In bacteria, the evolutionarily conserved Sec translocon and SecA play essential roles in protein translocation across and integration into the membrane (Rapoport et al., 2017). The translocon consists of three membrane-integrated proteins, SecY, SecE, and SecG (Mori and Ito, 2001). SecY forms a channel-like path for substrate secretory proteins (Van den Berg et al., 2004) and is stabilized by SecE (Taura et al., 1993). SecG peripherally binds to SecY (Tanaka et al., 2015) and stimulates the SecA-dependent protein translocation (Nishiyama et al., 1994). SecA, an essential ATPase (Lill et al., 1989), binds to cytoplasmic protrusions in SecY (Mori and Ito, 2006a; Zimmer et al., 2008) and pushes a polypeptide into the translocon (Economou and Wickner, 1994; Erlandson et al., 2008). Secretory proteins are generally synthesized as a precursor with an N-terminal signal sequence recognized by the translocation machinery, including the translocon and SecA, and is cleaved off during translocation. Although bacterial protein secretion to the periplasmic space is thought to occur post-translationally in many cases, some secretory proteins use the signal recognition particle (SRP) pathway, which targets the nascent precursor protein to the translocon (Huber et al., 2005; Shimohata et al., 2005). Notably, even in the latter cases, translocation requires the SecA motor function (Shimohata et al., 2005). The Sec translocon is also utilized for protein integration into the membrane. Membrane proteins typically have no cleavable signal sequence, but their transmembrane (TM) regions act as a signal for integration. SRP binds to the signal sequence equivalent (a TM segment) of a membrane protein and deliverers a ribosome-nascent protein complex (RNC) to the translocon (Steinberg et al., 2018). The RNC binds to the same SecY region as SecA (Frauenfeld et al., 2011; Kuhn et al., 2011). Bacteria also have the SecD/F complex that stimulates protein secretion in vivo (Pogliano and Beckwith, 1994) and facilitates the release of a polypeptide from the Sec translocon during a late step in translocation (Matsuyama et al., 1993). Structural and functional analyses have shown that SecD/F is a H⁺-driven motor, in which its periplasmic domain (P1 head) binds a translocon-engaged substrate (Furukawa et al., 2017) and undergoes a conformational change coupled with the H⁺ flow (Tsukazaki et al., 2011). The repetitive movements of the P1 head would allow a forward movement of a translocating chain until its release to the periplasm.

Protein translocation and membrane insertion are essential biological processes in all living organisms, and it is reasonable that factors required for the localization are tuned according to cellular demands. Bacteria have acquired unique regulatory mechanisms by which either the expression of one of duplicated translocation/integration factors is specifically induced (V.SecD2/F2 in Vibrio alginolyticus (Ishii et al., 2015) (see the next section) and YidC2 (a membrane chaperone) in Bacillus subtilis Rubio et al., 2005) or a unique factor is upregulated (SecA in Escherichia coli) (Oliver and Beckwith, 1982) in response to a decline in the relevant transport activity of the cell. To monitor the cellular protein translocation activities and regulate the expression of the relevant factors in real-time, these bacteria utilize a special class of nascent polypeptides, called monitoring substrates (Ito et al., 2018), a class of regulatory nascent polypeptide (Tenson and Ehrenberg, 2002), which undergoes translation elongation arrest. Currently, three proteins are known: V. alginolyticus VemP (Ishii et al., 2015), B. subtilis MifM (Chiba et al., 2009), and E. coli SecM (Nakatogawa and Ito, 2001). Translation of these proteins is arrested by the specific interaction of their intrinsic amino acid sequences, called arrest sequences or arrest motifs, with the components of the ribosome. Remarkably, the arrest motifs of different monitoring substrates are diverse, with no apparent sequence similarities.

A marine bacterium, V. alginolyticus, possesses two sets of the secD/F genes, designated V.secD1/F1 and V.secD2/F2, whose products utilize Na⁺- and presumably H⁺-motive forces, respectively. The bacterium adapts quickly to a salinity change by replacing these SecD/F paralogues (Ishii et al., 2015). Although V.secD1/F1 is expressed constitutively, the expression of V.secD2/F2 is tightly repressed under Na⁺-rich growth conditions, but induced under low Na⁺ growth conditions. The vemP gene located upstream of V.secD2/F2 on the same operon plays an essential role in the regulated expression of V.secD2/F2. VemP, a substrate of the Sec machinery, monitors the machinery's activity via its own translocation. It undergoes stable translation-arrest before the canonical termination step under a protein translocation-deficient condition. In this scenario, the stalled ribosome on a vemP-V.secD2/F2 mRNA destabilizes a stem-loop structure in the vemP-V.secD2 intergenic region, leading to the exposure of an otherwise masked ribosome-binding site for the V.secD2 gene. Thus, the elongation arrest allows entry of ribosomes to that site and consequent synthesis of V.SecD2/F2. Importantly, the VemP translation-arrest occurs even under a protein translocation competent condition, but it is rapidly canceled presumably by a translocation-coupled pulling force that drives translocation of VemP.

Our previous in vivo studies showed that the majority of VemP has its signal sequence processed even in the arrested state. This strongly suggests that the arrest-cancelation of VemP occurs after its translocation has proceeded beyond the signal sequence processing event on the Sec translocon (Mori et al., 2018). Although this feature of VemP seems suitable for monitoring the SecD/F function, molecular mechanisms of how this is accomplished remain to be elucidated. To understand the detailed mechanism of the VemP-arrest-mediated regulation of the V.secD2/F2 expression, it is crucial to know the dynamic interactions of VemP with other participating proteins during the arrest and its translocation-coupled cancelation processes.

Site-directed in vivo photo-crosslinking is a well-designed technique for analysis of inter- or intra-molecular interactions of proteins in living cells (Chin and Schultz, 2002), wherein a pair of a mutated tRNA and an engineered tyrosyl-tRNA synthetase allows for in vivo incorporation of a photo-reactive amino acid analog, p-benzoyl-L-phenylalanine (pBPA), into any selected positions of a target protein by amber suppression (Chin et al., 2002). Upon UV irradiation, pBPA in the target protein generates a covalent crosslinking with nearby molecules. Identification of crosslinked partners provides detailed information about interactions with them at an amino acid residue level resolution, proving useful for many studies (Miyazaki et al., 2020). To extend this technique to acquire temporal resolution, we recently developed the PiXie (pulse-chase and in vivo photo-crosslinking experiment) method. This new approach entails the use of an ultra-powerful UV irradiator that shortens the UV-irradiation time so that we can combine photo-crosslinking and pulse-chase approaches. It enables us to trace dynamic in vivo folding/assembly processes of newly synthesized proteins with high temporal (in the sub-minute order) and spatial (the amino acid residue level) resolutions (Miyazaki et al., 2018). Since a similar orthogonal tRNA/tRNA synthetase system for pBPA introduction is not available for V. alginolyticus, we cannot directly apply the PiXie method to the cognate bacterium to identify interactors of VemP. Vibrio species are closely related to enterobacteria including E. coli and possess a single copy of the secA, secY, secE, secG, and ffh (encoding the protein component of SRP) genes. Because each of the SecA (Kunioka et al., 1998), SecY (Bhattacharyya and Das, 1997), SecE (Nishiyama et al., 1998) and the two SecD/F (Ishii et al., 2015) homologs of Vibrio can function in E. coli cells, the basic structure and function of the Sec machinery and the interaction modes among these factors should be well conserved between these species. Therefore, photo-crosslinking analysis in E. coli would allow us to identify candidate proteins interacting with a VemP-nascent polypeptide in living cells.

