Autoproteolysis and Intramolecular Dissociation of Yersinia YscU Precedes Secretion of Its C-Terminal Polypeptide YscUCC

Abstract

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscUCC. Here we show that depletion of calcium induces intramolecular dissociation of YscUCC from YscU followed by secretion of the YscUCC polypeptide. Thus, YscUCC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscUCC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscUCC dissociation for Yop secretion. We propose that YscUCC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal

“…In addition, we recently showed that YscU CC is secreted by the T3SS when bacteria are grown in conditions allowing Yop secretion (26). These previous published studies with the data presented above in the present article, suggested that the C-terminal end of YscU CC would be essential for Yops secretion.…”

“…Previously, we showed by a combination of in vivo and in vitro experiments that YscU auto-proteolysis followed by dissociation and secretion of YscU CC is essential for Yop secretion in Y. pseudotuberculosis during growth in Ca 2ϩ -depleted medium (26). Indeed, we showed that depletion of calcium induced intra-molecular dissociation of YscU CC from the remaining part of YscU.…”

“…Samples were treated as described previously (26) and separated on Tris-Tricine polyacrylamid gels. Proteins were either stained with Coomassie R250 or, alternatively, transferred onto a PVDF membrane for immunoblotting.…”

“…Importantly, addition of ϳ1 mM Ca 2ϩ blocked secretion of both YscU CC and Yops. These results prompted us to suggest that YscU is the actual Ca 2ϩ -sensor in Yersinia T3SS (26). We have here studied the secretion of the early substrate YscU CC and showed that YscU CC encompasses a specific C-terminal T3S signal sequence corresponding to the last 15 residues of the protein (U 15 ).…”

“…Recently, we identified YscU CC as a new early substrate of Y. pseudotuberculosis T3SS (26). YscU is an inner membrane protein with a large cytoplasmic domain denoted YscU C (27).…”