Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope.

Wolfe, P B; Wickner, William; Goodman, Joel M.

doi:10.1016/s0021-9258(17)44342-0

Cited by 292 publications

(20 citation statements)

References 30 publications

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“…Outer membrane proteins of gram-negative bacteria are in a sense secretory proteins because they have to traverse the plasma membrane before reaching the outer membrane. As such, they are synthesized with cleavable signal sequences and require two forms of energy and active SecA and SecY proteins for their export, On the other hand, most inner membrane proteins (1, 7,17,18,22,30,32,46,52,60) are not proteolytically processed when incorporated into the lipid bilayer of the plasma membrane. The only representative of these proteins studied in detail so far is the bacterial signal peptidase (leader peptidase, signal peptidase I).…”

mentioning

confidence: 99%

In vitro membrane assembly of a polytopic, transmembrane protein results in an enzymatically active conformation.

Ahrem

Hoffschulte

Müller

1989

The Journal of Cell Biology

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Abstract. In vitro integration of the polytopic, transmembrane lactose permease into membrane vesicles from Escherichia coli is demonstrated. To this end the enzyme was synthesized in a homologous, cell-free transcription-translation system. In this system, synthesis occurred in an essentially membrane-free environment leading to the formation of lactose permease aggregates, which were resistant to protease digestion and detergent solubilization. However, if inverted membrane vesicles from E. coli were included in the synthesis reaction, most de novo-synthesized lactose permease could be recovered from a membranecontaining subfraction (enriched in leader [signal] peptidase activity). This membrane association of lactose permease was Na2CO3 resistant, detergent sensitive, and yielded a distinct pattern of proteolytic cleavage peptides. Moreover, membrane vesicles when present cotranslationally during synthesis of lactose permease, acquired the capability to accumulate lactose, strongly suggesting a correct in vitro assembly of the enzyme.Because of the extensive aggregation of lactose permease synthesized in the absence of membranes, only low amounts originating from the soluble enzyme pool integrated posttranslationally into the membrane vesicles. Unlike the translocation of the outer membrane protein LamB into membrane vesicles, integration of lactose permease was found to be independent of the H+-motive force.

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mentioning

confidence: 99%

In vitro membrane assembly of a polytopic, transmembrane protein results in an enzymatically active conformation.

Ahrem

Hoffschulte

Müller

1989

The Journal of Cell Biology

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“…1A. The backbone of the design is from E. coli LepB, an inner membrane protein with two N‐terminal TMHs and a periplasmic C‐terminal domain [19]. The model 19‐residue TMH of composition 4L/15A or 6L/13A (H‐segment) followed by the SecM( Ec ) AP is fused to the C terminus of the LepB part.…”

Section: Resultsmentioning

confidence: 99%

Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

et al. 2022

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During SecYEG‐mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N‐terminal end is ~ 45 residues away from the peptidyl transferase centre. However, we recently uncovered instances where the first contact is delayed by up to ~ 10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the Escherichia coli inner membrane protein BtuC: a positively charged loop and a re‐entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re‐entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion.

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“…Here, we have used a series of constructs harboring a force-generating model TMH (H-segment) of composition n L/(19- n )A ( n = 4, 6) placed L residues away from the C-terminal end of the 17-residue E.coli SecM (SecM( Ec )) AP followed by a 23-residue C-terminal tail [8], Figure 1a. The backbone of the design is from E. coli LepB, an inner membrane protein with two N-terminal TMHs and a periplasmic C-terminal domain [19]. The model 19-residue TMH of composition 4L/15A or 6L/13A (H-segment) followed by the SecM( Ec ) AP is fused to the C terminus of the LepB part.…”

Section: Resultsmentioning

confidence: 99%

Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

Nicolaus

Ibrahimi

Besten

et al. 2021

Preprint

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During SecYEG-mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N-terminal end is ~45 residues away from the peptidyl transferase center. However, we recently uncovered instances where the first contact is delayed by up to ~10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the BtuC protein: a positively charged loop and a re-entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re-entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion.

show abstract

Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope.

Cited by 292 publications

References 30 publications

In vitro membrane assembly of a polytopic, transmembrane protein results in an enzymatically active conformation.

In vitro membrane assembly of a polytopic, transmembrane protein results in an enzymatically active conformation.

Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

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