Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Hanako and colleagues demonstrated that glycolipid MPIase is essential for the TAT system, and they successfully reconstituted the TAT system in vitro for the first time. This will facilitate the understanding of the mechanism of the TAT system.
My major points are listed below for the authors to consider:
(1) The authors successfully reconstituted the TAT system using the purified TatA/B/C, but the translocation efficiency was much lower than that of native INV. The authors partly attributed this to the reason that "MPIase recovery would be too low to detect the TAT activity" in the Discussion part. So, what would happen to the translocation efficiency if you added more MPIase to the reconstituted system? How about the abundance of MPIase from the INV and reconstituted proteoliposomes?
Fig. 3A shows that proteoliposomes reconstituted with active INV were inactive. However, TAT activity was observed when the proteoliposomes were fused with MPIase-containing liposomes. This indicates that MPIase was not sufficiently recovered during reconstitution. Typically, we added MPIase to proteoliposomes at 5% to phospholipids. However, increasing the amount of MPIase to exceed that of INV did not significantly increase the activity. The amounts of MPIase were stated in the text (P17, L3-4).
(2) Why were only TatC levels measured in Figure 2C, whereas the expression levels of TatA were not detected? Also, from my observation, the amount of TatC in the third lane is lower than that in the previous two lanes.
We also wanted to check the TatA levels, but the antibody was no longer available. Here, we confirmed that inactive INVs still contain Tat components.
(3) The authors should explain why the TatA/B/C ratios in Figure 3C (1:1:1) and Figure 3D (10:1:1) are inconsistent.
We examined various TatA/B/C ratios. We found that the ratio of 10:1:1 was better than 1:1:1. This was added to the text (P8, L11-16).
(4) ~30% of the fluorescence was recovered in the membrane fraction (Figure 4A) both in the functional TAT signal sequence (RR) and in the inactivating mutant signal sequence (KK), which suggests that MPIase acts as a relatively broad recognition factor. Given that MPIase does not discriminate between RR and KK, why do un-translocated substrates remain in the cytoplasm rather than non-specifically adhering to the membrane when MPIase is depleted in vivo?
Our results strongly suggest that MPIase acts as a signal sequence receptor. Therefore, in the absence of MPIase, the targeting of TAT precursors should be inhibited. Furthermore, because the TorA signal is less hydrophobic than the Sec signal, the level of nonspecific membrane binding would decrease.
Reviewer #2 (Public review):
Summary:
In this manuscript, the authors investigated the relationship between the Tat system and MPIase, a glycolipid that facilitates protein integration into the bacterial cell membrane. The TAT (twin-arginine translocation) system is a unique membrane transport machinery that exports fully folded proteins containing a twin-arginine signal peptide. Using both in vivo and in vitro approaches, the authors demonstrated that a sufficient amount of MPIase is required for Tat-dependent protein translocation. Furthermore, the authors successfully reconstituted the Tat transport system by combining recombinant TatA, TatB, TatC, MPIase, and FoF1-ATP synthase.
Strengths:
The reconstituted system clearly demonstrated the requirement for each component, as substrate translocation occurred only when all components were present. Based on these findings, the authors proposed a mechanistic role for MPIase in facilitating Tat-mediated membrane translocation. Previous studies have shown that MPIase is involved in Sec-dependent protein translocation and membrane protein integration, as well as YidC-dependent membrane insertion. The present study further demonstrated that MPIase also plays an essential role in the Tat translocation pathway. Overall, this work highlights the central importance of MPIase in bacterial membrane protein biogenesis and provides new insights into the molecular mechanism of Tat-dependent protein transport.
Weaknesses:
(1) To show the importance of the Tat system in bacterial cells, it would be good to describe in the introduction how many proteins are translocated via the Tat system.
The E. coli K12 cells possess 27 TAT substrates. This was added to the text (P2, L14).
(2) Figure 2B and D show that a sufficient amount of MPIase is important in SufI translocation. However, the reason why MPIase level was upregulated in the BL21 strain but not in the KS46 strain remains unexplained. The authors should address this point.
CdsA is a rate-limiting enzyme in MPIase biosynthesis. When MPIase production needs to increase, the cdsA promoters are activated. In KS46, however, cdsA is under the control of the arabinose promoter on a plasmid, so CdsA cannot be induced even when MPIase is necessary. This was added to the text (P6, L26-27).
