function of a SEC14 protein in Arabidopsis and rice during P deficiency. Is SEC14 related to LPTD1?

    Many thanks for noticing this mistake. We removed the citation Yang et al. in the revised version of the manuscript.

    Reference Tangpranomkorn et al. 2022: In the text, it says that this is a preprint, but in the Reference list, this is indicated with "Plant Biology" as Journal. In the internet, I could only find this manuscript in bioRxiv.

    This manuscript was accepted in “New Phytologist” in December 2024 and is now cited accordingly in the new version of the manuscript.

    Reviewer #3 (Significance (Required)):

    The manuscript by Leterme et al describes the characterization of the lipid binding and transport protein VTPS13M1 from Arabidopsis. I think that the liposome assay needs to be done with a negative control. Furthermore, I have major concerns with the lipid data in Fig. 3C and Fig. S4. These lipid data of the current manuscript need to be redone. I do not agree that the lipid data allow the conclusion that "AtVPS13M1 is involved in lipid remodeling in low phosphate" as stated in the title.

    References cited in this document:

    Dziurdzik, S.K., and E. Conibear. 2021. The Vps13 Family of Lipid Transporters and Its Role at Membrane Contact Sites. Int J Mol Sci. 22:2905. doi:10.3390/ijms22062905.

    Hanna, M., A. Guillén-Samander, and P. De Camilli. 2023. RBG Motif Bridge-Like Lipid Transport Proteins: Structure, Functions, and Open Questions. Annu Rev Cell Dev Biol. 39:409–434. doi:10.1146/annurev-cellbio-120420-014634.

    Hanna, M.G., P.H. Suen, Y. Wu, K.M. Reinisch, and P. De Camilli. 2022. SHIP164 is a chorein motif lipid transfer protein that controls endosome–Golgi membrane traffic. Journal of Cell Biology. 221:e202111018. doi:10.1083/jcb.202111018.

    Jouhet, J., E. Alves, Y. Boutté, S. Darnet, F. Domergue, T. Durand, P. Fischer, L. Fouillen, M. Grube, J. Joubès, U. Kalnenieks, J.M. Kargul, I. Khozin-Goldberg, C. Leblanc, S. Letsiou, J. Lupette, G.V. Markov, I. Medina, T. Melo, P. Mojzeš, S. Momchilova, S. Mongrand, A.S.P. Moreira, B.B. Neves, C. Oger, F. Rey, S. Santaeufemia, H. Schaller, G. Schleyer, Z. Tietel, G. Zammit, C. Ziv, and R. Domingues. 2024. Plant and algal lipidomes: Analysis, composition, and their societal significance. Progress in Lipid Research. 96:101290. doi:10.1016/j.plipres.2024.101290.

    Jouhet, J., J. Lupette, O. Clerc, L. Magneschi, M. Bedhomme, S. Collin, S. Roy, E. Maréchal, and F. Rébeillé. 2017. LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation. PLoS ONE. 12:e0182423. doi:10.1371/journal.pone.0182423.

    Kumar, N., M. Leonzino, W. Hancock-Cerutti, F.A. Horenkamp, P. Li, J.A. Lees, H. Wheeler, K.M. Reinisch, and P. De Camilli. 2018. VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites. J Cell Biol. 217:3625–3639. doi:10.1083/jcb.201807019.

    Leonzino, M., K.M. Reinisch, and P. De Camilli. 2021. Insights into VPS13 properties and function reveal a new mechanism of eukaryotic lipid transport. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1866:159003. doi:10.1016/j.bbalip.2021.159003.

    Leterme, S., O. Bastien, R.A. Cigliano, A. Amato, and M. Michaud. 2023. Phylogenetic and Structural Analyses of VPS13 Proteins in Archaeplastida Reveal Their Complex Evolutionary History in Viridiplantae. Contact (Thousand Oaks). 6:1–23. doi:10.1177/25152564231211976.

    Levine, T.P. 2022. Sequence Analysis and Structural Predictions of Lipid Transfer Bridges in the Repeating Beta Groove (RBG) Superfamily Reveal Past and Present Domain Variations Affecting Form, Function and Interactions of VPS13, ATG2, SHIP164, Hobbit and Tweek. Contact. 5:251525642211343. doi:10.1177/25152564221134328.

    Valverde, D.P., S. Yu, V. Boggavarapu, N. Kumar, J.A. Lees, T. Walz, K.M. Reinisch, and T.J. Melia. 2019. ATG2 transports lipids to promote autophagosome biogenesis. J Cell Biol. 218:1787–1798. doi:10.1083/jcb.201811139.

