Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Lipid transfer proteins (LTPs) play a crucial role in the intramembrane lipid exchange within cells. However, the molecular mechanisms that govern this activity remain largely unclear. Specifically, the way in which LTPs surmount the energy barrier to extract a single lipid molecule from a lipid bilayer is not yet fully understood. This manuscript investigates the influence of membrane properties on the binding of Ups1 to the membrane and the transfer of phosphatidic acid (PA) by the LTP. The findings reveal that Ups1 shows a preference for binding to membranes with positive curvature. Moreover, coarse-grained molecular dynamics simulations indicate that positive curvature decreases the energy barrier associated with PA extraction from the membrane. Additionally, lipid transfer assays conducted with purified proteins and liposomes in vitro demonstrate that the size of the donor membrane significantly impacts lipid transfer efficiency by Ups1-Mdm35 complexes, with smaller liposomes (characterized by high positive curvature) promoting rapid lipid transfer.
This study offers significant new insights into the reaction cycle of phosphatidic acid (PA) transfer by Ups1 in mitochondria. Notably, the authors present compelling evidence that, alongside negatively charged phospholipids, positive membrane curvature enhances lipid transfer - an effect that is particularly relevant at the mitochondrial outer membrane. The experiments are technically robust, and my primary feedback pertains to the interpretation of specific results.
(1) The authors conclude from the lipid transfer assays (Figure 5) that lipid extraction is the rate-limiting step in the transfer cycle. While this conclusion seems plausible, it should be noted that the authors employed high concentrations of Ups1-Mdm35 along with less negatively charged phospholipids in these reactions. This combination may lead to binding becoming the rate-limiting factor. The authors should take this point into consideration. In this type of assay, it is challenging to clearly distinguish between binding, lipid extraction, and membrane dissociation as separate processes.
We have included a detailed consideration of this issue on page 11 of the revised manuscript.
(2) The authors should discuss that variations in the size of liposomes will also affect the distance between them at a constant concentration, which may affect the rate of lipid transfer. Therefore, the authors should determine the average size and size distribution of liposomes after sonication (by DLS or nanoparticle analyzer, etc.)
We have included DLS measurements for all lipid sizes (page 6) (SupFig. 2A). Due to the sensitivity of the intensity distribution in DLS measurements by larger particles, we also conducted cryo-EM analysis of vesicles with different sizes (page 6) (SupFig. 2B).
We also now discuss the challenges posed by a fixed membrane-binding surface, which can lead to variations in vesicle spacing when using liposomes of different sizes and its possible influence on the interpretation of results (page 10-11).
(3) The authors use NBD-PA in the lipid transfer assays. Does the size of the donor liposomes affect the transfer of NBD-PA and DOPA similarly? Since NBD-labeled lipids are somewhat unstable within lipid bilayers (as shown by spontaneous desorption in Figure 5B), monitoring the transfer of unlabeled PA in at least one setting would strengthen the conclusion of the swap experiments.
To experimentally address this comment, we explored several different approaches. We first performed transfer experiments using unlabelled lipids, following the general procedures described in the manuscript. After the transfer reaction, we attempted to separate donor and acceptor vesicles by centrifugation and subsequently analyzed the samples by high-resolution mass spectrometry and thin-layer chromatography. Despite considerable effort, we were not able to reliably separate the differently sized liposomes. In particular, small liposomes proved difficult to handle during centrifugation, which is a well-known challenge (Kučerka et al. 1994, BBA; Boucrot et al. 2012, Cell). In addition, liposomes exhibited a tendency to cross-link in the presence of protein, further complicating the separation. Even if this separation step were straightforward, an important limitation of such an approach is that it is very difficult to monitor lipid transfer with sufficient time resolution. Much of the relevant activity occurs within the first 20–30 seconds, and precise interruption at defined time points would be essential.
We therefore set out to establish a fluorescence-based assay that would allow us to follow lipid transfer in real time. For this, we adapted a dequenching-type assay based on a PE coupled fluorescein dye, whose fluorescence is quenched in the proximity of negative charges (e.g., negatively charged lipid headgroups). In principle, this assay should allow us to monitor the movement of negatively charged PA lipids away from donor membranes. Although a fluorescein-based passive lipid-transfer assay has been described previously (Richens et al., 2017), it is used only rarely in the lipid-transfer field. While establishing this assay, we encountered several technical challenges. For example, immediately after protein addition, fluorescence intensity changed in unexpected ways that could not be attributed to lipid transfer. Such effects have been reported in the literature (Wall et al., 1995) and are most likely caused by changes in membrane charge density upon protein binding. After extensive fine -tuning of the experimental conditions and careful evaluation of the data, we were ultimately able to demonstrate that lipid-transfer rates are significantly higher with smaller than with larger liposomes. These results confirm our initial observations, and importantly, they were obtained using unlabelled PA.
The revised manuscript now includes this independent lipid-transfer assay demonstrating the transfer of non-labelled PA (page 11) (SupFig. 4).
(4) The present study suggests that membrane domains with positive curvature at the outer membrane may serve as starting points for lipid transport by Ups1-Mdm35. Is anything known about the mechanisms that form such structures? This should be discussed in the text.
