The Tat Substrate CueO Is Transported in an Incomplete Folding State

Abstract

In Escherichia coli, cytoplasmic copper ions are toxic to cells even at the lowest concentrations. As a defense strategy, the cuprous oxidase CueO is secreted into the periplasm to oxidize the more membrane-permeable and toxic Cu(I) before it can enter the cytoplasm. CueO itself is a multicopper oxidase that requires copper for activity. Because it is transported by the twin-arginine translocation (Tat) pathway, which transports folded proteins, a requirement for cofactor assembly before translocation has been discussed. Here we show that CueO is transported as an apo-protein. Periplasmic CueO was readily activated by the addition of copper ions in vitro or under copper stress conditions in vivo. Cytoplasmic CueO did not contain copper, even under copper stress conditions. In vitro Tat transport proved that the cofactor assembly was not required for functional Tat transport of CueO. Due to the post-translocational activation of CueO, this enzyme contributes to copper resistance not only by its cuprous oxidase activity but also by chelation of copper ions before they can enter the cytoplasm. Apo-CueO was indistinguishable from holo-CueO in terms of secondary structural elements. Importantly, the binding of copper to apo-CueO greatly stabilized the protein, indicating a transformation from an open or flexible domain arrangement with accessible copper sites to a closed structure with deeply buried copper ions. CueO is thus the first example for a natural Tat substrate of such incomplete folding state. The Tat system may need to transport flexibly folded proteins in any case when cofactor assembly or quaternary structure formation occurs after transport.

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

“…In vitro translocation assays were carried out as described previously (60). As translocation substrate served a T61C variant of the HiPIP precursor, which was fluorescence-labeled by modification of the cysteine with fluorescein-5-maleimide using standard protocols for maleimidecoupling (61).…”

Copper resistance genes of Burkholderia cenocepacia H111 identified by transposon sequencing

“…It has been reported that the twin‐arginine translocation (Tat) system is required for copper resistance in P. syringae DC3000 (Bronstein et al ., ). The Tat system transports folded proteins from the cytoplasm to the periplasm and has been associated with the transport of multi copper oxidases to the periplasm in gram‐negative bacteria (Bronstein et al ., ; Rowland and Niederweis, ; Stolle et al ., ). Our experiments identified the tatB homologue BCAL0324 as a copper resistance gene.…”

Proofreading of substrate structure by the Twin-Arginine Translocase is highly dependent on substrate conformational flexibility but surprisingly tolerant of surface charge and hydrophobicity changes

“…For example, it was shown that several disulphide-bonded proteins (including human growth hormone, a single-chain variable fragment (scFv) and interferon α2β) can be exported in the reduced state in wild type cells [23] and the native Tat substrate CueO is exported in its apo form [24]. However, these proteins are believed to adopt near-native structures even in the absence of disulphide bond formation or Cu ligation, and so the extent of misfolding is likely to be minimal.…”