    Wang, J., N. Fang, J. Xiong, Y. Du, Y. Cao, and W.-K. Ji. 2021. An ESCRT-dependent step in fatty acid transfer from lipid droplets to mitochondria through VPS13D−TSG101 interactions. Nat Commun. 12:1252. doi:10.1038/s41467-021-21525-5.

    Zhou, R., L.M. Benavente, A.N. Stepanova, and J.M. Alonso. 2011. A recombineering-based gene tagging system for Arabidopsis. Plant J. 66:712–723. doi:10.1111/j.1365-313X.2011.04524.x.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Leterme et al. (2024) describes the characterization of VPS13M1 from Arabidopsis. VPS13 proteins have been analyzed in yeast and animals, where they establish lipid transfer connections between organelles, but not much is known about VPS13 proteins in plants. First, different splicing forms were characterized, and the form A was identified as the most relevant one with 92% of the transcripts. The protein (just N-terminal 335 amino acids out of ca. 3000 amino acids) was expressed in insect cells and purified. Next, the protein was used for lipid binding assays with NBD-labeled lipids followed by analysis in polyacrylamide gel electrophoresis. VPS13M1 bound to PC, PE, PS and PA. Then, the protein from insect cells was incubated with Arabidopsis callus lipids, and lipids bound to VPS13M1 analyzed by LC-MS/MS. Lipid transfer between liposomes was measured by the change in fluorescence in donor liposomes derived from two labeled lipids after addition of the protein caused by lipid transfer and dilution to acceptor liposomes. T-DNA insertion mutants were isolated and the lipids measured in callus derived from these mutants. Protein localization in different plant organs was recorded with a GUS fusion construct transferred into transgenic plants. The protein was localized to mitochondria using a VPS13M1-Yepet fusion construct transferred into mutant plants. The mutant plants show no visible difference to wild type, even when the plants were grown under stress conditions like low temperature. The main message of the title is that VPS13M1 localizes to the mitochondria which is well documented, and it is involved in lipid remodeling under low phosphate conditions. The lipid transfer assay shown in Figure 2F lacks a negative control. This would be the experiment with donor and acceptor liposomes in the presence of another protein like Tom20. The lipid data (Fig. 3 and Fig. S4) do not sufficiently support the second claim, i.e. that the protein is involved in lipid remodeling under low P. Data in Fig. 3C are derived from only 3 replicates and in Fig. S4 from only 2 replicas with considerable error bars. Having only 2 replicates is definitely not sufficient. Fig. 3C shows a suppression in the decrease in PE and PC at 4 d of P deprivation (significant for two mutants for PE, for only one for PC). Fig. S4A shows suppression of the decrease in PC at 6 d after P deprivation (significant for both mutants), but no significant effect on PE. Fig. 4SB shows no significant change in PE or PC at -P after 8 d of P deprivation. The data are not consistent. There are also problems with the statistics in Fig. 3 and Fig. S4. The authors used T-test, but place letters a, b, c on top of the bars. Usually, asterisks should be used to indicate significant differences. Data indicate medians and ranges, not mean and SD. In Fig. S4, how can you indicate median and range if you have only 2 replicates? Why did the authors use callus for lipid measurements? Why not use leaves and root tissues? What does adjusted nmol mean? What does the dashed line at 1.05 on the y axis mean? Taken together, I suggest to repeat lipid measurements with leaves and roots from plantets grown under +P and -P conditions in tissue culture with 5 replcates. Significant differences can be analyzed on the level of absolute (nmol per mg FW/DW) or relative (%) amounts. Page 9, line 15: Please use the standard form of abbreviations of lipid molecular species with colon, e.g. PC32:0, not PC32-0 Page 11, line 4, (atvps13m1.1 and m1.3: please indicate the existence of mutant alleles with dashes, i.e. (atvps13m1-1 and atvps13m1-3

    Page 14, line 21: which line is indicated by atvps13m1.2-4? What does -4 indicate here? Page 16, line 25: many abbreviations used here are very specific and not well known to the general audience e.g. ONT, IR, PTC, NMD etc. I think it is OK to mention them here, but still use the full terms, given that they are not used very frequently in the manuscript. Page 19, line 11. The authors cite Hsueh et al and Yang et al for LPTD1 playing a role in lipid homeostasis during P deficiency. But Yang et al. described the function of a SEC14 protein in Arabidopsis and rice during P deficiency. Is SEC14 related to LPTD1? Reference Tangpranomkorn et al. 2022: In the text, it says that this is a preprint, but in the Reference list, this is indicated with "Plant Biology" as Journal. In the internet, I could only find this manuscript in bioRxiv.

