A PopZ‐linked apical recruitment assay for studying protein–protein interactions in the bacterial cell envelope
Autor: | Hoong Chuin Lim, Thomas G. Bernhardt |
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Rok vydání: | 2019 |
Předmět: |
Cytoplasm
Centromere Cell Cycle Proteins Peptidoglycan Microbiology Article Bacterial cell structure Protein–protein interaction 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins Cell Wall Caulobacter crescentus Protein Interaction Mapping Escherichia coli Protein Interaction Maps Molecular Biology Cellular compartment 030304 developmental biology 0303 health sciences biology 030306 microbiology Cell Cycle Cell Membrane Chromosomes Bacterial biology.organism_classification Cell biology Bacterial Outer Membrane chemistry Cyclase activity Cell Division Biogenesis Bacterial Outer Membrane Proteins |
Zdroj: | Mol Microbiol |
ISSN: | 1365-2958 0950-382X |
Popis: | Most bacteria are surrounded by a complex cell envelope. As with many biological processes, studies of envelope assembly have benefited from cell-based assays for detecting protein-protein interactions. These assays use simple readouts and lack a protein purification requirement, making them ideal for early stage investigations. The most widely used two-hybrid interaction assay for proteins involved in envelope biogenesis is based on the reconstitution of adenylate cyclase activity from a split enzyme. Because adenylate cyclase is only functional in the cytoplasm, both protein fusions used in the assay must have a terminus located in this compartment. However, many envelope assembly factors are wholly extracytoplasmic. Detecting interactions involving such proteins using two-hybrid systems has therefore been problematic. To address this issue, we developed a cytological assay in Escherichia coli based on PopZ from Caulobacter crescentus. Here, we demonstrate the utility of this PopZ-Linked Apical Recruitment (POLAR) method for detecting interactions between proteins located in different cellular compartments. Additionally, we report that recruitment of an active peptidoglycan synthase to the cell pole is detrimental for E. coli and that interactions between proteins in the inner and outer membranes of the Gram-negative envelope may provide a mechanism for recruiting protein complexes to subpolar sites. |
Databáze: | OpenAIRE |
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