In this study, we performed a non-biased, systematic PiXie analysis of VemP and investigated the factors that interact with a VemP-nascent polypeptide during its biosynthesis in E. coli. The physiological significance of the interactions observed in E. coli was evaluated by genetic and biochemical analysis in Vibrio cells. In addition, we identified an amino acid residue of VemP that imposes PpiD-dependence and thereby participates in the force-sensing regulation. Based on these analyses, we discuss how VemP integrates its translocation and arrest regulation for the real-time feedback regulation of the late step motor protein, SecD/F.

VemP in Vibrio controls the SecD/F expression through its force-sensitive translation arrest. Although we previously suggested that the arrest cancelation of VemP is coupled to a late step of translocation (Mori et al., 2018), the details of the responsible force and how this regulatory process occurs remain largely unknown. To understand the molecular mechanisms that lead to the elongation arrest and its cancelation, we performed a systematic PiXie analysis of VemP, which allowed us to identify cellular factors that sequentially interact with the newly synthesized VemP polypeptide chain. We previously used the PiXie method, which allows precisely time-controlled and residue-specific crosslinking, to analyze the post-translational maturation processes of protein complexes with different cellular localizations in bacteria (Miyazaki et al., 2018). This method should also be useful for studying co-translational events (Kramer et al., 2019; Pechmann et al., 2013) that a nascent polypeptide experiences during translation. In particular, a class of polypeptides that undergoes translation arrest would be suitable targets of the PiXie analysis. The molecular interaction behaviors of such arrest peptides, including bacterial VemP, SecM, MifM, and mammalian XBP1u (Chiba et al., 2009; Ishii et al., 2015; Nakatogawa and Ito, 2002; Yanagitani et al., 2011), would be highly relevant for our understanding of these nascent polypeptides in the cell. Thus, in this work, we applied the PiXie method to VemP, which we have identified and characterized previously (Ishii et al., 2015).

We identified Ffh and PpiD as factors directly interacting with the arrested VemP in addition to the ribosomal protein uL22 and the translocon components (Figure 1). The kinetic analyses suggest their sequential interactions with VemP (Figure 2); VemP initially interacts with uL22 and the cytosolic Ffh, then with the membrane-embedded translocon, and lastly with PpiD, a periplasmically exposed chaperone. The crosslinking of VemP with Ffh and PpiD should have functional significance rather than representing a result of a non-specific collision of VemP with these proteins, as suggested by our genetic analyses using the Ffh-depletable and the ppiD deletion strains. The observation that the distinct regions in VemP interact with uL22, Ffh, the translocon, and PpiD (Figures 1 and 6A, left) is consistent with pre-targeting interactions of the arrested VemP with uL22 and Ffh and a post-targeting interaction with PpiD (Figure 6A, right) (see Appendix1: Interactions of a VemP nascent chain with Sec translocon and the related components). A cryo-EM study showed that a VemP nascent polypeptide in the ribosomal exit tunnel forms a compacted secondary structure (Su et al., 2017). In this structure, Trp-124 of VemP contacts to the uL22 protein. The observed crosslinking of pBPA at Trp-124 with uL22 is consistent with the reported structure. Interestingly, we found that the crosslinking representing the VemP-uL22 interaction was enhanced markedly when cells were treated with NaN₃ that should have compromised an early step of the VemP translocation by inhibiting SecA. Thus, the VemP's interaction with uL22 occurs before its initial insertion into the translocon (Figure 2—figure supplement 4).

Figure 6

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We suggest that VemP in the ribosomal tunnel changes its conformation from the compacted state to some other state, perhaps a more extended one, as the translocation process proceeds, because the structural determination had used a nascent chain-ribosome complex without any translocation factors (Su et al., 2017; Figure 6A, right). It is conceivable that a pulling force generated by translocation acts to disrupt the VemP secondary structure and, consequently, the molecular interactions required for the elongation arrest.

We propose the following scheme for the targeting and translocation processes of VemP accompanied by the translation-arrest and its cancelation (Figure 6B). (i) In the cytosol, SRP recognizes and binds to the signal sequence of VemP and targets the nascent chain to the translocon. During this co-translational targeting step, the ongoing translation of VemP is subject to elongation arrest. The use of the SRP pathway, in conjunction with the amino acid sequence features of its signal sequence (Yap and Bernstein, 2011) and the early mature part (see below), might ensure the timely release of the translation complex from the translation-arrested state. (ii) The signal sequence is then inserted into the lateral gate region while the mature region of VemP is going to span the translocon's polypeptide-conducting channel. This early step of translocation is facilitated by the SecA ATPase (Figure 2—figure supplement 3) and accompanied by the signal sequence cleavage. Therefore, the inhibition of SecA with NaN₃ causes accumulation of the AP-un form of VemP. (iii) SecD/F and PpiD then interact with VemP and cooperate in canceling the translation arrest of VemP while aiding its further translocation. (iv) Finally, the full-length, mature VemP polypeptide is exported to the periplasm.

In the arrest cancelation event, PpiD and SecD/F seem to cooperate either sequentially or in concert, and our crosslinking results revealed that they physically interact with each other (Figure 4E). PpiD was a member of the periplasmic chaperone network (Dartigalongue and Raina, 1998; Matern et al., 2010), which directly interacts with both SecY/E/G and ribosome-tethered polypeptides (Antonoaea et al., 2008; Sachelaru et al., 2014). Although an in vitro study revealed that PpiD stimulates translocation of substrate proteins (Fürst et al., 2018), the deletion of the ppiD gene caused no detectable defect of outer membrane protein biogenesis (Justice et al., 2005). We found that the translocation of MBP was compromised weakly in the ΔppiD strain (Figure 4A). At any rate, the exact role of PpiD in protein export remains obscure. We showed here that PpiD physically contacts SecD as well as the AP-pro form of VemP in vivo. Furthermore, our genetic and biochemical analyses indicated that PpiD has a role in the cancelation of the VemP translation arrest (Figure 4). Although our experiments failed to detect a VemP-SecD crosslinking, due possibly to the transient/unstable nature of their interaction, SecD/F also functions in the arrest cancelation of VemP (Figure 4D). We propose that PpiD and SecD/F cooperate to facilitate translocation and arrest-cancelation of VemP.

