MECHANISM OF ACTION OF MEMBRANE-BOUND TRANSPORT PROTEINS

G.T. Robillard
Groningen Biomolecular Sciences and Biotechnology Institute, Nyenborgh 4, 9747AG Groningen, The Netherlands

Active transport proteins, ie. those which catalyze the transport of individual molecules per turnover up a concentration gradient, qualify as molecular machines driven by molecular motors. In some cases, the motor is an integral structural part of the machine, in other cases it can be physically separate. In most cases, the motor and machine components can be "uncoupled". These aspects will be illustrated by considering the mannitol transport protein from E. coli.

For the purpose of these considerations, we can simplify the mannitol transporter into a protein consisting of a membrane-bound and a cytoplasmic domain. The membrane-bound domain is responsible for moving mannitol across the membrane and it uses at least three distinct sites (conformations) to do so. It binds mannitol at a site accessible from the periplasm, moves it to an occluded site and finally to a site accessible from the cytoplasm.

Isomerization between the exterior and the occluded site is catalyzed by the input of energy in the form of a phosphorylation/dephosphorylation cycle which enables the accumulation of mannitol against a concentration gradient. In the absence of phosphorylation, the rate of isomerization is a 1000-fold slower and only downhill movement of mannitol is possible.

The intriguing feature of this system is that the phosphorylation/dephosphorylation cycle occurs on the cytoplasmic domain, not the membrane-bound transporter domain and these two domains can be physically separated from one another. Therefore, the mechanism driving the conformational changes responsible for transport must be one of conformational coupling between the two domains.

Data supporting these conclusions will be presented along with the 3D structure of the cytoplasmic domain which provides lots of room for speculation about the nature of the domain interactions.