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  1. Author Response

    Reviewer #3 (Public Review):

    The import of soluble precursor proteins into the mitochondrial matrix is a complex process that involves two membranes, multiple protein interactions with the translocating substrate, and distinct forms of energetic input. The traditional approaches for in vitro measurement of protein translocation across membranes typically involve radiography or immunodetection-based assays. These end-point approaches, however, often lack optimal resolution to analyze the sequential processes of protein transport. Therefore, the development of techniques to dissect the kinetic steps of this process will be of great interest to the field of protein trafficking.

    This study by Ford et al. employs a novel bioluminescence-based technique to analyze the import of presequence-containing precursors (PCPs) into the mitochondrial matrix in real time. As a follow-up study to previous work from the Collinson group (Pereira et al. 2019), this approach makes use of the split NanoLuc luciferase enzyme strategy, whereby mitochondria are isolated from yeast expressing matrix localized 'LgBiT' (encoded by the mt-S11 gene) and used for import experiments with purified PCPs containing 'SmBiT' (the 11-residue pep86 sequence). The light intensity that results from the high-affinity interaction of pep86 with mt-S11 is convincingly shown in this study to be a reliable reporter of protein import into the matrix space. Therefore, from a technical stance, this appears to be a very promising approach for making high-resolution measurements of the different kinetic steps of protein translocation.

    The authors leverage this technology to seek insights into several features of mitochondrial protein import, with some observations challenging key longstanding paradigms in the field. Using series of PCP constructs differing in length and placement of the pep86 peptide, the authors perform luminescence-based import tests with varying protein concentration, energetic input, and presequence charge distribution. Fits to the time course data suggest two main kinetic steps that govern matrix-directed import: transit of the PCP across the TOM complex into the IMS and association of the PCP with the TIM23 motor complex. The results support some very interesting insights into TIM23-mediated protein import, including: that precursor accumulation is strongly dependent on length; that the kinetically limiting step of IM transport is engagement with the TIM23 complex, not transmembrane transport itself; and that presequence charge distribution differently affects import rate and matrix accumulation. The results of this study appear repeatable among samples and the mathematical fits to time courses are well explained. However, there remain some questions about the nature of the experimental approach and the interpretation of the kinetics data in terms of the underlying biological processes. These questions are as follows:

    Major points

    Overall system characterization and mathematical analysis

    1. The Western-based characterization of the amount of matrix-localized 11S (shown in Figure 1 - figure supplement 1) shows that the concentration of 11S varies significantly (> twofold concentration difference, quantified as a ratio to Tom40) among yeast/mitochondria preps. Is there a particular reason for this large variability? Perhaps more significantly, the import efficiency (judged by luminescence amplitude) shows high batch variability as well (> twofold efficiency difference). While this series of experiments makes the case that the luminescence readout of import is not limited by matrix-localized 11S, it does raise a potential concern of batch-to-batch variation in import competence. Could this have any implications for the reproducibility of results by this assay, particularly regarding the kinetic parameters reported?

    It is very difficult to know what causes this variability as it can be seen even between triplicate preparations carried out on the same day. It could be due to slight differences in the flasks used to grow cells (such as the size of the baffles). However, we have determined that the variability in 11S concentration does not correlate with import competence (Figure 1 – figure supplement 1C), and that the kinetics of import are not affected (Figure 1 – figure supplement 2C).

    1. My understanding from the Pereira 2019 JMB paper is that the yeast expressing the matrix-targeted 11S were engineered so that the 11S construct contained a 35 residue presequence from ATP1. In Figure 1 - figure supplement 1, panel A, it looks like the mitochondria-derived 11S constructs are significantly larger than the purified 11S constructs used to calibrate concentration. If the added residues on the mitochondrial 11S constitute a presequence, then they should be cleaved up on import to yield the mature sized protein. Why are the mitochondrial 11S constructs so much larger than the purified ones? Explicit labeling of MW markers would be useful here.