SecD/F works as a monovalent cation-driven motor that pulls up a translocating polypeptide by undergoing a conformational change of the P1 head (Tsukazaki et al., 2011). Such an action could generate a pulling force that cancels the VemP elongation arrest. By contrast, PpiD itself would not generate a pulling force against the nascent chain because it is an ATP-independent chaperone (Matern et al., 2010). It is conceivable that PpiD captures a VemP polypeptide emerging from the translocon and then hand it over to the P1 head domain, which is known as the substrate-binding site of SecD (Furukawa et al., 2017). Possibly, the direct contact between PpiD and SecD facilitates the substrate transfer from PpiD to SecD. It is also possible that the binding of VemP by PpiD helps to prevent the reverse movement of VemP. Since both SecD/F and PpiD are well conserved in enterobacteria including E. coli that possess no VemP protein, the co-operative function of these proteins could be utilized to stimulate late step translocation of some un-identified secretory proteins in these organisms. Although further studies including identification of contact sites of PpiD with the P1 head of SecD and the detailed in vitro study of the late step translocation of VemP and other substrates are needed, it is noteworthy that bacterial species appear to be equipped with a dedicated pulling system acting from the extra cytosolic location for protein secretion and translational control.

We identified Arg-85 as a cis-element important for the in vivo stability of the translation-arrest of VemP, through its role in the regulation of the translocation coupling of the arrest-cancelation. Arg-85 is highly conserved among the VemP orthologues (Figure 5—figure supplement 3), pointing to its importance in the VemP function. This residue does not contribute to the establishment, per se, of the translation-arrested state of VemP, when its secretion is blocked (Figure 5B). However, under the export-proficient conditions, Arg-85 somehow acts to retard the translocation of VemP independently of the translocation arrest of VemP, and consequently increase the PpiD-dependency of the VemP translocation (Figure 5C). Our crosslinking results show that a region encompassing the Arg-74 to Trp-86 interval interacts with the translocon's components in the arrested state of VemP (Figure 6A), indicating that Arg-85 resides in the SecY/E/G-contacting region of the VemP nascent chain (Figure 6A). This spatial arrangement leads us to assume that Arg-85 interacts with the translocon, which inhibits the forward movement of VemP and stabilizes its elongation-arrested state by antagonizing the translocation force that otherwise (when Arg85 is mutated) could lead to a rapid arrest cancelation. Thus, a force strong enough to overcome the arrest must be applied in the case of wild-type VemP to make it competent for regulation.

Our experiments demonstrate that the presence of Arg-85 acts as an obstacle for translocation. However, the trans-side motor SecD/F with the aide from PpiD would overcome the obstacle and manage to allow translocation and arrest-cancelation to take place with a physiological efficiency. When Arg-85 is mutated to other amino acid residues, translocation does not meet any obstacle and proceed very rapidly such that we do not see any significant arrest in the Sec-proficient cells. When the signal sequence of VemP is non-functional or the Sec system is inactivated, the elongation arrest occurs normally and continues stably whether or not Arg85 is mutated.

In any case, the VemP arrest cancelation may require a pulling force by the PpiD-SecD/F system in addition to the force generated by SecA, which alone is insufficient. From this line of considerations, we propose that translation and translocation of VemP are tuned to monitor the periplasmic motor activity of the bacterial Sec system. Remarkably, monitoring substrates employ different force-generation mechanisms to enable real-time monitoring of local events of polypeptide translocation, which is superimposed with temporal elements of translation. The notion that the conserved Arg-85 residue confers the PpiD-SecD/F dependency of the arrest-release of VemP requires further experimental verification, including in vitro recapitulation of arrest cancelation by SecA plus ATP vs PpiD-SecD/F plus proton/sodium-motive forces. A single-molecule analysis to measure the pulling force required for the arrest-release of VemP will also be informative.

Interestingly, SecM also possesses a cis-element that is located outside the ribosome in the arrested translation complex and required for the efficient arrest cancelation (Ito et al., 2018; Nakamori et al., 2014). The regulatory target of SecM is SecA (Nakatogawa and Ito, 2001), and its arrest is canceled at an early stage of translocation, before the cleavage of the signal sequence (Mori et al., 2018; Nakatogawa and Ito, 2001). SecM may monitor the SecA-stimulated early translocation event via its cis-element. Thus, a role of the cis-element in SecM should be different with that of Arg-85 in VemP. Our current results, taken together, reveal that different monitoring substrates monitor different sub-steps of translocation, and a cis-element aids the monitoring function by different mechanisms. Our results suggest that pulling forces applied to the ribosome-nascent-chain complexes can be diverse and differentiated to be sensed and processed precisely by regulatory nascent polypeptides, opening up a new dimension of the force-sensing translation arrest.

All data generated and analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 3 and 4, Figure 2-figure supplements 1, 3 and 4 and Figure 5-figure supplements 1 and 2.

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Acceptance summary:

The study investigates the Vibrio "secretion monitor" VemP, that monirots the secretion capacity of an organism using ribosome stalling. This study has discovered that translation stalling of the secretion monitor VemP in V. alginolyticus is relieved (or cancelled) by the action of PpiD, a periplasmic chaperone that acts with the proteins SecD/F. Failure to relieve the stall allows increased expression of SecD/F to complete this auto-regulatory circuit. The authors further identify a crucial conserved amino acid (R85) in VemP that imposes its dependence on PpiD for translocation.

Decision letter after peer review:

Thank you for submitting your article "Fine interaction profiling of VemP and mechanisms responsible for its translocation-coupled arrest-cancelation" for consideration by eLife. Your article has been reviewed by Suzanne Pfeffer as the Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

The study investigates the Vibrio "secretion monitor" VemP, a less well understood analog of the better studied SecM of E. coli. Although VemP and SecM are not homologous, they are conceptually similar in serving the function of monitoring the secretion capacity of an organism using ribosome stalling. In the SecM system, a translation stall that occurs early during synthesis is relieved when SecA "pushes" the nascent chain during translocation and thereby applies a stall-cancelling force. When SecA is limiting, the stall persists, which allows SecA to be translated as it is located in the same operon as SecM. The VemP system operates in a roughly analogous way, but exactly how its stall is relieved (i.e., what activity is being monitored by this system) is not as well understood. As VemP is in the SecD/F operon, presumably this is involved, but has not been shown.

To investigate this problem, the authors use a site-specific crosslinking approach that has good time resolution. By systematically placing the photo-crosslinking amino acid along the length of VemP, they nicely map all the physical interactions. Using molecular weight, IPs, and analysis in deletion strains, most (but not all) of the main crosslinking partners are identified. This analysis paints a picture of a series of interactions that involve the ribosome exit tunnel, Ffh (the bacterial SRP homolog), the SecYEG translocon, and the periplasmic protein PpiD. Genetic analysis provides support that Ffh is involved in targeting (an expected result), and that PpiD and SecD/F are involved in cancelling the VemP stall. Crosslinking suggests that PpiD directly interacts with SecD/F, suggesting that they cooperate in stall cancellation. An interesting side observation is that mutation of a conserved arginine in VemP (R85), that seems to be within the SecY channel in the stalled state, causes efficient stall cancellation.

The strengths of the study are the interesting question that is being addressed, the high technical quality of the data, and the very systematic analysis of physical interactions using their time-resolved crosslinking method (termed PiXie). The conclusion that SecD/F and PpiD are involved in cancelling the stall at a late stage of translocation is generally convincing. The study would benefit from some greater specificity controls in a couple of key areas. One item that is poorly examined is the role of R85. Although an extensive analysis of this intriguing finding is beyond the scope of the study, some very straightforward characterisation would substantially add to the study and should be considered.

