Immediate entrance to the export pathway after synthesis as a requirement for export of the sak gene product in Escherichia coli

Abstract

Export through the cytoplasmic membrane and processing of the sak product in Escherichia coli cells were investigated with E. eoli strains carrying pTS301, which produce large amounts of staphylokinase at 42C. High-level synthesis of the sak product caused transient accumulation not only of the staphylokinase precursor (pSAK) but also of the maltose-binding protein and outer membrane protein A precursors. Thus it was concluded that the sak product shares the export pathway with E. coli secreted proteins at least at a certain step. During high-level synthesis of the sak product, a sigiiificant amount of the newly synthesized pSAK remained unprocessed after a chase period, possibly causing the observed accumulation of pSAK. Accumulating pSAK did not mature for a long period, whereas the newly synthesized sak product was exclusively detected in the mature form. These results suggest that it is necessary for the sak product to enter the export pathway during or immediately after synthesis to be exported and processed normally.Many secreted and membrane proteins of both procaryotic and eucaryotic cells are synthesized as larger precursors which have amino-terminal extensions termed signal peptides or leader peptides. It has been suggested that signal peptides serve to transfer polypeptide chains to the membrane and translocate them across the membrane (2, 6, 9, 30, 31). The signal peptides of both procaryotic and eucaryotic secreted proteins have quite similar structural features, some of which indeed promote protein translocation across the membrane of heterologous host cells. In one case, recent works have shown that the product of the penP gene from Bacillus licheniformis is exported and modified at the cysteine residue of the amino terminus, as are lipoproteins of Escherichia coli, in both B. licheniformis and E. coli (4,8) and that the product is localized in the outer membrane of E. coli, as are lipoproteins (26), showing that at least these two bacteria have quite similar export and processing machinery for diglyceride-modified lipoproteins. There have been reported, at the same time, several examples in which heterospecific secreted proteins are exported into the periplasmic space in E. coli (15,28,29, 32). However, the molecular nature of these phenomena remains to be solved.Recently, we have cloned the structural gene for staphylokinase (SAK), which is an extracellular protein produced by certain strains of Staphylococcus aureus, onto plasmid vector pBR322 and have shown that functional SAK is produced and exported into the periplasmic space of E. coli cells (24). From the amino acid sequence of the gene product deduced from the nucleotide sequence (25) and SAK purified from E. coli cells (23), we have identified the signal peptide of 27 amino acids. The signal peptide is different from the lipoprotein signal peptide and functions in both S. aureus and E. coli. However, when the sak product was synthesized at high level, large amounts of the precursor form of SAK (pSAK) accumulated in E. coli cells (23). Si...

Novel prlA alleles defective in supporting staphylokinase processing in Escherichia coli

“…The staphylokinase gene, sak, was isolated from a staphylococcal bacteriophage (32), and the product was shown to be efficiently secreted into the periplasmic space in E. coli by means of its own signal sequence (30). The mechanism of secretion of staphylokinase in E. coli seems to be quite similar to that of endogenous secretory proteins because of its requirement for the secA and prlA (secY) functions (29a).…”

Distinct mutation sites in prlA suppressor mutant strains of Escherichia coli respond either to suppression of signal peptide mutations or to blockage of staphylokinase processing

“…However, since a prlA mutant allele which has been isolated as a suppressor of a particular signal peptide mutation can usually suppress many other signal peptide mutations in the same gene and in the other genes at the same time (3,6,23), it is uncertain that the SecY protein directly interacts with a signal peptide. We recently found that export of the staphylokinase (19) from Staphylococcus aureus is blocked in two of the prlA mutant strains examined (the prlA4 and prlA401 strains; Iino and Sako, submitted for publication), although staphylokinase is efficiently exported to the periplasmic space in the wild-type strain (18). Two causes can be proposed for this blockage: (i) the structure of the signal peptide and (ii) an alteration of the SecY protein by the particular prlA mutation.…”

Secretion of an Aeromonas hydrophila aerolysin by a mutant strain of Escherichia coli

“…The a-haemolysin produced by some strains seems to be one of the few exceptions [1]. When heterospecific secretable proteins are cloned in E. coli, they are mostly not secreted, and remain in the periplasmic space [1][2][3][4][5][6]. A few cases have recently been reported in which foreign proteins appear to be secreted by E. coli [7][8][9][10].…”