Functional characterisation of bacterial tripartite ATP-independent periplasmic (TRAP) transporters

Tripartite ATP-independent periplasmic (TRAP) transporters are extracytoplasmic
solute receptor (ESR)-dependent secondary transporters that are widespread in
bacteria, but not eukaryotes. TRAP transporters are composed of an ESR
component and 2 unequally sized integral membrane components. The large
membrane component has 12 predicted transmembrane helices (TMHs) and is
thought to form the translocation channel. The small membrane component is
composed of 4 TMHs and is of unknown, but essential function. TRAP transporters
combine an ESR, which is normally associated with ATP-binding cassette (ABC)
transporters, with the utilisation of electrochemical gradients across the membrane,
using an undefined mechanism.
Analysis of the data contained within the relational database, TRAPdb
(www.trapdb.org), has revealed a number of new insights into TRAP transporter
function, including the observation that marine-dwelling organisms have a
propensity for high numbers of TRAP transporters - possibly indicating a role for
Na+ in the transport cycle.
Structural and functional analysis of TRAP transporters has been limited due to the
recalcitrant nature of integral membrane proteins. The work presented here details
the first overexpression and purification of the integral membrane proteins of a
TRAP transporter. Using Escherichia coli and Lactococcus lactis expression
systems, the integral membrane proteins from the E. coli TRAP transporter,
YiaMNO, and the Haemophilus influenzae TRAP transporter, SiaPQM, have been
expressed and purified.
The entire sialic acid-specific TRAP transporter, SiaPQM, has been functionally
reconstituted into liposomes. The energetic requirements of transport by SiaPQM
have been elucidated revealing that a Na+ gradient in combination with an applied
membrane potential are required to achieve maximal transport rates. Under normal
conditions, SiaPQM is a unidirectional transporter, unlike most other secondary
transporters, however, the presence of excess unliganded ESR is able to induce
efflux of substrate from the proteoliposomes.