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 Yersinia enterocolitica has two internal hydrophobic segments which might promote its export and association with the cytoplasmic membrane. To determine whether part of HMWP2 could be exported beyond the periplasmic face of the cytoplasmic membrane, we used TnphoA mutagenesis to construct 10 hybrid proteins in which periplasmic alkaline phosphatase (PhoA) was fused to the end of C-terminally truncated HMWP1 (at amino acid positions 1751 and 1753 two independent isolates]) had high alkaline phosphate activity (close to that of the native enzyme), both in Escherichia coli and in Y. pseudotuberculosis, indicating that the PhoA segment of the hybrid reached the periplasm. Deletion studies showed that the export signal resides in the second hydrophobic segment of HMWP2. This result would be compatible with the topology of the protein in the cytoplasmic membrane predicted from the distribution of charged amino acids at either end of the two hydrophobic segments. However, two hybrids in which the junction was even further toward the C terminus of HMMWP2 (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 HMPW2 is mainly cytoplasmic, consistent with its presumed role in the ATP-dependent, nonribosomal synthesis of an unknown peptide. We propose that the high alkaline phosphatase activity associated with some of the HMWP-2-PhoA hybrids results from the unmasking of the cryptic export signal activity in the second hydrophobic segment of HMPW2.
Guilvout
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I
Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur, Paris France.
Carniel
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E
Pugsley
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AP
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000502:physiology; D001426:Bacterial Proteins / Q000032:analysis* / Q000502:physiology; D001483:Base Sequence; D003600:Cytosol / Q000737:chemistry; D033862:Iron-Binding Proteins; D008969:Molecular Sequence Data; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals / Q000032:analysis* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015007:Yersinia / Q000737:chemistry*; D015011:Yersinia pseudotuberculosis / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D033862:Iron-Binding Proteins; D033902:Periplasmic Binding Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase