Glycolipid MPIase is essential for the TAT (Twin-Arginine Translocation) pathway
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eLife Assessment
This study demonstrates a critical role of the glycolipid membrane protein insertase (MPIase) in the twin-arginine translocation (Tat) pathway, a protein translocation system that is conserved across all domains of life. The successful reconstitution of the bacterial Tat system in both bacteria-derived and artificial liposomes provides solid experimental evidence that MPIase has a broader role in membrane protein translocation than previously recognized. By revealing the essential function of a nonproteinaceous membrane component in catalyzing a core cellular process, this work offers fundamental insights into the molecular mechanisms of protein translocation.
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Abstract
TAT (Twin-Arginine Translocation) is a preprotein translocation system dedicated to membrane translocation of prefolded proteins in plants and bacteria. The TAT translocon, consisting of the TatABC subunits, drives translocation using proton motive force. However, there have been no reports on the successful reconstitution of the TAT system. In this report, we show that MPIase, a glycolipid that has known to catalyze membrane protein integration, is essential for the TAT system. Our findings in recombinant Escherichia coli demonstrate that overproducing TatABC increases MPIase levels and that depleting MPIase results in TAT precursor accumulation in the cytosol. Furthermore, co-reconstitution of MPIase with TatABC revealed the translocation activities of TAT substrates in a proton motive force-dependent manner. This is the first successful reconstitution of the TAT system and will be advantageous for understanding its mechanisms.
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eLife Assessment
This study demonstrates a critical role of the glycolipid membrane protein insertase (MPIase) in the twin-arginine translocation (Tat) pathway, a protein translocation system that is conserved across all domains of life. The successful reconstitution of the bacterial Tat system in both bacteria-derived and artificial liposomes provides solid experimental evidence that MPIase has a broader role in membrane protein translocation than previously recognized. By revealing the essential function of a nonproteinaceous membrane component in catalyzing a core cellular process, this work offers fundamental insights into the molecular mechanisms of protein translocation.
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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?
(2) Why were only …
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?
(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.
(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.
(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?
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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 …
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.
(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.
(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.
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