The role of the positively charged N-terminus of the signal sequence of E. coli outer membrane protein PhoE in export

Bosch, Dirk; Boer, Paul de; Bitter, Wilbert; Tommassen, Jan

doi:10.1016/0005-2736(89)90524-5

Cited by 46 publications

(19 citation statements)

References 28 publications

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“…Bacterial SPs with a negatively charged n-region have generally been found to be exported more slowly (Vlasuk et al, 1983;Iino, Takahashi & Sako 1987;Lehnhardt et al, 1988;Bosch et al, 1989;Puziss, Fikes & Bassford, 1989), but no absolute requirement for positive charge is apparent.…”

Section: A Positively Charged N-region Is Required For Efficient Tranmentioning

confidence: 96%

The signal peptide

1990

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Section: A Positively Charged N-region Is Required For Efficient Tranmentioning

confidence: 96%

The signal peptide

1990

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“…The carboxy-terminal fragment 111 (Ile-9 -A X 1 ) , which has no net charge, reduced the translocation activity, but 50% inhibition was not reached within the concentration range tested (up to 50 pM). Extension of the signal peptide with the first seven amino acids of the PhoE mature part did not change the inhibitory effect ( [32]. The analogously substituted [Asp-19.…”

Section: Effect Of Signal Peptides On Protein Translocationmentioning

confidence: 99%

Inhibition of PhoE translocation across Escherichia coli inner‐membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation

Vrije

Batenburg

Jordi

et al. 1989

European Journal of Biochemistry

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To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (po1y)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated Escherichia coli proteins destined for the periplasmic space or outer membrane are synthesized in the cytoplasm as precursor proteins with an N-terminal signal peptide. This signal peptide is essential for precursor protein translocation across the inner membrane (as reviewed in [l]). Furthermore, this translocation process is energy-dependent [2 -61 and requires in addition protein factors (as reviewed in [7]). The signal peptides do not show sequence homology, but they are characterized by a basic amino terminus, a hydrophobic core of 10 -15 amino acid residues and a C-terminal polar section [S]. In the eukaryotic secretion pathway the analogous signal peptides show molecular interactions with the signal recognition particle [9, lo], putative signal sequence receptors [ l l , 121 and signal peptidase [13]. In the prokaryotic system, apart from the leader peptidase [14], no protein components directly interacting with the signal peptide have yet been identified. The structure of the signal peptides seems to be very well suited for a direct interaction with the lipid component of the membrane. A negatively charged lipid-specific insertion of synthetic signal peptides has indeed been demonstrated in model membrane experiments [lS -171.Synthetic signal peptides have been used to gain insight into the mechanism of protein translocation. They are able to inhibit precursor protein translocation in vitro into microCorrespondence to T.

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“…5B and D), although the inhibition was stronger. In contrast to the lysine mutant proteins, the arginine constructs R [5,6] and R [13,14] had almost the same level of inhibition, followed by more sharp restoration of translocation for R [19,20]. Unlike the study of enzyme activity, the pulse-chase experiments did not reveal the inhibition for K [29,30] and R [29,30] mutant proteins 60 min after the pulse.…”

Section: Statistical Analysis Of Net Charge Distributionmentioning

confidence: 76%

“…It was also suggested that net positive or neutral charge difference between the N-and C-terminal regions of the signal peptide may be required for protein secretion (51). Further mutational analysis has shown that this N-C charge imbalance may not be obligatory for translocation of proteins across the cytoplasmic membrane of bacteria (5,37,43). Several experimental works suggested that the charge-sensitive mature sequence critical for protein export in bacteria may be extended to 15 (47), 20 (27), and even 30 residues (1).…”

mentioning

confidence: 99%

The Net Charge of the First 18 Residues of the Mature Sequence Affects Protein Translocation across the Cytoplasmic Membrane of Gram-Negative Bacteria

et al. 2000

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This statistical study shows that in proteins of gram-negative bacteria exported by the Sec-dependent pathway, the first 14 to 18 residues of the mature sequences have the highest deviation between the observed and expected net charge distributions. Moreover, almost all sequences have either neutral or negative net charge in this region. This rule is restricted to gram-negative bacteria, since neither eukaryotic nor grampositive bacterial exported proteins have this charge bias. Subsequent experiments performed with a series of Escherichia coli alkaline phosphatase mutants confirmed that this charge bias is associated with protein translocation across the cytoplasmic membrane. Two consecutive basic residues inhibit translocation effectively when placed within the first 14 residues of the mature protein but not when placed in positions 19 and 20. The sensitivity to arginine partially reappeared again 30 residues away from the signal sequence. These data provide new insight into the mechanism of protein export in gram-negative bacteria and lead to practical recommendations for successful secretion of hybrid proteins.

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The role of the positively charged N-terminus of the signal sequence of E. coli outer membrane protein PhoE in export

Cited by 46 publications

References 28 publications

The signal peptide

The signal peptide

Inhibition of PhoE translocation across Escherichia coli inner‐membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation

The Net Charge of the First 18 Residues of the Mature Sequence Affects Protein Translocation across the Cytoplasmic Membrane of Gram-Negative Bacteria

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