Essential revisions:

1) The authors need to test the relationship between R85 and the dependence for translocation on PpiD and/or SecDF. The reason this is the most important is because these are the newest and most interesting aspects of the study: R85 is the first example of a residue outside the ribosome tunnel that influences stalling/restarting, and this is the first paper to demonstrate that SecD/F and PpiD are involved in stall cancellation. Whether R85 is important because of this residue imposing PpiD or SecD/F dependence seemed to me essential to address.

The most interesting and satisfying result would be if R85 imposes dependence on SecD/F and PpiD. This would mean that VemP has evolved this otherwise unnecessary residue in order to sense functionality of these factors so it can respond accordingly when they are impaired.

2) Although the authors stated that "the results shown are representatives of three technical replicates" in the legend to Figure 2A, the reviewers ask the authors to show other data using biological replicates to confirm the reproducibility in a supplemental figure. If there is an interactor, the kinetics of which is not reproducible, the reviewers recommend omitting the interactor. Such omission would not change the essential point in this work. Also, overall data interpretation by the authors is not quantitative and thus very difficult to follow. In the revised manuscript, the authors should disclose how they assigned the bands and quantitated the intensities in more detail, not only for Figure 2 but also for other figures, in particular Figure 1.

3) In addition, proof that SecA is involved as their model suggests would complete the story. This should also be a straightforward experiment considering the expertise of the authors.

Reviewer 1:

1) All the VemP-interactors has been identified from the anticipation based on the molecular weights of the crosslinked products. Since the crosslinked products are so many other than those the authors selected, there would be other interactors that might be involved in the arrest/cancelation process. Please discuss.

2) Is the proposed mechanism to pull the VemP nascent chain applicable to that for SecM?

3) The deletion of the ppiD gene causes no detectable defect of outer membrane biogenesis (Justice et al., 2005). Is the contribution of PpiD on the translocation specific to VemP?

4) Some of the labels in Figures (e.g., Figure 1—figure supplement 4, gel for secY-his10 strain) are incorrect.

Reviewer 2:

1) The authors find that Ffh, SecD/F, and PpiD all impact stall cancellation. The specificity of these observations would be greater if the authors could show that a translocon component that is not involved in VemP translocation does not impact the stall or cancellation. For example, does acute depletion of YidC have any effect? This experiment, if feasible, is important to exclude the possibility that any perturbation to protein translocation would impact VemP.

2) The crosslinking result in Figure 4E showing the SecD-PpiD interaction would be more convincing and specific if the BpA were placed in another position in the periplasmic side of SecD/F and shown to NOT crosslink to PpiD. This would help exclude the possibility of promiscuous crosslinking from any periplasmic region of SecD/F due to its general proximity to the periplasmic chaperone PpiD.

3) The authors propose that SecA is involved in translocation based on the effect of N3. However, they do not document an interaction of substrate with SecA or a genetic requirement. A substrate interaction would seem to be a strong prediction of their model. As a number of unidentified high molecular weight crosslinking products are observed in Figure 1—figure supplement 1A, it seems plausible that a direct interaction with SecA is indeed detectable. Have the authors checked this? If so, this would strengthen their study because all physical interactions show in their model would have experimental support (and it would really demonstrate how their PiXiE method can be comprehensive).

4) The discovery of a specific role for R85 is the most intriguing finding in the study, but is hardly explored at all. While many experiments could be proposed that are beyond the scope of this study, the one experiment that I feel is within the scope is to document the (potential) relationship between R85 and translocation-dependence on SecD/F/PpiD. If I have understood the author's model, R85 partially 'clogs' in the SecYEG translocon, which makes translocation dependent on SecD/F/PpiD. It is this dependence on SecD/F/PpiD that makes VemP a monitor of a late step in translocation. It seems important to test whether R85 makes VemP translocation dependent on PpiD, independent of arrest. In this experiment, one could analyse W143A versus W143A/R85A for translocation in WT versus ∆PpiD strains. The aim here is to test whether R85 imposes PpiD-dependence to translocation (by monitoring signal cleavage). If so, this would indicate that VemP has evolved and retained R85 in order to make its translocation and arrest cancellation dependent on PpiD, as speculated by the authors.

5) The colour schemes in Figure 1A and Figure 1—figure supplement 1B are not concordant (for example, uL22 is green in Figure 1A, but in Figure 1—figure supplement 1B green is a 100 kD crosslink; same for other partners). It would be more helpful to a reader if these colours matched up. Perhaps a good solution is to make a hybrid figure that combines all of these observations into one nice panel that is provided in the main figure as a revised Figure 1A. This way, a reader can appreciate an overview of all the crosslinks observed (i.e., Figure 1—figure supplement 1B) together with the subset that were verified by IPs (i.e., Figure 1A). One way to do this is to simply take Figure S1B, and under the respective sized crosslinks, put the corresponding protein and dots where this was verified by IPs (i.e., the information Figure 1A).

6) In the Discussion, the authors claim that their uL22 crosslink is in vivo validation of specific interactions seen by cryo-EM. This conclusion is probably overstated. The photo-cross-linking documents close proximity, but cannot be used to infer specific residue contacts. Perhaps reword this to say that the crosslinking "are consistent with" the previous structure.

Reviewer 3:

1) Most of the experiments were performed by expressing V. alginolyticus VemP in E. coli as heterologous host. I assume that this is necessary because an orthogonal tRNA/tRNA synthetase system for pBpa insertion does not exist for V. alginolyticus. This should be mentioned. It could also help if the authors would briefly summarize what is known about SecYEG, SecA, SRP etc in V. alginolyticus. This could strengthen their arguments that the data they observe in E. coli indeed reflect what likely is happening in the native host.

2) Ffh was found to cross-link to only one position within the signal sequence (A15), which I found surprising (Figure 1E), considering that approx. 15 residues of the signal sequence were tested (Figure 1—figure supplement 1). Residue A15 and H22 also show cross-linking products of 150 kDa and > 150 kDa, what are those? The predicted Ffh cross-link is also very weak and not detected by the VemP antibodies. Only after pull down with Ni-NTA reacting with His-tagged Ffh, the cross-link is visible. The predicted Ffh-VemP cross-link migrates at 55-60 kDa, which does not seem to fit the expected size (50 kDa + 15-17 kDa)? Are the authors really sure that this reflects an SRP cross-link?

3) It would have been expected that the authors also observe XL to SecA, which based on their model should interact with the mature domain of VemP. Has this been tested and have the high MW XL at 150 kDa and > 150 kDa (Figure 1—figure supplement 1) been probed with anti-secA antibodies?

4) Considering their model, it would have been expected that SecY cross-links also to the signal sequence, if this is trapped at the lateral gate during translocation. Trapping of the signal sequence and a hairpin-like insertion of the nascent VemP would explain the cross-links to PpiD at residues 30-60 and cross-links to SecY of down-stream residues. This could suggest that these down-stream residues are inside the SecY channel. Then why do we see SecG? How does this fit to the general geometry of the SecYEG translocon?

5) The sequential interaction profile of VemP is very interesting, although I think one has to be very careful with the interpretation: there is a substantial variation in the amount of AP-un, AP-pro and FL VemP for the different pBpa variants displayed in Figure 2. Therefore, the rather small differences in the decrease of XL products could simply reflect these variations.

