Blackwell Publishing Journal Backfiles 1879-2005
As an initial approach in the study of the mechanism of secretion of the extracellular heat-stable enterotoxin of Escherichia coli (STA), and in order to use this polypeptide as an extracellular carrier we previously constructed a fusion between the complete STA toxin (pre-pro-STA) and the mature B subunit of the periplasmic heat-labile enterotoxin (LTB); the resulting STA-LTB hybrid was not secreted to the extracellular environment, and cells expressing the hybrid lysed at temperatures above 35°C. In this work we have established that the hybrid is initially detected as pre-pro-STA-LTB and converted to pro-STA-LTB, which lacks the 19amino acids that share the properties of a signal peptide; the sequenced 17 amino-terminal residues of pro-STA-LTB defined the processing site of pre-pro-STA-LTB at pro_3phe_2ala_1 ↓ gln+1. This process was sensitive to an energy uncoupler (CCCP) and was correlated with translocation of pro-STA-LTB across the inner membrane. Additionally, we are able to show that although pre-pro-STA-LTB is processed at 37°C and 29°C, it is more efficiently processed at the latter temperature. At 37°C, pro-STA-LTB was poorly released into the periplasm, resulting in accumulation of this protein, pre-pro-STA-LTB, and pre-β-lactamase in the inner membrane, and in cell lysis. In contrast, at 29°C pro-STA-LTB was localized in the periplasm and in the inner membrane, and pre-pro-STA-LTB and pre-β-lactamase did not accumulate; however, translocation of periplasmic pro-STA-LTB across the outer membrane still did not occur, and a second processing step that would eliminate the pro segment from pro-STA-LTB was never observed. Thus, the fusion of pre-pro-STA and LTB resulted in a polypeptide that, while incompatible with secretion to the extracellular medium, is exported to the periplasm in a temperature-conditional fashion. This latter observation is consistent with an STA secretion pathway whereby pre-pro-STA is first processed to periplasmic pro-STA by the removal of 19-amino-acid signal peptide.
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