Twin-arginine translocase mutations that suppress folding quality control and permit export of misfolded substrate proteins
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Author list: Rocco M.A., Waraho-Zhmayev D., DeLisa M.P.
Publisher: National Academy of Sciences
Publication year: 2012
Journal: Proceedings of the National Academy of Sciences (0027-8424)
Volume number: 109
Issue number: 33
Start page: 13392
End page: 13397
Number of pages: 6
ISSN: 0027-8424
eISSN: 1091-6490
Languages: English-Great Britain (EN-GB)
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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 3B - 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 3A 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.
Keywords
Library screening, membrane protein, Protein folding, Protein translocation, Secretory pathway
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