(3) In Figures 4A and B, the authors explain that MPIase first works as a receptor of TorA-GFP without recognizing the RR motif. This conclusion is based on the results of the fractionation assays, where "sup" indicates the cytoplasmic and periplasmic fractions, and "ppt" indicates the membrane fraction. In Figure 4B, under the TatABC+++, (RR), +MPIase condition, the substrate is secreted most efficiently via the Tat pathway and should therefore be recovered in the periplasm fraction (sup). However, the authors point out that efficiently processed substrate was recovered in the ppt fraction rather than the sup fraction. The authors should explain why this occurred.
We found a mistake in the processing of the results for the sample of the TatABC+++, (RR), +MPIase condition, in Fig. 4B. Therefore, we remeasured the sample and corrected the figure. The relevant text was also modified (P9, L16-29). It was found that a large part of fluorescence was recovered in the supernatant fraction. We also uploaded the raw data for the fluorescence values.
Recommendations for the authors:
Reviewing Editor Comments:
Overall, I agree with the reviewers that the conclusions are reasonably well supported by the data. I have additional scientific/editorial concerns and recommendations:
(A) - Scientific:
(1) P.4 lines 10-15 and Fig. 1 top-left (the effect of overexpressed TatABC).
The authors state that the extent of Surf1 maturation increased upon overexpression of TatABC, but the band intensities for mature Surf1 in the TatABC-induced vs uninduced lanes are very similar.
The expression was weakened: ' The level of the mature form increased....' was changed to ' The level of the mature form slightly increased....'
(2) P.7 lines 23-24, Figure 3A. (the TAT system reconstituted in liposomes).
The authors state that a small portion of Surf1 is successfully translocated and protected by ProK. But unlike the assay results on IMVs, the size of the mature form (i.e., translocated form) appears to be the same as the untranslocated form. Some explanation seems to be needed.
In INV, the signal sequence is cleaved off by Lep, however, the cleavage is not coupled with translocation. In the reconstituted proteoliposomes, such cleavage is hardly observed since the membrane proteins were diluted by fusing with MPIase-containing liposomes. This was added to the text (P7, L26-29).
(3) Based on the model (Figure 5), the interaction between the RR motifs and MPI appears non-electrostatic as the substrate binds to the sugar moieties of the MPIase, not to the pyrophosphate part. Then what is the molecular nature of the substrate-MPI interaction? Some explanation/speculation seems to be needed.
MPIase has been identified as a factor that drives membrane protein insertion. Through the analysis of the mechanisms of insertion, we found that the glycan chain interacts directly with the transmembrane region of the membrane proteins through the numerous acetyl residues on the glycan. Moreover, we found that the positive charges of substrate membrane proteins interact with the pyrophosphate residue through the electrostatic interaction. Therefore, it is reasonable that the h region of the TAT signal binds to the MPIase glycan through the hydrophobic interactions, and the n region including the RR motif binds to the pyrophosphate residue. This was added to the text (P8, L25-27; P11, L14-17).
(B) - Editorial:
(1) P.2. lines 5-10. (Introduction)
The description of the TAT-targeting signal needs to be more clearly described for a broader readership. For example, what are "h" and "c"?
The signal sequence is composed of three regions, 'n', 'h', and 'c' from N-terminus. The n region contains positive charges including the RR motif. The next h region contains a hydrophobic stretch. The c region contains a cleavage site. This was added to the text (P2, L4-11).
(2) P.7 lines 23-24.
What is the rationale for using Pm-Fob-His as a control?
This is a control for a membrane protein unrelated to the TAT system to reveal that MPIase is specifically interacts with TatABC.
(3) P.8 lines 7-8.
CCCP is not defined.
CCCP (Carbonyl cyanide 3-chlorophenylhydrazone) is a protonophore. This was added to the text (P8, L18).
Reviewer #1 (Recommendations for the authors):
(1) The conclusions derived from Figures 1-3 heavily rely on representative immunoblotting images. Given that representative tracks can inherently introduce selection bias, how do the authors ensure the statistical robustness of these findings without quantitative bar graphs and rigorous significance analysis? To fully substantiate these interpretations, it is recommended that these immunoblotting assays be quantified across at least three independent biological replicates.
We did not quantify the results in sections where we presented qualitative discussions. However, we performed all experiments at least three times.
(2) Is it possible that the error values be added after the statistical values of translocation efficiency in Figures 2 and 3?
We added the SD values to some results in Fig. 3.
(3) "MPIase depletion of was then confirmed", an extra "of".
Corrected.
(4) The capitalization style of "TAT" in the whole text is suggested to be unified.
Corrected.