    We noted that it seemed likely that the presequence was not getting cleaved off. There may also be some kind of SDS-PAGE mobility issues for 11S (common for beta-barrels), such that the purified version has a different mobility to the matrix localised version. Therefore, the possibility remains that the MTS is cleaved off, but the mature product migrates anomalously on gels. For this reason we carried out experiments to show that 11S is matrix localised, which turned out to be the case (Figure 1 – figure supplement 1D). So irrespective non-MTS cleavage, or unexpected gel mobility of correctly processed 11S, the reporter is where it should be – in the matrix. These points are elaborated in the text.

    Labels have been added to molecular weight markers, as requested.

    1. From Figure 1D, given that the amplitude linearly increases with added Acp1pep86 up to ~45 nM, this suggests that matrix-localized 11S is in stoichiometric excess of imported peptide within this range of added substrate. Given a matrix [11S] of 2.8 uM, a stoichiometrically equivalent amount of Acp1-pep86 would be equivalent to an import of <0.5% of added substrate, and it is suggested that import efficiency is actually much lower than that. How can this very low import efficiency be explained?

    Import is single turnover under our assay conditions and is therefore limited by the number of import sites rather than matrix [11S]. Under standard conditions, we intentionally add substrate in vast excess and only anticipate that a very small proportion will be imported.

    1. Apropos of point #3 above: Given the low efficiency of import observed for the purified PCP substrates in this study, one wonders if this due to the formation of off-pathway (translocation incompetent) precursors established during the import reaction, before substrates have a chance to engage OM receptors (e.g., due to aggregation, etc.) In this case, the interpretation of single-turnover conditions may instead be caused by a vast majority of PCP losing translocation competence, rather than the requirement for energetic resetting that is suggested. Might this be a possibility?

    We anticipate that some PCP will aggregate and add substrate in excess to allow for that. Our interpretation of the reaction as single turnover was drawn from a comparison of PCP-pep86-DHFR import amplitude in the presence versus absence of MTX, rather than amplitudes from absolute amounts of PCP. We cannot think of a reason why MTX would affect protein solubility.

    1. Import time courses in many cases show a progressive drop in luminescence at later time points after a maximum value has been reached. This reduction in signal cannot be accounted for by the two rate constants in the equation used in two-step kinetic model. How were such luminescence deviations accounted for when fitting data to obtain these kinetics parameters? What might be the reason for this downward drift in signal once maximum amplitude has been reached?

    We almost always see this gradual drop in luminescence in both the mitochondrial and bacterial systems. The data points acquired after the amplitude are excluded for the fitting. The assay is based on an enzymatic reaction and we think that the downward drift is due to a combination of substrate depletion and accumulation of reaction products.

    Import kinetics: dependence on total protein size

    1. In Figure 3 - figure supplement 1, some of the kinetic parameters from the PCP concentration-dependent responses are quite noisy. For instance, responses for the shortest constructs (L and DL) show a lot of variability in the k1 and k2 parameters. Is this (partly) due to difficulty in resolving these two parameters during the nonlinear least-squares fitting protocol for these particular constructs?

    It is difficult to resolve k1 and k2 perfectly, so the numbers are only estimates.

    1. The data in Figure 3, panels E and F (derived from Figure 3 - figure supplement 1) in some cases show non-linear dependence of kinetic parameters on the 'N to pep86 distance' for the length (panel E) and position (panel F) variants. For instance, from the length series, the k1 mean goes from 132 to 385 to 237 nM for the DL, DDL, and DDDL constructs, respectively. The variances suggest that these differences are real. Is there a reason that kinetic parameters would have such non-monotonic dependence on length?

    We don’t know the reason for this variance, but it could be investigated in future studies.

    Import kinetics: dependence on energetic input

    1. The data of Figure 4A show the results of partial dissipation of the membrane potential by 10 nM valinomycin. Most studies designed to cause a gradual dissipation of membrane potential do so by protonophore (e.g., CCCP) titration. Given that matrix-directed import is completely blocked by low micromolar amounts of this potent ionophore, it would be useful to have an independent readout (e.g., TMRM measurements) of the residual membrane potential that exists upon treatment with the lower concentrations of valinomycin used here.

