Hydrophobic mismatch is a key factor in protein transport across lipid bilayer membranes via the Tat pathway

Hao, Binhan; Zhou, Wenjie; Theg, Steven M.

doi:10.1016/j.jbc.2022.101991

Cited by 12 publications

(16 citation statements)

References 83 publications

(106 reference statements)

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“…We then screened these pep86-tagged SufI constructs to determine their suitability for the transport assay. A conventional in vivo transport assay (25) was carried out to evaluate the performance of both pep86 variants by isolating the periplasmic fraction of cells co-expressing either wild-type TatABC (i.e., wtTat) or ΔtatA, together with SufI, SufI-Cpep86, or Npep86-SufI, followed by SDS-PAGE and immunoblotting (Figure 1C-E). Cells with the wild-type Tat expressing either SufI-Cpep86 or Npep86-SufI achieved more than 50% of transport relative to the wild-type Tat system transporting non-pep86-tagged SufI (Figure 1D).…”

Section: Resultsmentioning

confidence: 99%

“…Plasmids pTat101 or ΔtatA were co-transformed with pQE80l (SufI-FLAG), pQE80l (Npep86-SufI-FLAG), or pQE80l (SufI-Cpep86-FLAG), which is under the control of the T5 promoter, into the Tat knockout strain, DADE-A. Details of the in vivo transport assay was described elsewhere (25). Overnight cultures were diluted into the fresh LB medium.…”

Section: In Vivo Transport Assaymentioning

confidence: 99%

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A Real-Time Analysis of Protein Transport via the Twin Arginine Translocation Pathway in Response to Different Components of the Protonmotive Forcein vivo

Zhou

Hao

Bricker

et al. 2023

Preprint

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The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the Δψ component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology (NanoBiT) in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions, ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

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Section: Resultsmentioning

confidence: 99%

Section: In Vivo Transport Assaymentioning

confidence: 99%

A Real-Time Analysis of Protein Transport via the Twin Arginine Translocation Pathway in Response to Different Components of the Protonmotive Forcein vivo

Zhou

Hao

Bricker

et al. 2023

Preprint

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“…The following TMH of TatA is very unusual: Being too short to span a membrane of normal thickness, it generates a hydrophobic mismatch with the membrane [ 75 ][ 77 ]. It consists of only 12 hydrophobic residues, flanked by the above-mentioned polar residue at the N-terminal side and a strictly conserved glycine residue on its C-terminal side ( Figure 4B ).…”

Section: Learning From Tata a Tat Translocon Component That Resembles...mentioning

confidence: 99%

“…None of the hydrophobic residues is essential as they all can be exchanged by cysteine without losing functionality [ 73 ]. The hydrophobic mismatch of this TMH has been shown to generate membrane stress by thinning the membrane in case of multiple clustered TatA protomers [ 78 ][ 75 ][ 77 ]. MD simulations indicated that the adjacent APH plays an important role for this membrane thinning, as laterally aligned APHs remove lipid head groups from the region, which disturbs the lipid bilayer and causes the formation of a deep V-shaped groove in the membrane [ 75 ].…”

Section: Learning From Tata a Tat Translocon Component That Resembles...mentioning

confidence: 99%

“…Non-lytic holin-mediated transport could in principle be achieved also by gated pore mechanisms or by other types of substrate-selective hydrophilic pores that are formed by defined arrangements of holins only. Such usual mechanisms have also been postulated for TatA in early years [ 98 , 99 ], before the “membrane-weakening and pulling” hypothesis was published [ 79 ], and before TatA truncational analyses [ 69 ], the demonstration of transport with signal peptides cross-linked to TatC [ 100 ], NMR structures [ 66 ], the demonstration of membrane-weakening [ 78 ][ 77 ], and MD simulations [ 75 ] argued against these older models. To achieve an autonomous transport of folded proteins, usual transporters would require a large size with domains for a large channel, gating, energy coupling etc., and as even the largest holins are rather small membrane proteins, it seems unlikely that holins are large enough to enable such mechanisms.…”

Section: Tpee-family Holins Need To Catalyze Transport Only Once Wher...mentioning

confidence: 99%

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Occurrence and potential mechanism of holin-mediated non-lytic protein translocation in bacteria

Brüser¹,

Mehner-Breitfeld²

2022

Microb Cell

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Holins are generally believed to generate large membrane lesions that permit the passage of endolysins across the cytoplasmic membrane of prokaryotes, ultimately resulting in cell wall degradation and cell lysis. However, there are more and more examples known for non-lytic holin-dependent secretion of proteins by bacteria, indicating that holins somehow can transport proteins without causing large membrane lesions. Phage-derived holins can be used for a non-lytic endolysin translocation to permeabilize the cell wall for the passage of secreted proteins. In addition, clostridia, which do not possess the Tat pathway for transport of folded proteins, most likely employ non-lytic holin-mediated transport also for secretion of toxins and bacterioc-ins that are incompatible with the general Sec pathway. The mechanism for non-lytic holin-mediated transport is unknown, but the recent finding that the small holin TpeE mediates a non-lytic toxin secretion in Clostridium perfringens opened new perspec-tives. TpeE contains only one short transmembrane helix that is followed by an amphipathic helix, which is reminiscent of TatA, the membrane-permeabilizing component of the Tat translocon for folded proteins. Here we review the known cases of non-lytic holin-mediated transport and then focus on the structural and functional comparison of TatA and TpeE, resulting in a mechanistic model for holin-mediated transport. This model is strongly supported by a so far not recognized naturally occurring holin-endolysin fusion protein.

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Bacterial Protein Transport Pathways and Analogous Conserved Pathways in Eukaryotes

Kauffman,

Kaushik,

Kuhn

et al. 2024

Endosymbiotic Organelle Acquisition

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Hydrophobic mismatch is a key factor in protein transport across lipid bilayer membranes via the Tat pathway

Cited by 12 publications

References 83 publications

A Real-Time Analysis of Protein Transport via the Twin Arginine Translocation Pathway in Response to Different Components of the Protonmotive Forcein vivo

A Real-Time Analysis of Protein Transport via the Twin Arginine Translocation Pathway in Response to Different Components of the Protonmotive Forcein vivo

Occurrence and potential mechanism of holin-mediated non-lytic protein translocation in bacteria

Bacterial Protein Transport Pathways and Analogous Conserved Pathways in Eukaryotes

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