Thus, although their interpretation is probably correct and fits to the general concept of co-translational targeting, the quantification shown in Figure 2 likely overestimates the accuracy of the cross-linking method.

6) I understand that the authors found no XL to YidC, although it is in close contact to SecY and has like PpiD a large periplasmic domain. The authors might want to refer to the papers of Götzke et al., (2014) and Jauss et al., (2019), who suggested that PpiD and YidC associate with distinct SecYEG populations. This could explain why PpiD but not YidC is found in their cross-linking approach.

7) The authors should explain why PpiD shows two cross-linking products. Does this reflect XL to AP-un and AP-pro? The same applies to Figure 4E: why does SecD359 shows two PpiD cross-linking products of significant size difference (approx. 25 kDa)?

8) The use of the secY25/pSTV28-syd strain as translocation defective strain is probably not the best choice. Why not using a more defined SecY mutant, like the SecY39 or SecY40 strains that have been characterized in Koreaki Itos lab and other labs. The partial suppression of the secY24 mutant by overexpression of the largely uncharacterized protein Syd introduces an ambiguity that could be easily prevented. Along the same line: if the secY25/pSTV28-syd is indeed translocation deficient, isn't it surprising that full-length VemP and processed VemP/MBP are detectable at all?

9) A general comment regarding the figures and XLs: the color of the arrow heads is difficult to see (with the exception of red) and the color code in the main text and Figure 1—figure supplement 1B does not match.

Essential revisions:

1) The authors need to test the relationship between R85 and the dependence for translocation on PpiD and/or SecDF. The reason this is the most important is because these are the newest and most interesting aspects of the study: R85 is the first example of a residue outside the ribosome tunnel that influences stalling/restarting, and this is the first paper to demonstrate that SecD/F and PpiD are involved in stall cancellation. Whether R85 is important because of this residue imposing PpiD or SecD/F dependence seemed to me essential to address.

The most interesting and satisfying result would be if R85 imposes dependence on SecD/F and PpiD. This would mean that VemP has evolved this otherwise unnecessary residue in order to sense functionality of these factors so it can respond accordingly when they are impaired.

We greatly appreciate this highly helpful advice for increasing significance of our finding. According to the suggestion (comment #4) form the reviewer 2, we addressed whether the mutation of the R85 residue cancels the dependency of the VemP translocation on PpiD in the absence of the translation arrest. We first examined effects of the R85W and the W143A mutations, individually or in combination, on the VemP translocation in the wild type and the ΔppiD strains (the data is shown as Figure 5—figure supplement 2). Although the data apparently support that Arg-85 imposes PpiD-dependence in translocation of VemP, it was difficult to obtain a clear conclusion from the data, because the VemP(W143A) underwent reduced, but still significant elongation arrest in the ΔppiD strain. Thus, we next used a VemP(F.S.) mutant, in which the VemP arrest motif sequence had been replaced with an completely different amino acid sequence by a frame shift (F.S.) mutation (Mori et al., (2018)), which allowed us to examine the effect of the R85W mutation on PpiD dependent-VemP translocation under the condition where contribution of the elongation arrest is completely eliminated. The results clearly showed that Arg-85 confers PpiD-dependence to VemP (the experimental data are presented as new Figure 5C). We have described these results and modified the Discussion section accordingly in the revised manuscript. In addition, we have mentioned these results in the Abstract as they should be one of the most important findings obtained in this study, as suggested by the reviewers.

Please note that we have removed the original Figure 5B that represents quantified results of the original Figure 5A, because the data shown in the original Figure 5A was quite clear and the graph in the Figure 5B seemed to be not so informative.

2) Although the authors stated that "the results shown are representatives of three technical replicates" in the legend to Figure 2A, the reviewers ask the authors to show other data using biological replicates to confirm the reproducibility in a supplemental figure. If there is an interactor, the kinetics of which is not reproducible, the reviewers recommend omitting the interactor. Such omission would not change the essential point in this work.

Thank you for the suggestion. We repeated the PiXie experiments for all the VemP(pBPA) derivatives used in the original Figure 2 and Figure 2—figure supplement 1 by using 3 different transformants (biological replicates) to confirm the reproducibility. Although we obtained essentially the same results as the original ones for all the samples examined, the SecY-VemP XLs migrated somewhat broadly on SDS-PAGE and were overlapped with background bands. Unfortunately, in these experiments, the intensities of the background signals substantially were high with unknown reasons. This made exact quantification of the SecY-VemP XLs and their kinetic analysis difficult because they overlap with some of rather strong background signals. Thus, according to the reviewing editor's suggestion, we omitted the kinetic analysis data for the SecY-VemP XLs. Finally, we have removed the original Figure 2 and a part of the original Figure 2—figure supplement 1, and instead included the new Figure 2, in which all the new results of the PiXie experiments obtained with 6 kinds of VemP(pBPA) derivatives are presented.

Also, overall data interpretation by the authors is not quantitative and thus very difficult to follow. In the revised manuscript, the authors should disclose how they assigned the bands and quantitated the intensities in more detail, not only for Figure 2 but also for other figures, in particular Figure 1.

In the experiments shown in the new Figure 1 and the new Figure 1 related supplementary figures, we first assigned bands as the XLs when they were specifically pulled-down by the anti-VemP antibody in the VemP(pBPA)-dependent manner. Regarding the VemP-Ffh XL, we could not detect XLs by using the anti-VemP antibody because a strong background band migrated at the overlapped position with the XL. We thus detected the XL as the one that was specifically pulled-down by Ni-NTA agarose in the VemP(A15pBPA)-dependent manner, when cells expressing the Ffh-His₁₀ was used (Figure 1E, right panel). The assignments were confirmed by one or some of the following experimental results, (i) reactivity of the XLs to the antibody against partner proteins (uL22 (Figure 1B), SecG (Figure 1C), and PpiD (Figure 1—figure supplement 3B)), (ii) disappearance of the XLs in strains lacking the gene for the partner protein (SecG (Figure 1—figure supplement 3A) and PpiD (Figure 1—figure supplement 3B)), and (iii) pull-down of the XLs from cells expressing His₁₀-tagged versions of the partner proteins (SecY(Figure 1D and Figure 1—figure supplement 4A) and PpiD (Figure 1F)). We believe that these results clearly validated our assignment of the XLs.

To show the correctness of the assignment of the XLs in Figure 2, we have added a new Figure as Figure 2—figure supplement 1B, in which the whole gel images of all the pulse-chase experiments shown in the new Figure 2 are presented. The data show that the XLs were generated in a UV-irradiation dependent manner, further confirming that they are pBPA-mediated XLs.

An example (a representative result) of the actual quantification procedure for the immunoprecipitated (IP) bands is shown in the new Figure 2—figure supplement 2 to help understanding by readers. All the raw data used for the quantification are presented in the Source data files. Regarding the Figure 2, all the quantified regions containing IP bands are shown by squares in the representative gel image on the second page of the file. All the quantified and static data of the experimental results have been summarized in the Excel files.

3) In addition, proof that SecA is involved as their model suggests would complete the story. This should also be a straightforward experiment considering the expertise of the authors.