    We have now included data that shows the partial effect of 10 nM valinomycin on membrane potential (TMRM measurements) and protein import (Figure 4 – figure supplement 1A-B).

    1. The step associated with k1, designated as transport across the TOM complex, is suggested to go to completion before starting the step associated with k2, engagement of the TIM23 complex. The k1 step shows a strong dependence on membrane potential (Fig. 4A, middle), particularly for the length series. Why would this be, given that no part of translocation across the OM should be associated with a valinomycin-sensitive electric potential?

    This effect is relatively small and mainly affects shorter PCPs. Our interpretation is that passage of the PCP through TOM is reversible, and committing PCP to import across the IMM (which requires ∆ψ) prevents this reversibility. However, it is also possible that transport through TOM and TIM23 are partially coupled. Both these possibilities are discussed in the discussion.

    Working model

    1. One of the most surprising outcomes of this study is that passive transport of substrates across the TOM complex and energy-coupled transport via the TIM23 complex are kinetically separable and independent events. As the authors note in the Discussion, the current paradigm of the field is that matrix-targeted substrates concurrently traverse the OM and IM via the TIM-TIM23 supercomplex, and this model is supported by quite a bit of experimental evidence. Even in this study, the fact that the PCP-pep86-DHFR construct exposes the pep86 sequence to the matrix in the presence of MTX (Figure 2) is evidence of a two membrane-spanning intermediate. Key mechanistic questions arise regarding the model proposed in this study. For example, if PCPs traverse the TOM complex as a stand-alone step, what is the driving force (e.g., a simple pathway of protein interactions with increasing affinity)? And would soluble, matrix-directed substrates be expected to accumulate in the very restricted space of the IMS? If so, how would TIM23directed membrane proteins keep from aggregating in the aqueous IMS? These questions would be worth addressing in the discussion of the model.

    We have included a discussion of the experimental evidence for TOM-TIM23 supercomplexes. The acid chain hypothesis has been proposed as the driving force for PCP transport though TOM ‒ an interaction between positive charges of the presequence and negatively charged residues within the TOM40 channel. Proteins that are targeted to the IMS are imported through TOM without the participation of TIM23 and we think that matrix-targeted proteins can do the same. This could explain why TOM is in excess over TIM23. We also think that some matrix-targeted PCPs can accumulate in the IMS, although this may not be true of membrane proteins.

    Import kinetics: dependence on MTS charge distribution

    1. The fact that import rates are increased with a more electropositive presequence makes sense in terms of the electrophoretic pull exerted on the PCP (matrix, negative). However, the greater accumulation of precursors containing more electronegative presequences remains puzzling. In the manuscript, this is explained based on the concept that accumulation of positive charges will cause partial collapse the membrane potential. However, I am still uncertain about this explanation for a few reasons. First, for each PCP, the presequence will constitute just a small fraction of the total length of the precursor, and therefore contribute a small fraction of the total charge density of imported protein. Would such a small change in total PCP charge be expected to have the dramatic effect observed among samples?

    The majority of the total PCP charge is from the mature region, and whilst the positive charges in the presequence undoubtedly deplete ∆ψ, the differences in extent of ∆ψ depletion that we see between PCPs that vary in charge, is due to the difference in charge of the mature regions (as their presequences are identical).

    Second, given the small amount of protein imported under these conditions, would the total charge of imported PCPs be expected to affect transmembrane ion distribution so significantly? For instance, as I recall, it takes up to micromolar amounts of mitochondria-targeted lipophilic cations (e.g., TPP+) to cause a major change in the TMRM-detected membrane potential.

    The effect was indeed unexpected. Despite the seemingly small number of PCPs that are imported, the total number of charged residues will be much greater.

    Finally, I would expect isolated mitochondria to be capable of respiratory control. It is well known, for example, that isolated mitochondria can respond to temporary draw-down of the membrane potential (e.g., by ADP/Pi addition) by going into state 3 respiration and restoring membrane gradients. Why would that not be the case here (Figure 5D)?

