Effect of membrane phospholipid composition and charge of the signal peptide of Escherichia coli alkaline phosphatase on efficiency of its secretion.

The secretion of the Escherichia coli alkaline phosphatase with a different charge of signal peptide due to replacement of positively charged Lys(-20) has been studied depending on the phospholipid composition of the membranes and the activity of the translocational ATPase--protein SecA. Changing the signal peptide charge, along with a change in phospholipid composition, has been shown to reduce the efficiency of secretion. In the absence of phosphatidylethanolamine the membrane contains anionic phospholipids only, and the dependence of secretion on the signal peptide charge decreases.

Membrane phospholipid composition of Escherichia coli affects secretion of periplasmic alkaline phosphatase into the medium.

Secretion of alkaline phosphatase (PhoA) encoded by a gene constituent of plasmids has been studied in Escherichia coli strains with controlled synthesis of anionic phospholipids (phosphatidylglycerol and cardiolipin, strain HDL11) and zwitterionic phospholipid (phosphatidylethanolamine, strain AD93). Changing the phospholipid composition of the membrane of these strains leads to an increase in secretion of PhoA, which is usually localized in the periplasm, into the culture medium. This correlates with a higher secretion of exopolysaccharides and lower content of lipopolysaccharide in the outer membrane.

Processing of Escherichia coli alkaline phosphatase. Sequence requirements and possible conformations of the -6 to -4 region of the signal peptide.

Analysis of the precursors of bacterial exported proteins revealed that those having bulky hydrophobic residues at position -5 have a high incidence of Pro residues at positions -6 and -4, Val at position -3, and Ser at positions -4 and -2. This led to a hypothesis that the previously observed inhibition of processing by bulky residues at position -5 can be suppressed by introduction of Pro, Ser, or Val in the corresponding nearby positions. Subsequent mutational analysis of Escherichia coli alkaline phosphatase showed that, as it was predicted, Pro on either side of bulky hydrophobic -5 Leu, Ile, or Tyr completely restores efficiency of the maturation.

Study of interaction of export initiation domain of Escherichia coli mature alkaline phosphatase with membrane phospholipids during secretion.

The efficiency of secretion of alkaline phosphatase from Escherichia coli depending on the primary structure of its N-terminal region and the content of zwitterionic phospholipid phosphatidylethanolamine and anionic phospholipids in membranes has been studied in this work to establish the peculiarities of interaction of mature protein during its secretion with membrane phospholipids. It has been shown that the effect of phosphatidylethanolamine but not anionic phospholipids on the efficiency of alkaline phosphatase secretion is determined by the primary structure of its N-terminal region. The absence of phosphatidylethanolamine appreciably reduces the efficiency of secretion of wild type alkaline phosphatase and its mutant forms with amino acid substitutions in positions +5+6 and +13+14.