As pointed out by the reviewer 2, we previously showed that the NaN₃ (a SecA inhibitor) treatment of cells severely compromised both translocation and arrest-cancelation of VemP (Mori et al., (2018)). Although it strongly suggests involvement of SecA in the translocation of VemP, we understand that we have not demonstrated direct interaction between VemP and SecA, nor provided genetic evidence about involvement of SecA in the VemP translocation. According to the reviewer 2’s comment, we examined export of VemP in a secA51(Ts) mutant cell by pulse-chase experiments. Our results clearly showed that the translocation and arrest cancelation of VemP was severely and specifically retarded at high temperature in the secA51(Ts) mutant cells, but not in its isogenic wild type cells, indicating that SecA is crucial for VemP biogenesis and its arrest cancelation. We have added the new data as Figure 2—figure supplement 3 and inserted description of these results in the Results.

About the VemP-SecA interaction, several unidentified high molecular weight bands that might represent the VemP-SecA XLs have been observed in Figure 1—figure supplement 1. We examined these bands and found that an XL of ~150 kDa, observed with the VemP(L40pBPA) variant, was precipitated with an anti-SecA antibody. However, the PiXie analysis showed that the pulse-labeled SecA-VemP XL remained stable during the chase periods, in sharp contrast to XLs with other proteins shown in the Figure 2. These results suggest that, while the observed SecA-VemP cross-linking could show the ability of SecA to directly recognize VemP, it may not reflect their physiological interaction that occurs during the translocation of VemP. It would be possible that the off-pathway interaction between VemP and SecA can be stable and more easily detected than the physiological one that could occur in a very short time scale. Considering the above points, we did not analyze the XL of ~150 kDa any further. We have added these results as new Figure 1—figure supplement 2 and explained them in the Results in the revised manuscript.

Reviewer 1:

1) All the VemP-interactors has been identified from the anticipation based on the molecular weights of the crosslinked products. Since the crosslinked products are so many other than those the authors selected, there would be other interactors that might be involved in the arrest/cancelation process. Please discuss.

Thank you for the comment. We basically agree with the reviewer's opinion. We understand that a number of extra bands (possible XLs) were detected with many VemP(pBPA) derivatives. It would be possible that some of the uncharacterized bands might represent the XLs with un-identified partner proteins that contribute to the arrest/cancelation process of VemP. We have added description on this point in the Results in the revised manuscript.

2) Is the proposed mechanism to pull the VemP nascent chain applicable to that for SecM?

As described in the manuscript, the arrest cancelation of VemP occurs at a late step in its translocation after the processing of its signal sequence, as a result of co-operative functioning of SecDF and PpiD. On the other hand, the arrest cancelation of SecM seems to occur at an earlier step of translocation, that is, before the processing of the signal sequence. At this step, SecDF and PpiD would not be able to participate in the translocation/arrest-cancelation of SecM. The regulation system for the SecM arrest cancelation would be suitable for directly monitoring function of SecA ATPase whose expression is controlled by SecM. In addition, SecM seems not to possess a residue corresponding to Arg-85 of VemP that plays a crucial role in the cancelation. Therefore, we have stated that the mechanism proposed in this paper would be utilized for the arrest-cancelation of VemP but not for that of SecM. We have briefly discussed the difference between VemP and SecM in theDiscussion in the revised manuscript.

3) The deletion of the ppiD gene causes no detectable defect of outer membrane biogenesis (Justice et al., 2005). Is the contribution of PpiD on the translocation specific to VemP?

Obviously PpiD is not essential for cell viability and biogenesis of major OMPs, as shown by Justice et al. However, their results would not contradict possible contribution of PpiD to transport of some minor secretory proteins. Since PpiD is well-conserved among enterobacteria including E. coli that possess no VemP protein, we think it possible that PpiD plays physiological/regulatory roles in biogenesis of some kinds of secretory proteins even in E. coli, like in the case of VemP. This point has been discussed in the Discussion in the revised manuscript.

4) Some of the labels in Figures (e.g., Figure 1—figure supplement 4, gel for secY-his10 strain) are incorrect.

We apologize for the mistakes. We have corrected the errors.

Reviewer 2:

1) The authors find that Ffh, SecD/F, and PpiD all impact stall cancellation. The specificity of these observations would be greater if the authors could show that a translocon component that is not involved in VemP translocation does not impact the stall or cancellation. For example, does acute depletion of YidC have any effect? This experiment, if feasible, is important to exclude the possibility that any perturbation to protein translocation would impact VemP.

We completely agree with the above comment. It would be important to obtain data showing that a factor not involving in the VemP translocation does not contribute to the arrest cancelation of VemP. Although the reviewer requested us to examine the effect of YidC depletion on the VemP arrest cancelation, we have not performed this experiment as we think that it would be difficult to obtain a clear conclusion by this approach, from the following reasons. (i) A long-term cultivation (about 5-6 hours) will be required for effective depletion of YidC. Because YidC depletion will affect the biogenesis of many membrane proteins including the translocon components, we might observe side effects caused by a reduction of translocon-components after such longer depletion, even if YidC itself is not directly involved in VemP translocation. (ii) On the other hand, even when we observe no effect on the VemP translocation/arrest cancelation upon YidC-depletion, we cannot exclude the possibility that a small amount of YidC is sufficient for the above processes. (iii) indeed, though we have not detected a YidC-VemP XL so far, we cannot exclude the possibility that YidC contributes to VemP translocation.

Instead, we examined the effect of a deletion of the secB gene that encodes a secretion specific chaperone on the VemP arrest cancelation. It is well known that SecB is a member of the Sec translocation pathway and contributes to the post-translational target of substrates to the SecYEG translocon. Our data clearly show that the knock-out of the secB gene severely retards processing of the signal sequence of MBP, a SecB-dependent substrate, but not impact the VemP translocation and its arrest-cancelation. These results not only indicate that perturbation of some aspects of protein translocation does not necessarily affect the VemP translcation/arrest-cancelation, but also reinforce our conclusion that VemP is co-translationally targeted to the translocon via the SRP pathway. We have added these data as the new Figure 3—figure supplement 1 and its description in the Results in the revised manuscript. We thank the reviewer again for the productive suggestion.

2) The crosslinking result in Figure 4E showing the SecD-PpiD interaction would be more convincing and specific if the BpA were placed in another position in the periplasmic side of SecD/F and shown to NOT crosslink to PpiD. This would help exclude the possibility of promiscuous crosslinking from any periplasmic region of SecD/F due to its general proximity to the periplasmic chaperone PpiD.

Thank you for pointing out an important point. We have performed systematic in vivo pBPA cross-linking experiments targeted to many residues locating at the molecular surface of the first periplasmic domain of SecD, and found that only a specific set of the residues (Asp-359 presented in this paper and its neighboring residues) were cross-linked with PpiD, suggesting that a specific region in SecD is involved in its interaction with PpiD.

Since we want to publish the above cross-linking results elsewhere as a part of a separate paper, we would not like to include the results in this manuscript, if possible. However, if you need to see the data in order to review this work, we will be happy to disclose the unpublished data as a confidential information. If so, please let me know.