    The isolated mitochondria that we used for the import assays demonstrate increased O2 consumption in response to ADP addition, as expected (Figure 5 – figure supplement 1A-B). In addition to this new figure, we have now included TMRM data (Figure 6 – figure supplement 2B) that shows a depletion of ∆ψ in response to ADP addition, that is temporary and dependent on the amount of ADP added. We are therefore confident that our isolated mitochondria are capable of respiratory control as expected. We think that the lack of restoration of ∆ψ, following import-induced dissipation, is a consequence of the import process in vitro. Perhaps the import process compromises the channel resulting in concomitant ion/ charge dissipation during the active process. Moreover, this is likely to be exacerbated in vitro upon acute exposure to PCP, causing a sudden saturation of the import sites – thereby compromising the ∆ψ and the mitochondria’s ability to rapidly recover (this possibility has been noted in the MS).

    General

    1. Although the spectral approach in this study is developed as an alternative to the more traditional import assays, it would be useful to have some control import tests (done with Westerns or autoradiography) as complements to the luminescence-based imports. For example, control tests to accompany Figure 1 that show import efficiency or tests that accompany Figure 3 to show import of the different length and position series constructs. Perhaps this could be done with immunodetection of Acp1 or the pep86 epitope, showing protease-protected, processed import substrates that appear in a membrane potential/ATP-dependent manner. Even if the results from the more traditional techniques ran contrary to the results using the NanoLuc system, this would still allow the authors to compare which effects are consistent and which are dissimilar between different approaches.

    We have now included a Western blot import assay for the PCP-pep86-DHFR substrate and show that import is ∆ψ-dependent (Figure 2 ‒ figure supplement 1).

    1. The authors might also consider conducting imports with mitoplasts as a way to test the kinetic model that includes the TIM23-mediated step alone.

    We conducted import assays with mitoplasts and have now included this as a main Figure 5.

    1. It is difficult to follow the logic in the Discussion regarding the number of TIM23 sites limiting the number of 11S imported into mitochondria in live cells (page 15, lines 23-27). Are the authors suggesting that in vivo, one TIM23 complex serves to transport a single protein? This needs to be clarified.

    This has been removed, and this section of the discussion has been clarified.

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  2. Evaluation Summary:

    This study employs a novel bioluminescence-based technique to analyze the import of precursor proteins into the mitochondrial matrix in real time. This is an innovative technical advance that can provide mechanistic detail on the kinetic steps of mitochondrial protein import. It has potential applications in other membrane protein transport systems and it could be applicable to studies in applied science such as screening for drugs targeting the mitochondrial import apparatus.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Ford et al. investigated protein import into the mitochondrial matrix via the presequence pathway using an innovative NanoLuc translocation assay. In this assay, a model precursor tagged C-terminally with a small fragment of the NanoLuc enzyme is imported into purified mitochondria. The mitochondria were prepared from a yeast strain overexpressing a NanoLuc enzyme lacking the small fragment that contains a mitochondrial presequence that directs the protein into the matrix. Upon import of the model precursor, the active NanoLuc enzyme is formed and produces a luminescense signal in the present of a dye. The authors used this assay to study the effect of ATP and loss of the membrane potential on the kinetics of protein import. The kinetic profiles indicate the presence of two rate-limiting steps. The authors propose that the first step corresponds to binding of the precursor protein to the TOM complex. The second step could represent the initiation of transport across the inner membrane. They further found that precursor properties such as net charge and size have an impact on these steps. Based on the findings the authors proposed a kinetic model including two rate-limiting steps. The used assay could be an interesting to study the dynamics and import kinetics of different types of mitochondrial precursor proteins.

