YERSINIA SPP HMWP2, A CYTOSOLIC PROTEIN WITH A CRYPTIC INTERNAL SIGNAL SEQUENCE WHICH CAN PROMOTE ALKALINE-PHOSPHATASE EXPORT

The iron starvation-induced, 2,042-amino-acid protein HMWP2 of Yersini a enterocolitica has two internal hydrophobic segments which might pro mote its export and association with the cytoplasmic membrane. To dete rmine whether part of HMWP2 could be exported beyond the periplasmic f ace of the cytoplasmic membrane, we used TnphoA mutagenesis to constru ct 10 hybrid proteins in which periplasmic alkaline phosphatase (PhoA) was fused to the end of C-terminally truncated HMWP2. Only the three hybrids in which the fusion junction was just after the second hydroph obic segment of HMWP2 (at amino acid positions 1751 and 1753 [two inde pendent isolates]) had high alkaline phosphatase activity (close to th at of the native enzyme), both in Escherichia coli and in Y. pseudotub erculosis, indicating that the PhoA segment of the hybrid reached the periplasm. Deletion studies showed that the export signal resides in t he second hydrophobic segment of HMWP2. This result would be compatibl e with the topology of the protein in the cytoplasmic membrane predict ed from the distribution of charged amino acids at either end of the t wo hydrophobic segments. How-ever, two hybrids in which the junction w as even further toward the C terminus of HMWP2 (at positions 1793 and 1999) had only weak alkaline phosphatase activity, suggesting that the predicted topology is incorrect. The location of HMWP2 was therefore determined by subcellular fractionation. The results indicate that HMW P2 is mainly cytoplasmic, consistent with its presumed role in the ATP -dependent, nonribosomal synthesis of an unknown peptide. We propose t hat the high alkaline phosphatase activity associated with some of the HMWP2-PhoA hybrids results from the unmasking of the cryptic export s ignal activity in the second hydrophobic segment of HMWP2.