title: Cytosolic selection systems to study protein stability.
abstract: Here we describe biosensors that provide readouts for protein stability in the cytosolic compartment of prokaryotes. These biosensors consist of tripartite sandwich fusions that link the in vitro stability or aggregation susceptibility of guest proteins to the in vivo resistance of host cells to the antibiotics kanamycin, spectinomycin, and nourseothricin. These selectable markers confer antibiotic resistance in a wide range of hosts and are easily quantifiable. We show that mutations within guest proteins that affect their stability alter the antibiotic resistances of the cells expressing the biosensors in a manner that is related to the in vitro stabilities of the mutant guest proteins. In addition, we find that polyglutamine tracts of increasing length are associated with an increased tendency to form amyloids in vivo and, in our sandwich fusion system, with decreased resistance to aminoglycoside antibiotics. We demonstrate that our approach allows the in vivo analysis of protein stability in the cytosolic compartment without the need for prior structural and functional knowledge.
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Ajamaluddin
authors.initials: A
authors.lastname: Malik
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Antje
authors.initials: A
authors.lastname: Mueller-Schickert
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA jbardwel@umich.edu.
authors.forename: James C A
authors.initials: JC
authors.lastname: Bardwell
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015374:Biosensing Techniques / Q000379:methods; D003600:Cytosol / Q000737:chemistry*; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005827:Genetics, Microbial / Q000379:methods; D008967:Molecular Biology / Q000379:methods*; D055550:Protein Stability; D012641:Selection, Genetic
chemical_list: D000900:Anti-Bacterial Agents