Abstract
Tripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria but poorly characterized. They contain three subunits, a small membrane protein, a large membrane protein, and a substrate-binding protein (SBP). Although the function of the SBP is well established, the membrane components have only been studied in detail for the sialic acid TRAP transporter SiaPQM from Haemophilus influenzae, where the membrane proteins are genetically fused. Herein, we report the first in vitro characterization of a truly tripartite TRAP transporter, the SiaPQM system (VC1777-1779) from the human pathogen Vibrio cholerae. The active reconstituted transporter catalyzes unidirectional Na(+)-dependent sialic acid uptake having similar biochemical features to the orthologous system in H. influenzae. However, using this tripartite transporter, we demonstrate the tight association of the small, SiaQ, and large, SiaM, membrane proteins that form a 1:1 complex. Using reconstituted proteoliposomes containing particular combinations of the three subunits, we demonstrate biochemically that all three subunits are likely to be essential to form a functional TRAP transporter.
Highlights
Vibrio cholerae has a need for sialic acid transport and catabolism for colonization
We have described the in vitro characterization of the sialic acid-specific tripartite ATP-independent periplasmic (TRAP) transporter, VcSiaPQM, from V. cholerae
The siaPQM genes are encoded within the Vibrio pathogenicity island 2 (VPI-2), which is present in strains of V. cholerae that can cause epidemic cholera outbreaks
Summary
Vibrio cholerae has a need for sialic acid transport and catabolism for colonization. Results: The VC1777–1779 genes encode a functional tripartite ATP-independent periplasmic (TRAP) transporter for sialic acid in which the membrane subunits form a tight complex. The active reconstituted transporter catalyzes unidirectional Na؉-dependent sialic acid uptake having similar biochemical features to the orthologous system in H. influenzae Using this tripartite transporter, we demonstrate the tight association of the small, SiaQ, and large, SiaM, membrane proteins that form a 1:1 complex. Often the Q and M genes are fused genetically into a longer gene, QM, and this is the case in the sialic acid-specific TRAP transporter, SiaPQM, from Haemophilus influenzae, where the membrane domains are encoded by the siaQM gene ( called siaT by other authors) [15, 16] In these fused systems, which are not uncommon, there is a predicted extra “linker helix” that is needed to connect the CIN end of the Q subunit with the predicted NOUT location of the M subunit (Fig. 1 and supplemental Fig. 1). That the Q and M subunits form a tight stoichiometric complex in the membrane and present the first biochemical data suggesting that all three subunits are essential for transporter function
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