We included a detailed consideration of this interesting point in the discussion section on page 13-14.
Reviewer #2 (Public review):
Summary:
Lipid transfer between membranes is essential for lipid biosynthesis across different organelle membranes. Ups1-Mdm35 is one of the best-characterized lipid transfer proteins, responsible for transferring phosphatidic acid (PA) between the mitochondrial outer membrane (OM) and inner membrane (IM), a process critical for cardiolipin (CL) synthesis in the IM. Upon dissociation from Mdm35, Ups1 binds to the intermembrane space (IMS) surface of the OM, extracts a PA molecule, re-associates with Mdm35, and moves through the aqueous IMS to deliver PA to the IM. Here, the authors analyzed the early steps of this PA transfer - membrane binding and PA extraction - using a combination of in vitro biochemical assays with lipid liposomes and purified Ups1-Mdm35 to measure liposome binding, lipid transfer between liposomes, and lipid extraction from liposomes. The authors found that membrane curvature, a previously overlooked property of the membrane, significantly affects PA extraction but not PA insertion into liposomes. These findings were further supported by MD simulations.
Strengths:
The experiments are well-designed, and the data are logically interpreted. The present study provides an important basis for understanding the mechanism of lipid transfer between membranes.
Weaknesses:
The physiological relevance of membrane curvature in lipid extraction and transfer still remains open.
We thank the reviewer for the constructive feedback on our work. We agree that the physiological relevance of membrane curvature in lipid extraction and transfer remains an open question. Our data show that Ups1 binding to native-like OM membranes under physiological pH conditions is curvature-dependent, supporting the idea that this mechanism may optimize lipid transfer in vivo. While the intricate biophysical basis of this behaviour can only be dissected in vitro, these findings offer valuable insight into how curvature may functionally regulate Ups1 activity in the cellular context. To directly test this, it will be important in future studies to identify Ups1 mutants that lack curvature sensitivity and assess their performance in vivo, which will help clarify the physiological importance of this mechanism.
Reviewer #3 (Public review):
The manuscript by Sadeqi et al. studies the interactions between the mitochondrial protein Ups1 and reconstituted membranes. The authors apply synthetic liposomal vesicles to investigate the role of pH, curvature, and charge on the binding of Ups1 to membranes and its ability to extract PA from them. The manuscript is well written and structured. With minor exceptions, the authors provide all relevant information (see minor points below) and reference the appropriate literature in their introduction. The underlying question of how the energy barrier for lipid extraction from membranes is overcome by Ups1 is interesting, and the data presented by the authors could offer a valuable new perspective on this process. It is also certainly a challenging in vitro reconstitution experiment, as the authors aim to disentangle individual membrane properties (e.g., curvature, charge, and packing density) to study protein adsorption and lipid transfer. I have one major suggestion and a few minor ones that the authors might want to consider to improve their manuscript and data interpretation:
Major Comments:
The experiments are performed with reconstituted vesicles, which are incubated with recombinant protein variants and quantitatively assessed in flotation and pelleting assays. According to the Materials and Methods section, the lipid concentration in these assays is kept constant at 5 µM. However, the authors change the size of the vesicles to tune their curvature. Using the same lipid concentration but varying vesicle sizes results in different total vesicle concentrations. Moreover, larger vesicles (produced by freeze-thawing and extrusion) tend to form a higher proportion of multilamellar vesicles, thus also altering the total membrane area available for binding. Could these differences in the experimental system account for the variation in binding? To address this, the authors would need to perform the experiments either under saturated (excess protein) conditions or find an experimental approach to normalize for these differences.
To experimentally address this comment, we have conducted a detailed structural analysis of liposomes of different sizes using cryo-EM to determine the degrees of multi-lamellarity and to estimate how much membrane surface is available for protein binding. We found that while indeed as expected liposomes extruded through a 400 nm sized filter showed about 75 % of the initially calculated membrane surface is still available (SupFig. 3A). For 50 nm extruded liposomes, this number went up to about 93 % and for sonicated liposomes the number was about 94 %. Given the fact that we found about 70 % binding of Ups1 to sonicated liposomes, while this number went down to about 40 % with 50 nm liposomes and to about 30 % for 400 nm extruded liposomes, we can rule out that the effects we observe are due to an increased or decreased available membrane binding area.
Additionally, we performed experiments with increasing amounts of lipids to analyse the impact of lipid concentration on Ups1 membrane binding, when comparing 400 nm extruded liposomes with sonicated liposomes. Interestingly, while we do observe an increased binding of Ups1 to sonicated liposomes with concentrations varying between 2.5 mM to 10 mM no major increase in binding was observed with 400 nm extruded liposomes. Ups1 membrane binding to sonicated liposomes highly exceeded binding to 400 nm extruded liposomes under all tested conditions (page 7) (SupFig. 3B).