3) The authors propose that SecA is involved in translocation based on the effect of N3. However, they do not document an interaction of substrate with SecA or a genetic requirement. A substrate interaction would seem to be a strong prediction of their model. As a number of unidentified high molecular weight crosslinking products are observed in Figure 1—figure supplement 1A, it seems plausible that a direct interaction with SecA is indeed detectable. Have the authors checked this? If so, this would strengthen their study because all physical interactions show in their model would have experimental support (and it would really demonstrate how their PiXiE method can be comprehensive).

Thank you for the valuable comments. Please see our response to the reviewing editor’s comment 3.

4) The discovery of a specific role for R85 is the most intriguing finding in the study, but is hardly explored at all. While many experiments could be proposed that are beyond the scope of this study, the one experiment that I feel is within the scope is to document the (potential) relationship between R85 and translocation-dependence on SecD/F/PpiD. If I have understood the author's model, R85 partially 'clogs' in the SecYEG translocon, which makes translocation dependent on SecD/F/PpiD. It is this dependence on SecD/F/PpiD that makes VemP a monitor of a late step in translocation. It seems important to test whether R85 makes VemP translocation dependent on PpiD, independent of arrest. In this experiment, one could analyse W143A versus W143A/R85A for translocation in WT versus ∆PpiD strains. The aim here is to test whether R85 imposes PpiD-dependence to translocation (by monitoring signal cleavage). If so, this would indicate that VemP has evolved and retained R85 in order to make its translocation and arrest cancellation dependent on PpiD, as speculated by the authors.

Thank you for the productive proposal. Please see our response to the reviewing editor’s comment 1.

5) The colour schemes in Figure 1A and Figure 1—figure supplement 1B are not concordant (for example, uL22 is green in Figure 1A, but in Figure 1—figure supplement 1B green is a 100 kD crosslink; same for other partners). It would be more helpful to a reader if these colours matched up. Perhaps a good solution is to make a hybrid figure that combines all of these observations into one nice panel that is provided in the main figure as a revised Figure 1A. This way, a reader can appreciate an overview of all the crosslinks observed (i.e., Figure 1—figure supplement 1B) together with the subset that were verified by IPs (i.e., Figure 1A). One way to do this is to simply take Figure 1—figure supplement 1B, and under the respective sized crosslinks, put the corresponding protein and dots where this was verified by IPs (i.e., the information Figure 1A).

Thank you for your suggestions to improve our presentation. According to the suggestion, the colors of XLs in the Figure 1—figure supplement 1 was coordinated with the corresponding colors used in Figure 1A to increase clarity. In addition, we have modified the Figure 1 by adding a dashed red line that represents positions at which possible cross-linking with PpiD was observed in the Figure 1—figure supplement 1.

Although we deeply appreciate your helpful suggestion, we would like to show the data in the Figure 1A and those in the Figure 1—figure supplement 1B as separate figures, after introduction of several modifications described above. It is because some XLs observed in the Figure 1—figure supplement 1 would represent non-physiological interactions, as we explained above for the SecA-VemP XLs. We think that the separate presentation of the overall crosslinking profile as revealed by anti-VemP IP (Figure 1—figure supplement 1B) and the XLs that would reflect physiological interaction with the conformed partners (Figure 1A) would facilitate correct understanding of the crosslinking data by readers.

6) In the Discussion, the authors claim that their uL22 crosslink is in vivo validation of specific interactions seen by cryo-EM. This conclusion is probably overstated. The photo-cross-linking documents close proximity, but cannot be used to infer specific residue contacts. Perhaps reword this to say that the crosslinking "are consistent with" the previous structure.

Thank you for the suggestion. It is true that the crosslinking does not necessarily indicate the direct physical contact between the crosslinked proteins. We have changed the text in the Discussion in the revised manuscript, as suggested.

Reviewer 3:

1) Most of the experiments were performed by expressing V. alginolyticus VemP in E. coli as heterologous host. I assume that this is necessary because an orthogonal tRNA/tRNA synthetase system for pBpa insertion does not exist for V. alginolyticus. This should be mentioned. It could also help if the authors would briefly summarize what is known about SecYEG, SecA, SRP etc in V. alginolyticus. This could strengthen their arguments that the data they observe in E. coli indeed reflect what likely is happening in the native host.

Thank you for the valuable comments. According to the suggestions, we have made several additions and modifications to the text in the last part of Introduction, which includes explanation of our heterologous experimental system, brief introduction of the Sec system in Vibrio species and a statement about significance of this study even using the heterologous system.

2) Ffh was found to cross-link to only one position within the signal sequence (A15), which I found surprising (Figure 1E), considering that approx. 15 residues of the signal sequence were tested (Figure 1—figure supplement 1). Residue A15 and H22 also show cross-linking products of 150 kDa and > 150 kDa, what are those? The predicted Ffh cross-link is also very weak and not detected by the VemP antibodies. Only after pull down with Ni-NTA reacting with His-tagged Ffh, the cross-link is visible. The predicted Ffh-VemP cross-link migrates at 55-60 kDa, which does not seem to fit the expected size (50 kDa + 15-17 kDa)? Are the authors really sure that this reflects an SRP cross-link?

With respect to the VemP-Ffh crosslinking, we quantitated the signal intensities of the regions corresponding to the migrating position of the VemP(A15pBPA)-Ffh XL for all the lanes in the gel shown in Figure 1—figure supplement 4B and found that several VemP derivatives containing pBPA at the positions 11, 18, 31, 36 and 46 gave slight but significant signals, suggesting that crosslinking with Ffh occurs not only at the A15 position but also at the other positions within or adjacent to the signal sequence. Thus, we suppose that the Ffh can interact with the VemP signal sequence at several positions (although the interaction at the position A15 might play a major role in this interaction).

It is sometimes observed that apparent molecular sizes of XLs estimated from their mobilities on SDS-PAGE don’t well match their sizes calculated as the sum of molecular sizes of the cross-linked proteins. We think that mobilities of XLs on SDS-PAGE depend on not only molecular sizes but also their shapes (ex. a T-like shape or a X-like shape). Probably, the Ffh-VemP XL would exhibit an anomalous mobility due to the above reason. We concluded the XL observed for VemP (A15pBPA) to be the XL with SRP(Ffh), from the following reasons. (i) The XL was reproducibly and specifically pulled down by Ni-NTA agarose, only when we used the cells expressing His-tagged version of Ffh. (ii) The XL was significantly enhanced by compromising the targeting step of VemP using NaN3 treatment of the cells (See Figure 2—figure supplement 4), which is expected to stabilize the VemP-SRP interaction. (iii) The generation of the VemP-SRP XL is consistent with our genetic/biochemical data that SRP contributes to both VemP translocation and its arrest-cancelation.

3) It would have been expected that the authors also observe XL to SecA, which based on their model should interact with the mature domain of VemP. Has this been tested and have the high MW XL at 150 kDa and > 150 kDa (Figure 1—figure supplement 1) been probed with anti-secA antibodies?

Thank you for the comments. The reviewing editor and the reviewer 2 also raised similar questions. Please see our response to the reviewer editor’s comment 3.

