Change in the cellular localization of alkaline phosphatase by alteration of its carboxy-terminal sequence (original) (raw)
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Mgg Molecular General Genetics, 1990
Alkaline phosphatase (AP) is secreted into the medium when the carboxy-terminal 25 amino acids are replaced by the 60 amino acid carboxy-terminal signal peptide (HlyAs) of Escherichia coli haemolysin (HlyA). Secretion of the AP-HlyA~ fusion protein is dependent on HlyB and HlyD but independent of SecA and SecY. The efficiency of secretion by HlyB/HlyD is decreased when AP carries its own N-terminal signal peptide. Translocation of this fusion protein into the periplasm is not observed even in the absence of HlyB/HlyD. The failure of the Sec export machinery to transport the latter protein into the periplasm seems to be due in part to the loss of the carboxy-terminal sequence of AP since even AP derivatives which do not carry the HlyA signal peptide but lack the 25 C-terminal amino acids of AP are localized in the membrane but not translocated into the periplasm.
A comparative study on the secretion of alkaline phosphatase in Escherichia coli
Journal of the Taiwan Institute of Chemical Engineers, 2009
A B S T R A C T Secretion of alkaline phosphatase (AP), a homodimeric and disulfide bond-containing protein, fused with the YfhG and the TorA signal peptides via the Sec and the Tat pathways, respectively, in Escherichia coli were investigated. The efficiency of AP translocation via the Sec pathway in MC4100, 75.5 AE 0.7%, was higher than that via the Tat pathway, 66.5 AE 0.7%. The amount of active, periplasmic AP, as suggested by the results of AP activity analysis, translocated via the Sec pathway was more than threefold that via the Tat pathway. The efficient secretion of AP via the Sec pathway alleviated protein aggregations, thus reducing the formation of alkaline phosphatase inclusion bodies in the cytoplasm. The translocation of AP in E. coli strain DR473 with relatively oxidative cytoplasm confirms the dual specificity of the YfhG signal peptide for the Sec and Tat machineries. Secretion of AP via the Tat pathway in MC4100, on the other hand, suggests that the formation of disulfide bonds may not be necessary for AP secretion via the Tat pathway. The failure of coexpressing TorD, a molecular chaperone, in enhancing the translocation of AP via the Tat pathway in DR473, and the results of in vitro on-column binding experiments suggest that proper binding to the signal peptide by TorD is a prerequisite for enhancement in protein translocation.
Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion
Proceedings of the National …, 1985
We have constructed a series of plasmids containing a modified form of the phoA gene ofEscherichia coli K-12 that have general utility for studies of protein secretion. In these plasmids, the promoter and signal sequence-encoding region of the phoA gene have been deleted; thus, expression of the gene, giving rise to active alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.11, is absolutely dependent upon fusion in the correct reading frame to DNA containing a promoter, a translational start site, and a complete signal sequence-encoding region. Alkaline phosphatase, which is normally located in the periplasm of E. coli, is efficiently secreted to the periplasm when fused either to a signal sequence from another periplasmic protein, .3-lactamase (penicillin amido-fi-lactamhydrolase, EC 3.5.2.6), or to signal sequences from the outer membrane proteins LamB and OmpF. These heterologous signal sequences are processed during secretion. In the absence of a complete signal sequence, phosphatase becomes localized in the cytoplasm and is inactive. Phosphatase fusion proteins lacking up to 13 amino-terminal amino acids beyond the signal sequence show the same specific activity as that of the wild-type enzyme. However, a significant decrease in activity is seen when 39 or more amino-terminal amino acids are deleted. Addition of approximately 150 amino acids from the enzyme .3-lactamase to the amino terminus of alkaline phosphatase has little effect on the specific activity of the enzyme. The ability to change the amino terminus of phosphatase without altering its activity makes the enzyme particularly useful for construction of protein fusions. The fact that phosphatase is designed for transport across the cytoplasmic membrane makes it an ideal tool for study of protein secretion.
Signal sequence of alkaline phosphatase of Escherichia coli
Journal of Bacteriology, 1982
The amino acid sequence of the signal sequence of phoA was determined by DNA sequencing by using the dideoxy chain termination technique (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, 1977). The template used was single-stranded DNA obtained from M13 on f1 phage derivatives carrying phoA, constructed by in vitro recombination. The results confirm the sequence of the first five amino acids determined by Sarthy et al. (J. Bacteriol. 139:932-939, 1979) and extend the sequence in the same reading frame into the amino terminal region of the mature alkaline phosphatase (Bradshaw et al., Proc. Natl. Acad. Sci. U.S.A., 78:3473-3477, 1981). As was predicted (Inouye and Beckwith, Proc. Natl. Acad. Sci. U.S.A. 74:1440-1444, 1977), the signal sequence was highly hydrophobic. The alteration of DNA sequence was identified for a promoter mutation that results in the expression of phoA independent of the positive control gene phoB and in insensitivity to high phosphate.
Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli
Journal of bacteriology, 1983
A phoA-lacZ gene fusion was used to isolate mutants altered in the alkaline phosphatase signal sequence. This was done by selecting Lac+ mutants from a phoA-lacZ fusion strain that produces a membrane-bound hybrid protein and is unable to grow on lactose. Two such mutant derivatives were characterized. The mutations lie within the phoA portion of the fused gene and cause internalization of the hybrid protein. When the mutations were genetically recombined into an otherwise wild-type phoA gene, they interfered with export of alkaline phosphatase to the periplasm. The mutant alkaline phosphatase protein was found instead in the cytoplasm in precursor form. DNA sequence analysis demonstrated that both mutations lead to amino acid alterations in the signal sequence of alkaline phosphatase.
Phosphorescence of alkaline phosphatase of E. coli in vitro and in situ
Biochimica et biophysica acta, 1981
Escherichia coli K-12, which is rich in alkaline phosphatase, exhibits phosphorescence characteristic of tryptophan at room temperature. E coli mutants which do not have alkaline phosphatase do not show long-lived phosphorescence. The phosphorescence spectrum and lifetime of E. coli K-12 was similar to that of purified alkaline phosphatase from E. coli. These results indicate that the long-lived tryptophan phosphorescence in E. coli is likely to be derived from alkaline phosphatase in situ. The temperature dependence of tryptophan phosphorescence life-time of purified alkaline phosphatase and E. coli K-12 differ; this may imply that alkaline phosphatase in E. coli may be associated with the cell envelope and is therefore protected against structural changes in the protein which result in increased phosphorescence decay rates.
Conditions leading to secretion of a normally periplasmic protein in Escherichia coli
Journal of bacteriology, 1987
The phosphate-binding protein (PhoS) is a periplasmic protein which is part of the high-affinity phosphate transport system of Escherichia coli. Hyperproduction of PhoS in strains carrying a multicopy plasmid containing phoS led to partial secretion of the protein. By 6 h after transfer to phosphate-limiting medium, about 13% of the total newly synthesized PhoS was secreted to the medium. Kinetic studies demonstrated that this secretion consists of newly synthesized PhoS. This secretion occurs in PhoS-hyperproducer strains but not in a PhoS-overproducer strain. Another type of secretion concerning periplasmic PhoS was observed in both PhoS-hyperproducer and PhoS-overproducer strains. This mode of secretion depended upon the addition of phosphate to cells previously grown in phosphate-limiting medium.