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  4. Reviewer #2 (Public Review):

    In this study, the authors use a luminescence-based method (NanoLuc) to investigate the kinetics of protein import by the main mitochondrial protein translocases, the TOM and the TIM23 complexes. In a recently published paper, the same group had described the NanoLuc approach to dissect the mechanisms of protein transport across biological membranes, including import into mitochondria. Compared to other methods that have been traditionally used to study protein import into mitochondria, the NanoLuc approach offers elevated time resolution and rapid data quantification, providing a powerful means to dissect the mechanisms that drive the protein transport reactions.

    In this new paper, the authors exploit the NanoLuc approach to obtain precise and time-resolved information on the transport of mitochondrial, matrix-targeted presequence-containing precursors (PCPs). By dissecting the import of a relatively small PCP, they observe an import kinetics characterized by two rate limiting steps. Taking into account the dependency of the import reaction on the main energy sources that drive transport by TIM23, the inner membrane electrochemical potential (delta psi) and the hydrolysis of ATP, they attribute the slowest rate-limiting step to transport by the TOM complex. The authors also suggest that PCPs are fully transported across the OM prior to engaging with the TIM23 complex. This result is somewhat in discordance with a mechanistic model based on the transport of larger PCPs, which generate two-membrane spanning translocation intermediates tethering the TOM and TIM23 complexes. Importantly the authors also investigate how charges in the amino acid sequence of the mature protein, i.e. the portion of the PCPs C-terminal to their MTS, influence the import reaction. This aspect of the study is particularly intriguing as the role of PCP mature segments in determining import efficiency is only marginally understood. The authors conclude that positively charged precursors are imported with a very fast kinetics and cause rapid depletion of the membrane potential, which limits the final import amplitude. Instead, negatively charged precursors "consume" less delta psi but reach higher import amplitudes.

    The conclusions of this study are well supported by the experimental data. However, I am not fully convinced about once specific claim related to the fact that the import reaction may be largely single turnover.

    Taken together, the findings presented in this manuscript advance our mechanistic understanding of mitochondrial protein import by the TOM and TIM23 complexes. Most notably, this study also sets an important benchmark for the investigation of other mechanistic aspects of the mitochondrial import reactions. Furthermore, this approach can be useful to screen for (and characterize) drugs targeting the mitochondrial import apparatus. In summary, this study is of high relevance for the broad scientific community.

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  5. Reviewer #3 (Public Review):

    The import of soluble precursor proteins into the mitochondrial matrix is a complex process that involves two membranes, multiple protein interactions with the translocating substrate, and distinct forms of energetic input. The traditional approaches for in vitro measurement of protein translocation across membranes typically involve radiography or immunodetection-based assays. These end-point approaches, however, often lack optimal resolution to analyze the sequential processes of protein transport. Therefore, the development of techniques to dissect the kinetic steps of this process will be of great interest to the field of protein trafficking.

    This study by Ford et al. employs a novel bioluminescence-based technique to analyze the import of presequence-containing precursors (PCPs) into the mitochondrial matrix in real time. As a follow-up study to previous work from the Collinson group (Pereira et al. 2019), this approach makes use of the split NanoLuc luciferase enzyme strategy, whereby mitochondria are isolated from yeast expressing matrix localized 'LgBiT' (encoded by the mt-S11 gene) and used for import experiments with purified PCPs containing 'SmBiT' (the 11-residue pep86 sequence). The light intensity that results from the high-affinity interaction of pep86 with mt-S11 is convincingly shown in this study to be a reliable reporter of protein import into the matrix space. Therefore, from a technical stance, this appears to be a very promising approach for making high-resolution measurements of the different kinetic steps of protein translocation.