    Significance

    The manuscript by Leterme et al describes the characterization of the lipid binding and transport protein VTPS13M1 from Arabidopsis. I think that the liposome assay needs to be done with a negative control. Furthermore, I have major concerns with the lipid data in Fig. 3C and Fig. S4. These lipid data of the current manuscript need to be redone. I do not agree that the lipid data allow the conclusion that "AtVPS13M1 is involved in lipid remodeling in low phosphate" as stated in the title.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The manuscript "AtVPS13M1 is involved in lipid remodelling in low phosphate and is located at the mitochondria surface in plants" by Leterme et al. identifies the protein VPS13M1 as a lipid transporter in Arabidopsis thaliana with important functions during phosphate starvation. The researchers were able to localise this protein to mitochondria via GFP-targeting in Arabidopsis. Although VPS13 proteins are well described in yeast and mammals, highlighting their importance in many vital cellular processes, there is very little information on them in plants. This manuscript provides new insights into plant VPS13 proteins and contributes to a better understanding of these proteins and their role in abiotic stress responses, such as phosphate starvation.

    Major points:

    • Please describe and define the domains of the VPS13M1 protein in detail, providing also a figure for that. Figure 1 is mainly describing possible splice variants, whereas the characteristics of the protein are missing.
    • Please compare the expression level of VPS13M1 in the presence and in the absence of phosphate.
    • Page 9, second paragraph: Here, the lipid transport capability of AtVPS13M1 is described. Varying concentrations of this recombinant protein should be used in this test. Further, it is not highlighted, that a truncated version of VSP13M1 is able to transport lipids. This is surprising, since this truncated version is less than 10% of the total protein (only aa 1-335).
    • Also, for phenotype analysis, T-DNA insertion mutants are used that still contain VPS13M1 transcripts. Although protein fragments where not detected by proteomic analysis, this might be due to low sensitivity of the proteomic assay. Further the lipid transport domain of VPS13M1 (aa 1-335) might not be affected by the T-DNA insertions at all. Here more detailed analysis needs to be done to prove that indeed loss-of protein function occurs in the mutants.
    • Localisation in mitochondria: As the Yepet signal is very weak, a control image of not transfected plant tissue needs to be included. Otherwise, it might be hard to distinguish the Yepet signal from background signal. The localisation data presented in Figure 5 does not allow the conclusion that VPS13M1 is localized at the surface of mitochondria as stated in the title. It only indicates (provided respective controls see above) that VPS13M1 is in mitochondria. Please provide more detailed analysis such as targeting to tobacco protoplasts, immunoblots or in vitro protein import assays. Also test +Pi vs. -Pi to see if VPS13M1 localisation is altered in dependence of Pi.
    • Phenotype analysis needs to be done under Pi stress and not under cold stress! Further, root architecture and root growth should also be done under Pi depletion. Here the title is also misleading, it is not at all clear why the authors switch from phosphate starvation to cold stress.

    Minor points:

    Page 3, line 1: what does the abbreviation VPS stand for?

    Page 3, line 1: change "amino acids residues" to "amino acid residues"

    Page 3, line 8 - 12: please rewrite this sentence. You write, that because of their distribution VPS13 proteins do exhibit many important physiological roles. The opposite is true: They are widely distributed in the cell because of their involvement in many physiological processes.

    Page 6, line6: change "cDNA obtained from A. thaliana" to "cDNA generated from A. thaliana.

    Page 6, line 10: change" 7.6kb" to "7.6 kb"

    Page 7: address this question: can the isoforms form functional VPS13 proteins? This might help to postulate whether these isoforms are a result of defective splicing events.

    Figure 2 B: Change "AtVPS13M1"to "AtVPS13M1(1-335)"

    Figure 2, legend:

    • put a blank before µM in each case.
    • Change 0,125µM to 0.125 µM
    • what does "in absence (A-0µM)" mean?
    • Which statistical analysis was employed?
    • Further, rewrite the sentence "Mass spectrometry (MS) analysis of lipids bound to AtVPS13M1(1-335) or Tom20 (negative control) after incubation with calli total lipids. Results are expresses in nmol of lipids per nmol of proteins (C) or in mol% (D)".
    • "C" and "D" are not directly comparable, as in "C" no Tom20 was used and in "C" no insect cells were used.
    • Further, in "D" the experimental setup is not clear. AtVPS13(1-335) is supposed to be purified protein after incubation with calli lipids (figure 2, A). Further, in the same figure, lipid composition of "insect cells" and "calli-Pi" are compared why? Please clarify this. Figure 3:
    • t-test requires a normal distribution of the data. This is not possible for an n=3. Please use an adequate analysis.
    • Please clarify the meaning of the letters on the top of the bars in the legend. Please, make it clear that two figures belong to C.
    • Reorganise the order of figure 3 (ABCD)

