Related Experiment Videos
Yersinia spp. HMWP2, a cytosolic protein with a cryptic internal signal sequence which can promote alkaline
I Guilvout1, E Carniel, A P Pugsley
1Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur, Paris France.
Abstract:
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.
Insights
The Yersinia enterocolitica protein HMWP2, involved in iron starvation response, is primarily cytoplasmic, not membrane-associated as initially hypothesized. Its export signal is located in the second hydrophobic segment.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Biochemistry
Background:
- The iron starvation-induced protein HMWP2 from Yersinia enterocolitica possesses hydrophobic segments suggesting membrane association and potential export.
- Understanding HMWP2's localization is crucial for elucidating its role in bacterial physiology.
Purpose of the Study:
- To investigate the subcellular localization of Yersinia enterocolitica's HMWP2 protein.
- To determine if HMWP2 is exported beyond the cytoplasmic membrane and identify potential export signals.
Main Methods:
- Construction and analysis of hybrid proteins using TnphoA mutagenesis, fusing periplasmic alkaline phosphatase (PhoA) to truncated HMWP2.
- Deletion studies to pinpoint the region responsible for export signals.
- Subcellular fractionation to determine the precise location of HMWP2.
Main Results:
- Hybrid proteins with PhoA fused to HMWP2 (at positions 1751 and 1753) exhibited high alkaline phosphatase activity, indicating periplasmic localization of PhoA.
- Deletion studies identified the second hydrophobic segment of HMWP2 as containing an export signal.
- Subcellular fractionation revealed HMWP2 is predominantly cytoplasmic, contradicting initial predictions of membrane association.
Conclusions:
- The predicted topology of HMWP2 in the cytoplasmic membrane appears incorrect.
- HMWP2 is mainly a cytoplasmic protein, likely involved in ATP-dependent, nonribosomal peptide synthesis.
- The observed alkaline phosphatase activity in hybrids may result from the unmasking of a cryptic export signal within HMWP2's second hydrophobic segment.