    The authors leverage this technology to seek insights into several features of mitochondrial protein import, with some observations challenging key long-standing paradigms in the field. Using series of PCP constructs differing in length and placement of the pep86 peptide, the authors perform luminescence-based import tests with varying protein concentration, energetic input, and presequence charge distribution. Fits to the time course data suggest two main kinetic steps that govern matrix-directed import: transit of the PCP across the TOM complex into the IMS and association of the PCP with the TIM23 motor complex. The results support some very interesting insights into TIM23-mediated protein import, including: that precursor accumulation is strongly dependent on length; that the kinetically limiting step of IM transport is engagement with the TIM23 complex, not transmembrane transport itself; and that presequence charge distribution differently affects import rate and matrix accumulation. The results of this study appear repeatable among samples and the mathematical fits to time courses are well explained. However, there remain some questions about the nature of the experimental approach and the interpretation of the kinetics data in terms of the underlying biological processes. These questions are as follows:

    Major points

    Overall system characterization and mathematical analysis

    1. The Western-based characterization of the amount of matrix-localized 11S (shown in Figure 1 - figure supplement 1) shows that the concentration of 11S varies significantly (> twofold concentration difference, quantified as a ratio to Tom40) among yeast/mitochondria preps. Is there a particular reason for this large variability? Perhaps more significantly, the import efficiency (judged by luminescence amplitude) shows high batch variability as well (> twofold efficiency difference). While this series of experiments makes the case that the luminescence readout of import is not limited by matrix-localized 11S, it does raise a potential concern of batch-to-batch variation in import competence. Could this have any implications for the reproducibility of results by this assay, particularly regarding the kinetic parameters reported?

    2. My understanding from the Pereira 2019 JMB paper is that the yeast expressing the matrix-targeted 11S were engineered so that the 11S construct contained a 35 residue presequence from ATP1. In Figure 1 - figure supplement 1, panel A, it looks like the mitochondria-derived 11S constructs are significantly larger than the purified 11S constructs used to calibrate concentration. If the added residues on the mitochondrial 11S constitute a presequence, then they should be cleaved up on import to yield the mature sized protein. Why are the mitochondrial 11S constructs so much larger than the purified ones? Explicit labeling of MW markers would be useful here.

    3. From Figure 1D, given that the amplitude linearly increases with added Acp1-pep86 up to ~45 nM, this suggests that matrix-localized 11S is in stoichiometric excess of imported peptide within this range of added substrate. Given a matrix [11S] of 2.8 uM, a stoichiometrically equivalent amount of Acp1-pep86 would be equivalent to an import of <0.5% of added substrate, and it is suggested that import efficiency is actually much lower than that. How can this very low import efficiency be explained?

    4. Apropos of point #3 above: Given the low efficiency of import observed for the purified PCP substrates in this study, one wonders if this due to the formation of off-pathway (translocation incompetent) precursors established during the import reaction, before substrates have a chance to engage OM receptors (e.g., due to aggregation, etc.) In this case, the interpretation of single-turnover conditions may instead be caused by a vast majority of PCP losing translocation competence, rather than the requirement for energetic resetting that is suggested. Might this be a possibility?

    5. Import time courses in many cases show a progressive drop in luminescence at later time points after a maximum value has been reached. This reduction in signal cannot be accounted for by the two rate constants in the equation used in two-step kinetic model. How were such luminescence deviations accounted for when fitting data to obtain these kinetics parameters? What might be the reason for this downward drift in signal once maximum amplitude has been reached?

    Import kinetics: dependence on total protein size

    1. In Figure 3 - figure supplement 1, some of the kinetic parameters from the PCP concentration-dependent responses are quite noisy. For instance, responses for the shortest constructs (L and DL) show a lot of variability in the k1 and k2 parameters. Is this (partly) due to difficulty in resolving these two parameters during the nonlinear least-squares fitting protocol for these particular constructs?

    2. The data in Figure 3, panels E and F (derived from Figure 3 - figure supplement 1) in some cases show non-linear dependence of kinetic parameters on the 'N to pep86 distance' for the length (panel E) and position (panel F) variants. For instance, from the length series, the k1 mean goes from 132 to 385 to 237 nM for the DL, DDL, and DDDL constructs, respectively. The variances suggest that these differences are real. Is there a reason that kinetic parameters would have such non-monotonic dependence on length?

    Import kinetics: dependence on energetic input

    1. The data of Figure 4A show the results of partial dissipation of the membrane potential by 10 nM valinomycin. Most studies designed to cause a gradual dissipation of membrane potential do so by protonophore (e.g., CCCP) titration. Given that matrix-directed import is completely blocked by low micromolar amounts of this potent ionophore, it would be useful to have an independent readout (e.g., TMRM measurements) of the residual membrane potential that exists upon treatment with the lower concentrations of valinomycin used here.

    2. The step associated with k1, designated as transport across the TOM complex, is suggested to go to completion before starting the step associated with k2, engagement of the TIM23 complex. The k1 step shows a strong dependence on membrane potential (Fig. 4A, middle), particularly for the length series. Why would this be, given that no part of translocation across the OM should be associated with a valinomycin-sensitive electric potential?

    Working model

    1. One of the most surprising outcomes of this study is that passive transport of substrates across the TOM complex and energy-coupled transport via the TIM23 complex are kinetically separable and independent events. As the authors note in the Discussion, the current paradigm of the field is that matrix-targeted substrates concurrently traverse the OM and IM via the TIM-TIM23 supercomplex, and this model is supported by quite a bit of experimental evidence. Even in this study, the fact that the PCP-pep86-DHFR construct exposes the pep86 sequence to the matrix in the presence of MTX (Figure 2) is evidence of a two membrane-spanning intermediate. Key mechanistic questions arise regarding the model proposed in this study. For example, if PCPs traverse the TOM complex as a stand-alone step, what is the driving force (e.g., a simple pathway of protein interactions with increasing affinity)? And would soluble, matrix-directed substrates be expected to accumulate in the very restricted space of the IMS? If so, how would TIM23-directed membrane proteins keep from aggregating in the aqueous IMS? These questions would be worth addressing in the discussion of the model.

    Import kinetics: dependence on MTS charge distribution

    1. The fact that import rates are increased with a more electropositive presequence makes sense in terms of the electrophoretic pull exerted on the PCP (matrix, negative). However, the greater accumulation of precursors containing more electronegative presequences remains puzzling. In the manuscript, this is explained based on the concept that accumulation of positive charges will cause partial collapse the membrane potential. However, I am still uncertain about this explanation for a few reasons. First, for each PCP, the presequence will constitute just a small fraction of the total length of the precursor, and therefore contribute a small fraction of the total charge density of imported protein. Would such a small change in total PCP charge be expected to have the dramatic effect observed among samples? Second, given the small amount of protein imported under these conditions, would the total charge of imported PCPs be expected to affect transmembrane ion distribution so significantly? For instance, as I recall, it takes up to micromolar amounts of mitochondria-targeted lipophilic cations (e.g., TPP+) to cause a major change in the TMRM-detected membrane potential. Finally, I would expect isolated mitochondria to be capable of respiratory control. It is well known, for example, that isolated mitochondria can respond to temporary draw-down of the membrane potential (e.g., by ADP/Pi addition) by going into state 3 respiration and restoring membrane gradients. Why would that not be the case here (Figure 5D)?

    General

    1. Although the spectral approach in this study is developed as an alternative to the more traditional import assays, it would be useful to have some control import tests (done with Westerns or autoradiography) as complements to the luminescence-based imports. For example, control tests to accompany Figure 1 that show import efficiency or tests that accompany Figure 3 to show import of the different length and position series constructs. Perhaps this could be done with immunodetection of Acp1 or the pep86 epitope, showing protease-protected, processed import substrates that appear in a membrane potential/ATP-dependent manner. Even if the results from the more traditional techniques ran contrary to the results using the NanoLuc system, this would still allow the authors to compare which effects are consistent and which are dissimilar between different approaches.

    2. The authors might also consider conducting imports with mitoplasts as a way to test the kinetic model that includes the TIM23-mediated step alone.

    3. It is difficult to follow the logic in the Discussion regarding the number of TIM23 sites limiting the number of 11S imported into mitochondria in live cells (page 15, lines 23-27). Are the authors suggesting that in vivo, one TIM23 complex serves to transport a single protein? This needs to be clarified.

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