title: A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo.
abstract: Escherichia coli thioredoxin is normally a cytoplasmic protein involved in the reduction of disulfide bonds. However, thioredoxin can be translocated to the periplasm when it is attached to a cotranslational signal sequence. When exported to the periplasm, it can partially replace the activity of DsbA in promoting the formation of disulfide bonds. In contrast, when thioredoxin is fused to a posttranslational signal sequence, very little of it appears in the periplasm. We propose that this absence of posttranslational export is due to the rapid folding of thioredoxin in the cytoplasm. We sought mutants of thioredoxin that retarded its folding in the cytoplasm, which we accomplished by fusing thioredoxin to a posttranslational signal sequence and selecting for mutants in which thioredoxin was exported to the periplasm, where it could replace DsbA. The collection of mutants obtained represents a limited number of amino acid changes in the protein. In vitro studies on purified mutant proteins show that all but one are defective in the kinetics and thermodynamics of protein folding. We propose that the slower folding of the thioredoxin mutant proteins in the cytoplasm allows their export by a posttranslational pathway. We discuss some implications of this class of mutants for aspects of the folding pathway of thioredoxin and for its mechanism of export. In particular, the finding that a folding mutant that allows protein translocation alters an amino acid at the C terminus of the protein suggests that the degree to which thioredoxin folds during its translation must be severely restricted.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Damon
authors.initials: D
authors.lastname: Huber
authors.forename: Myoung-Il
authors.initials: MI
authors.lastname: Cha
authors.forename: Laurent
authors.initials: L
authors.lastname: Debarbieux
authors.forename: Anne-Gaëlle
authors.initials: AG
authors.lastname: Planson
authors.forename: Nelly
authors.initials: N
authors.lastname: Cruz
authors.forename: Gary
authors.initials: G
authors.lastname: López
authors.forename: María Luisa
authors.initials: ML
authors.lastname: Tasayco
authors.forename: Alain
authors.initials: A
authors.lastname: Chaffotte
authors.forename: Jon
authors.initials: J
authors.lastname: Beckwith
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D015153:Blotting, Western; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism; D030562:Databases, Protein; D004220:Disulfides; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry; D005821:Genetic Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D013347:Subcellular Fractions; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D013879:Thioredoxins; D019704:Protein Disulfide-Isomerases; D010100:Oxygen