Recommendations for the authors:
Reviewer #2 (Recommendations for the authors:):
(1) Figures 1, 2, and 3 - In the flotation assays, the Ups1-containing fractions differ between experiments. The presence of liposomes in these fractions should be confirmed, for example, by fluorescence measurements. In relation to this, the broad low MW bands in Supplementary Figure 3 may reflect liposomes (mixed micelles of lipids and SDS?), as their fractionation patterns coincide with those of Ups1 at pH 5.5 -6.7 but deviate at pH 7.0 and 7.5. Could the authors clarify this discrepancy?
Flotation profiles vary with changing conditions of the experiment. We have included a picture of a gel showing the Coomassie staining and the fluorescence of the used lipids side by side to show that the protein bands co-migrate together with liposomes (SupFig. 5).
(2) Figures 2, 3, and 5 - The sizes of the liposomes (400 nm and 50 nm) should be experimentally confirmed, e.g., by dynamic light scattering (DLS).
We have included DLS measurements confirming the differences of liposome sizes. Please see answer to point 2 of Reviewer 1.
(3) Figure 4C - The free energy landscape for different phospholipids is interesting. What about other acidic phospholipids, such as PS?
This is indeed an interesting point. Our molecular dynamics simulations show that PE has a similar free energy landscape to PA while PC is significantly different. This might point into the direction that the headgroup size plays a major role. For intra-mitochondrial PS transport a specific protein complex consisting of Ups2/Mdm35 has been identified, and it will be an interesting question for future studies if PS transfer is regulated by similar factors.
(4) Supplementary Figure 2 - The deformation of liposomes by Ups1 is interesting. Does this depend on the presence of PA or other acidic phospholipids?
We asked ourself the same question throughout the project. As pointed out in the manuscript, the membrane-deforming activity of Ups1 is relatively mild when compared to proteins found for example in endocytosis. This made a proper static analysis challenging. We weren’t able to unambiguously show whether other acidic phospholipids showed comparable effects to PA.
(5) It may not be easy to assess experimentally, but the OM in mitochondria should have scramblase activity. Then, such scramblase activity could influence the observed effects of membrane curvature on Ups1-mediated PA transfer.
(6) It would be helpful to discuss this possibility in the manuscript.
In the revised version of the manuscript, we now discuss the existence of scramblases, such as Sam50 and VDAC, in the outer mitochondrial membrane with regard to their likely effect on membrane packing (page 13 - 14). As for a co-reconstitution experiment we considered the in vitro analysis of the impact that a scramblase in liposomes might have on lipid transfer outside the scope of this study.
(7) Figure 6 is not referenced in the main text.
Thank you, this oversight was corrected.
(8) The non-abbreviated forms of LUV and SUV should be defined in the text upon first use.
We now include a definition in the manuscript.
(9) The term "transfer velocity" would be better expressed as "transfer rate".
We agree, and we changed the wording accordingly.
Reviewer #3 (Recommendations for the authors):
(1) As flotation assays are a central technique of the study, readers who are not familiar with this method could benefit from a few explanatory sentences and appropriate references in the introduction section.
Figure 1B now contains an updated version of a cartoon outlining the flotation assay and a description in the manuscript (page 4) that should make it easier to understand the assay. We have also included a direct reference within the methods section to a paper describing this assay in more detail.
(2) Related to the major point, but also to improve the manuscript overall, the authors could add DLS (for size distribution and zeta potential) and cryo-EM (for multilamellarity analysis) data. This would aid future efforts to reproduce their observations.
In the revised version of the manuscript we include DLS and zeta potential measurements as well as a detailed analysis of liposome multilamellarity by cryo-EM (also see answer to point 2 by Reviewer 1) (SupFig. 2A & B; SupFig. 3E).
(3) Could the authors state the specific zeta potentials of the negatively charged (under varying pH) and neutral liposomes and relate these to natural membranes?
We have included zeta potential measurements of differently charged liposomes in and changed the text accordingly (page 8) (SupFig. 3E).
(4) Changes in pH affect several characteristics of membranes (including lipid dipoles, charge, packing density, fluidity, and phase separation), particularly charge density. This experimental system does not allow all of these factors to be disentangled and studied separately. Some of the observations presented in Figures 2 and 5 could also be explained by these effects.
The effects of pH on various membrane properties, such as lipid headgroup dipoles, lipid packing, interfacial tension, and others, are well described in the literature. For example, it was implied that increasing pH leads to phosphatidic acid (PA) becoming more negatively charged when in proximity to phosphatidylethanolamine (PE). We already discuss this effect in the manuscript, as our observation that Ups1 binding to membranes depends on negatively charged lipids but nevertheless increases with decreasing pH is unexpected.
As pointed out, many of the parameters mentioned above are beyond control in our assays, and a systematic analysis of each of these factors with respect to Ups1 membrane binding and lipid transfer would be well beyond the scope of this manuscript. We have therefore included a passage discussing this issue in more detail (page 4-5).
(5) Is the curvature simulated in the theoretical models comparable to the curvature of the liposome systems (e.g., a sphere of 100 nm diameter)?
The simulated curvature spans a defined range, with the highest curvature corresponding to vesicles with diameters of approximately 15 nm. This corresponds reasonably well to the vesicle size distribution as analyzed by cryo-EM.
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