    Page 10, 3. Paragraph: since the finding, that no peptides were found in the VSP13M1 ko lines, although transcription was not altered, is surprising, please include the proteomic data in the supplement

    Page 11, line 17: The in vitro experiments showed a low affinity of VSP13M1 towards galactolipids. It is further claimed that this is consistent with the finding of the AtVSP13M1 Ko line in vivo, that in absence of PI, no change in DGDG content could be observed. However, the "absence" of VSP13M1 in vivo might still result in a bigger VSP13M1 protein, than the truncated form (1-335) used for the in vitro experiments

    Page 13, lane 8: you should reconsider the use of a triple Yepet tag: If two or more identical fluorescent molecules are in close proximity, their fluorescence emission is quenched, which results in a weak signal (as the one that you obtained). See: Zhuang et al. 2000 (PNAS) Fluorescence quenching: A tool for single-molecule protein-folding study

    Page 13, line 14: change 1µm to 1 µm

    Page 13, line 29: please reduce the sentence to the first part: if A does not colocalize with B, it is not necessary to mention that B does not colocalise with A.

    Page 14, 2. Paragraph: it is not conclusive that phenotype analysis is suddenly conducted with plants under cold stress, since everything was about Pi-starvation and the role of VSP13M1. Lipid remodelling under Pi stress completely differs from the lipid remodelling under cold stress.

    Page 14, line 20: change figure to Figure

    Page 07, line 17: change artifact to artefact

    Significance

    General assessment:

    The paper is well written and technically sound. However, some points could be identified, that definitely need a revision. Overall, we got the impression that so far, the data gathered are still quite preliminary and need some more detailed investigations prior to publication (see major points).

    Advance: The study definitely fills a gap of knowledge since not much is known on the function of plant VPS13 proteins so far.

    Audience: The study is of very high interest to the plant lipid community but as well of general interest for Plant Molecular Biology and intracellular transport.

    Our expertise: Plant membrane transport and lipid homeostasis.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary: An analysis of an Arabidopsis VSP13 presumed lipid transport is provided. The analysis pretty much follows similar studies done on yeast and human homologs. Key findings are the identification of multiple products from the locus due to differential splicing, analysis of lipid binding and transport properties, subcellular location, tissue specific promoter activity, mutant analysis suggesting a role in lipid remodeling following phosphate deprivation, but no physiological or growth defects of the mutants.

    Major points: The paper is generally written and documented, the experiments are well conducted and follow established protocols. The following major points should be considered:

    1. There are complementary lipid binding assays that should be considered such as liposome binding assays, or lipid/western dot blots. All of these might give slightly different results and may inform a consensus. Of course, non-membrane lipids such as TAG cannot be tested in a liposome assay.
    2. Similarly, lipid transfer based only on fluorophore-labeled lipids may be misleading because the fluorophore could affect binding. It is mentioned that the protein in this assay is tethered by 3xHiis to the liposomes. Un less I ma missing something, I do not understand how that should work. This needs to be better explained.
    3. The in vivo lipid binding assay could be obscured by the fact that the protein was produced in insect cells and lipid binding occurs during the producing. What is the evidence that added plants calli lipids can replace lipids already present during isolation.
    4. The effects on lipid composition of the mutants are not very drastic from what I can tell. Furthermore, how does this fit with the lipid composition of mitochondria where the protein appears to be mostly located?
    5. For the localization of the fusion protein, has it been tested whether the furoin is functional? This should be tested (e.g. by reversion of lipid composition).
    6. It is speculated that different splice forms are located to different compartments. Can that be tested and used to explain the observed subcellular location patterns?
    7. GUS fusion data only probe promoter activity but not all levels of gene expression. That caveat should be discussed.

    Minor points:

    1. Extraplastidic DGDG and export from chloroplasts following phosphate derivation was first reported in PMID: 10973486.
    2. Check throughout the correct usage of gene expression as genes are expressed and proteins produced.
    3. In general, the paper is too long. Redundancies between introduction, results and discussion should be removed to streamline.
    4. I suggest to redraw the excel graphs to increase line thickness and enlarge font size to increase presentation and readability.

    Significance

    Interorganellar lipid trafficking is an important topic and especially under studied in plants. Identifying components involved represents significant progress in the field. Similarly, lipid remodeling following phosphate derivation is an important phenomenon and the current advances our understanding.

  5. Version published to 10.1101/2024.05.22.594332v1 on bioRxiv