4) Considering their model, it would have been expected that SecY cross-links also to the signal sequence, if this is trapped at the lateral gate during translocation. Trapping of the signal sequence and a hairpin-like insertion of the nascent VemP would explain the cross-links to PpiD at residues 30-60 and cross-links to SecY of down-stream residues. This could suggest that these down-stream residues are inside the SecY channel. Then why do we see SecG? How does this fit to the general geometry of the SecYEG translocon?

As discussed above, the signal sequence of VemP should interact with the lateral gate of SecY at an early step of its translocation and could generate a XL, when pBPA is introduced into the VemP signal sequence. However, the signal sequence of VemP is mostly removed at an early time point of translocation, where translation of VemP is still arrested (note that even after pulse-labeling, the arrested VemP molecules are mostly in the processed form). Thus, even if the pBPA-containing signal sequence of VemP, which had been cleaved off form AP-un, is crosslinked with the SecY, the XLs cannot be isolated by immunoprecipitation using the anti-VemP antibody raised against the epitope in the mature part of VemP. Although the XLs might be able to be isolated by using a His-tag attached to SecY, a small size of the signal sequence will cause only a slight retardation in the mobility of the XL bands, making it difficult to distinguish them from a broad SecY band. Also, a low amount of AP-un having pBPA in the signal sequence will hinder detection of its crosslinking with SecY.

Concerning the cross-linking with SecG, the crystal structure of the SecYEG complex determined by Tanaka et al., (2015) reveals that the transmembrane region of SecG is located on the exterior surface of the N-terminal half of the SecY channel, and the cytoplasmic loop between the TMs 1 and 2 of SecG covers the cytoplasmic cavity of the SecY channel at a resting state. It would be thus conceivable that even at the active state, the cytoplasmic loop of SecG exists near the cytoplasmic cavity of SecY. This would provide one explanation for why SecG as well as SecY is cross-linked with a translocating VemP.

Although we appreciate your comments, which should be useful and very interesting to the researchers working in this field of study, we are afraid that inclusion of the discussion on this point in the main text may interrupt the central story of this study and thus hinder its understanding by many of readers of this journal that would be mostly non-specialists. Thus, we would like to include the above discussions as the Appendix 1, if possible.

5) The sequential interaction profile of VemP is very interesting, although I think one has to be very careful with the interpretation: there is a substantial variation in the amount of AP-un, AP-pro and FL VemP for the different pBpa variants displayed in Figure 2. Therefore, the rather small differences in the decrease of XL products could simply reflect these variations.

Thus, although their interpretation is probably correct and fits to the general concept of co-translational targeting, the quantification shown in Figure 2 likely overestimates the accuracy of the cross-linking method.

We appreciate your comments. We agree that we cannot completely exclude the possibility that difference in the rates of disappearance of these XLs is not due to difference in the timing of the interactions, but is due to variations in the amounts of the VemP(pBPA) derivatives. However, the amounts of AP-un, AP-pro and FL VemP are comparable among the VemP(pBPA) derivatives used in the kinetic study. Also, while we analyzed only one XL for each of uL22 and Ffh as we unfortunately detected a clear crosslinking at a single position for these proteins, two different XLs were used for kinetic analysis of each of SecG and PpiD, and they gave consistent results. In addition, as pointed out by the reviewer, our results are nicely fit with the general concept of co-translational targeting. We thus think that our interpretation is well grounded, but modified the relevant text to make it a more careful description. Specifically, we have changed the words “We demonstrated” in the original manuscript to “The kinetic analyses suggest” in the Discussion in the revised manuscript.

6) I understand that the authors found no XL to YidC, although it is in close contact to SecY and has like PpiD a large periplasmic domain. The authors might want to refer to the papers of Götzke et al., (2014) and Jauss et al., (2019), who suggested that PpiD and YidC associate with distinct SecYEG populations. This could explain why PpiD but not YidC is found in their cross-linking approach.

Thank you for the information. It could be a reason why we did not detect a VemP-YidC XL. We have discussed this point in the Appendix 1 described above.

7) The authors should explain why PpiD shows two cross-linking products. Does this reflect XL to AP-un and AP-pro? The same applies to Figure 4E: why does SecD359 shows two PpiD cross-linking products of significant size difference (approx. 25 kDa)?

We think that pBPA introduced at position 359 in SecD can be cross-linked with two different positions in PpiD that could be located distantly in the primary structure. As described above, it is often observed that XLs generated from the same two proteins exhibit different gel mobilities when their crosslinking sites are different. This is briefly explained in the Results.

8) The use of the secY24/pSTV28-syd strain as translocation defective strain is probably not the best choice. Why not using a more defined SecY mutant, like the SecY39 or SecY40 strains that have been characterized in Koreaki Itos lab and other labs. The partial suppression of the secY24 mutant by overexpression of the largely uncharacterized protein Syd introduces an ambiguity that could be easily prevented. Along the same line: if the secY25/pSTV28-syd is indeed translocation deficient, isn't it surprising that full-length VemP and processed VemP/MBP are detectable at all?

Thank you for the critical comments. We have previously demonstrated that the secY24/psyd system used in this study as well as the secY39 mutation severely compromised translocation of VemP and stabilized its arrested form (Mori et al., (2018)). We indeed observed small amount of full-length VemP and processed VemP/MBP in the secY24/psyd system. However, similar results were obtained, even when the secY39 mutation was used (Please compare of Figure 1B, lanes 1 to 3 and 10 to 12 (secY24/psyd) with Figure S2A (secY39) in the above Mori et al., paper), indicating that complete inhibition of protein translocation is quite difficult using these systems (actually the secY24/psyd system appears to exert slightly stronger effects than secY39). As you mentioned, inactivation of the Sec system using the secY24/psyd was not complete, but we think it sufficient to evaluate the effects of the mutations of VemP on its translocation. Indeed, we were able to detect clear difference between the behaviors of VemP(W143A) and VemP(R85W) under the SecY24/Syd-induced translocation-compromised condition. These previous results and observations, we think, justifies the utilization of the secY24/psyd system.

9) A general comment regarding the figures and XLs: the color of the arrow heads is difficult to see (with the exception of red) and the color code in the main text and Figure 1—figure supplement 1B does not match.

We have changed the color of the arrow heads. Details have been described in the response to the reviewer 2’s comment 5. Please see above.

In addition, please note that we have corrected both the average values of the Ara (-) sample (shown as purple squares) and the lengths of the corresponding error bars in the graph of the Figure 3A, because we found mistakes in the quantified data used for preparation of the original Figure 3A. The corrections do not affect the conclusion. We apologize for the careless mistakes.

We have also included the source data for Figure 2, Figure 3 and Figure 4 and Figure 2—figure supplement 1, Figure 2—figure supplement 3 and Figure 2—figure supplement 4 and Figure 5—figure supplement 1 and Figure 5—figure supplement 2.

Author details

1. Ryoji Miyazaki

   Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan

   Contribution

   Conceptualization, Resources, Data curation, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

2. Yoshinori Akiyama

   Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan

   Contribution

   Resources, Supervision, Funding acquisition, Validation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4483-5408

3. Hiroyuki Mori

   Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Writing - review and editing

   For correspondence

   hiromori@infront.kyoto-u.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0429-1269

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