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Biogenesis of YidC cytoplasmic membrane substrates is required for positioning of autotransporter IcsA at future poles.

ABSTRACT: Localization of proteins to specific sites within bacterial cells is often critical to their function. In rod-shaped bacteria, proteins involved in diverse and important cell processes localize to the cell poles. The molecular mechanisms by which these proteins are targeted to the pole, however, are poorly understood. The Shigella autotransporter protein IcsA, which is localized to the pole on the surface of the bacterium, is targeted to the pole in the cytoplasm by a mechanism that is conserved across multiple Gram-negative bacterial species and has thus served as an important and informative model for studying polar localization. We present evidence that in Escherichia coli, the establishment of polar positional information recognized by IcsA requires the activity of the cytoplasmic membrane protein insertase YidC. We show that the role of YidC in IcsA localization is independent of the cell septation and cytokinesis proteins FtsQ and FtsEX. FtsQ is required for polar localization of IcsA and, based on cross-linking studies, is inserted in the vicinity of YidC, but, we find, is not dependent on YidC for membrane insertion. FtsEX is a YidC substrate, but we find that it is not required for polar localization of IcsA. These findings indicate that polar positional information recognized by IcsA depends on one or more membrane proteins that require YidC for proper membrane insertion.

SUBMITTER: Gray AN

PROVIDER: S-EPMC3911155 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Biogenesis of YidC cytoplasmic membrane substrates is required for positioning of autotransporter IcsA at future poles.

Gray Andrew N AN Li Zaoping Z Henderson-Frost Josephine J Goldberg Marcia B MB

Journal of bacteriology 20131122 3

Localization of proteins to specific sites within bacterial cells is often critical to their function. In rod-shaped bacteria, proteins involved in diverse and important cell processes localize to the cell poles. The molecular mechanisms by which these proteins are targeted to the pole, however, are poorly understood. The Shigella autotransporter protein IcsA, which is localized to the pole on the surface of the bacterium, is targeted to the pole in the cytoplasm by a mechanism that is conserved ...[more]

PMID: 24272775

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Project description:BackgroundAutotransporters are attractive cell surface display vehicles as they lack complex adaptor proteins necessary for protein export. Recent reports have suggested that the native effector domain (? domain) and translocation domain (? domain) interact with each other to drive translocation of the effector domain to the outer membrane. In this report we compared the expression, surface localisation and folding of TEM-1 ?-lactamase (Bla) and maltose binding protein (MalE or MBP) fused to either full length Shigella flexneri IcsA (IcsA) autotransporter or to the ? domain alone (IcsA?) to determine the contribution of the native IcsA ? domain in presenting the fusion proteins on the surface of E. coli K-12 UT5600 (?ompT).ResultsExpression of IcsA-Bla was greater than IcsA?-Bla. High levels of IcsA-MalE were detected but IcsA?-MalE was not expressed. All fusion proteins other than IcsA?-MalE were localised to the outer membrane and were detected on the surface of UT5600 via immunofluorescence microscopy. All bacteria expressing IcsA-MalE were labelled with both ?-IcsA and ?-MBP. UT5600 expressing IcsA?-MalE was not labelled with ?-MBP. A third of UT5600 expressing IcsA-Bla were detectable with ?-Bla but only 5% of UT5600 (IcsA?-Bla) were labelled with ?-Bla. The correct folding of the Bla moiety when fused to IcsA and IcsA? was also retained as UT5600 expressing either fusion protein exhibited a decreased zone of inhibition in the presence of ampicillin. UT5600 expressing IcsA-Bla was more resistant compared to UT5600 expressing IcsA?-Bla.ConclusionsThe export mechanism of autotransporters is not well understood but accumulating evidence suggest a critical role for the native effector or ? domain in facilitating its own export via interactions with the translocation or ? domain. This is the first report directly comparing expression of heterologous proteins fused to the full length IcsA autotransporter and fusion to the ? domain alone. Protein expression and surface presentation of the fusion proteins were dramatically improved when fused to IcsA rather than IcsA?. Future studies involved in designing autotransporters as cell surface display vehicles would benefit from including the native ? domain. This work also provides further evidence for a key interaction between the autotransporter ? and ? domains.

Project description:The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.

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Biogenesis of YidC cytoplasmic membrane substrates is required for positioning of autotransporter IcsA at future poles.

Publications

Biogenesis of YidC cytoplasmic membrane substrates is required for positioning of autotransporter IcsA at future poles.

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