Author response:
Reviewer #1:
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 thank the reviewer for the constructive and positive evaluation of our manuscript. We agree that, while our data support the interpretation that the rate-limiting step occurs at the donor membrane, it is difficult to dissect in our assay which of the individual steps at the donor membrane - such as binding of Ups1, lipid extraction into the binding pocket, or dissociation of Ups1 - is rate-limiting. Nevertheless, although we cannot exclude contributions from membrane binding or dissociation, several observations suggest that lipid extraction is a rate-limiting step under our experimental conditions.
The acceptor membrane has a similar lipid composition to the donor membrane (in tendency, the donor membrane is even a bit richer in binding-promoting lipids). If binding was ratelimiting, similar constraints would be expected at the acceptor membrane during lipid insertion. However, this is not observed.
Regarding dissociation, if this step were rate-limiting, one would expect similar constraints to be evident at the acceptor vesicles as well. Nevertheless, membrane dissociation might be mechanistically coupled to lipid extraction and thus difficult to evaluate as an independent step.
Based on our data and the considerations described above, we suggest that lipid extraction is the dominant rate-limiting step at the donor membrane under our conditions. However, we agree that a clear separation of these individual steps is not possible with the current experimental design. We will revise the corresponding passage to clarify that the rate-limiting step occurs at the donor membrane and, based on our observations, likely involves lipid extraction. Future studies aiming on dissecting these steps, will be important for elucidating the mechanism and regulation of Ups1-mediated lipid transfer both in vitro and in vivo.
(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 agree that variations in liposome size will influence the average distance between vesicles at a given lipid concentration, which may in turn affect the rate of lipid transfer. As suggested, we will include DLS measurements to characterize the size distribution of our different liposome preparations.
Our setup was designed to keep the total membrane surface area comparable across conditions. This approach ensures a comparable overall binding capacity for Ups1 and enables the comparison of membrane binding and lipid extraction from different membranes. However, we agree that vesicle spacing, which is affected by liposome size at constant lipid concentration, could potentially influence certain steps in the transfer process, such as the time required for Ups1 to travel between donor and acceptor membranes. Whether this intermembrane travel time contributes to rate limitation is indeed an interesting question, and we will address this point through further discussion in the revised manuscript.
Investigating such effects in our current experimental system would require altering the vesicle concentration, which would in turn change the total membrane surface area and introduce additional variables. Nevertheless, exploring the influence of vesicle spacing and intermembrane distance on lipid transfer represents a promising direction for future studies aimed at dissecting the rate-limiting steps of the transfer cycle.
(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.
Ups1-mediated transfer of PA has been demonstrated both by mass spectrometry analysis of donor and acceptor vesicles (Connerth et al., 2012) and by NBD-fluorescence-based lipid transfer assays (Lu et al., 2020; Miliara et al., 2015; Miliara et al., 2019; Miliara et al., 2023; Potting et al., 2013; Watanabe et al., 2015). The fluorescence-based approach has been the most widely applied across multiple studies and has enabled detailed analysis of various aspects of lipid transfer by Ups1. It has been used to investigate mutants of key structural elements—such as the lipid-binding pocket and the α2–loop region. It has also been used to analyze fusion constructs between Ups1 and Mdm35, the influence of Mdm35 variants, and competition with excess Mdm35. Additionally, by comparing the transfer of NBD-labeled PA and NBD-labeled PS, this assay has provided insights into the determinants of the lipid specificity of Ups1. Hence, our experiments are based on the standard assay used to analyse lipid transfer in the field and thus can be corralated with the majority of published data.
Nevertheless, we agree that it is important to keep in mind that NBD labeling may alter the biophysical properties of lipids and, consequently, affect their transfer efficiency. Moreover, NBD-labeled lipids are not suitable for comparing the transfer efficiency of different PA species, as the label itself may mask differences in acyl chain composition. Therefore, it will be valuable to establish complementary methods that do not rely on NBD-labeled PA. We aim to develop these non-standard methods for possible inclusion in the present study, but even if not fully implemented at this stage, they will certainly form an important part of future investigations.
(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.
The origin of positively curved membrane domains is indeed highly relevant in the context of our findings, and while not the primary focus of this work, we will place more emphasis on discussing how such curvature may arise. Mechanisms include the action of curvature-generating proteins, asymmetric lipid composition and curvature induced at membrane contact sites. We have so far included examples of proteins in the outer mitochondrial membrane that are expected to influence curvature in their vicinity, and we will expand on this aspect and other contributing factors more thoroughly in the revised text.
Reviewer #2:
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:
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 wrifen 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 saturation (excess protein) conditions or find an experimental approach to normalize for these differences.
We thank the reviewer for the constructive and positive comments. We agree that, since the total number of lipids was kept constant, the number of vesicles varied with vesicle size in our experiments. However, the setup was specifically designed to maintain a comparable total membrane surface area across conditions, ensuring a comparable number of available binding sites for Ups1. Because membrane surface area decreases with the square of the vesicle radius, keeping vesicle number constant would have led to a marked reduction in binding surface. Our approach was therefore aimed at preserving comparable binding capacity while varying membrane curvature.
With respect to multilamellarity, we thank the reviewer for addressing this important point. As described above, we aimed to maintain a constant total membrane surface area across all conditions to ensure an equal number of potential binding sites. We agree that multilamellarity in large liposomes could restrict accessibility to part of the membrane surface. However, we see in our experiments that even when the total membrane surface area of the small liposomes is reduced to one quarter of the standard amount, binding to the small liposomes remained stronger than to the larger liposomes at the higher concentration. This strongly indicates that restricted accessibility cannot account for the curvature-specific effect observed. Nonetheless, we will further address this aspect experimentally and in the discussion of the revised manuscript.
References
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