title: Twin-arginine translocase mutations that suppress folding quality control and permit export of misfolded substrate proteins.
abstract: The bacterial twin-arginine translocation (Tat) pathway facilitates the transport of correctly folded proteins across the tightly sealed cytoplasmic membrane. Here, we report the isolation and characterization of suppressor mutations in the Tat translocase that allow export of misfolded proteins, which form structures that are not normally tolerated by the wild-type translocase. Selection of suppressors was enabled by a genetic assay that effectively linked in vivo folding and stability of a test protein with Tat export efficiency of a selectable marker protein, namely TEM-1 β-lactamase. By using a test protein named α(3)B-a designed three-helix-bundle protein that forms collapsed, stable molten globules but lacks a uniquely folded structure-translocase mutants that rescued export of this protein were readily identified. Each mutant translocase still efficiently exported folded substrate proteins, indicating that the substrate specificity of suppressors was relaxed but not strictly altered. A subset of the suppressors could also export other misfolded proteins, such as the aggregation-prone α(3)A protein and reduced alkaline phosphatase. Importantly, the isolation of genetic suppressors that inactivate the Tat quality-control mechanism provides direct evidence for the participation of the Tat translocase in structural proofreading of substrate proteins and reveals epitopes in the translocase that are important for this process.
authors: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA.
authors: Mark A
authors: MA
authors: Rocco
authors: Dujduan
authors: D
authors: Waraho-Zhmayev
authors: Matthew P
authors: MP
authors: DeLisa
mesh_terms: D000469:Alkaline Phosphatase; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016153:Genes, Suppressor; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D050505:Mutant Proteins / Q000302:isolation & purification; D009154:Mutation / Q000235:genetics*; D017510:Protein Folding; D021382:Protein Sorting Signals; D021381:Protein Transport; D012641:Selection, Genetic; D015398:Signal Transduction / Q000235:genetics; D013379:Substrate Specificity; D013489:Suppression, Genetic
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; D021382:Protein Sorting Signals; C408585:twin-arginine translocase complex, E coli; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli