Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or β‐galactosidase fused to the Hly C‐terminal signal domain

Abstract

Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA, when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the beta-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain.

Determinants of extracellular protein secretion in gram-negative bacteria

“…Not surprisingly, PddA, -B, -C, and -D are homologs of PulG, -H, -I, and -J. Amino-terminal cleavage of the leader sequence from the precursor of PulG by PulO has also been confirmed (47). Other homologs of type IV pilins involved in extracellular secretion of enzymes from E. chrysanthemi and X. campestris have also recently been described (7,15 mammalian prochymosin (25). In each case, only a fraction of hybrid protein synthesized was released from E. coli; however, the observed extracellular secretion was always dependent on the presence of the HlyB-HlyD translocator proteins.…”

Secretion of the Caulobacter crescentus S-Layer Protein: Further Localization of the C-Terminal Secretion Signal and Its Use for Secretion of Recombinant Proteins

“…Figure 2A shows that aggregated protein could be recovered from the culture fluids of cells synthesizing RsaA242C, RsaA166C, RsaA134C, and RsaA119C, indicating that all of these C-terminal peptides of RsaA were capable of autonomous secretion. The autonomous secretion of the 119-aa C-terminal peptide indicated that an RTX motif was not absolutely required for secretion, a conclusion reached for other proteins secreted by type I systems (10,12,16,18).…”

“…Although the presence or absence of the RTX sequences is unlikely to be responsible for the aggregation of hybrid proteins, these sequences are likely to affect the kinetics and efficiency of the secretion process (11). It has also been shown that the importance of including RTX motifs in a hybrid protein varies with the particular passenger protein (10,18,19).…”

“…Proteins secreted by type I systems typically exhibit two features: (i) an extreme C-terminal secretion signal located within the last 60 amino acids (aa) which is not cleaved as part of the secretion process and (ii) more-Nterminal glycine-rich ("RTX") motifs, nine-residue sequences which include the consensus sequence GGXGXD. These motifs are responsible for Ca 2ϩ binding and are also thought to be important for proper presentation of the secretion signal to the secretion machinery (3,10,18,19). RsaA possesses six RTX motifs which exactly match the consensus sequence, but the exact location of the secretion signal is unclear (13).…”

“…Figure 2A shows that aggregated protein could be recovered from the culture fluids of cells synthesizing RsaA242C, RsaA166C, RsaA134C, and RsaA119C, indicating that all of these C-terminal peptides of RsaA were capable of autonomous secretion. The autonomous secretion of the 119-aa C-terminal peptide indicated that an RTX motif was not absolutely required for secretion, a conclusion reached for other proteins secreted by type I systems (10,12,16,18).The culture fluids of cells synthesizing RsaA242C, RsaA166C, RsaA134C, and RsaA119C were subjected to ice-cold trichloroacetic acid (TCA) precipitation (final concentration, 10%, wt/vol) following sequential course filtration through nylon mesh. When the TCA precipitate was examined by SDS-* Corresponding author.…”