The bacterial plasma membrane, consisting of a lipid bilayer with associated proteins, is the prime permeability barrier for the bacterial cell, separating the cytoplasm from the bacterium's environment. During the life of the bacterial cell, new proteins must be inserted into the membrane, either partially (in the case of membrane proteins) or completely (in the case of secretory proteins that are translocated across the membrane). It is recognized that different proteins utilize different mechanisms to achieve this insertion or translocation, and a variety of protein translocation mechanisms have been described. The overall goal of Dr. Dalbey's laboratory is to understand how proteins insert into or across membranes and achieve their correct asymmetric topologies.
The bacterial plasma membrane is in fact a charged (energized) capacitor, with both an electrical potential and a hydrogen ion (proton) concentration gradient across it. The energy stored in this capacitor is termed the proton motive force, or PMF, and serves as the energy source for a variety of bacterial metabolic activities that take place at the membrane. This project addresses the role of the PMF in the insertion of newly synthesized proteins into bacterial membranes. This role is presently poorly understood. There are a number of possible roles the PMF may play: it may directly affect the translocating membrane protein; it may activate a protein machinery to promote insertion; or some combination of the two. Recently, it has been shown in bacteria that the translocation of negatively charged amino acids ("residues") across the membrane can be driven by the PMF, and that negatively charged residues within a protein or peptide can play an active role in the translocation process. These results provide evidence for an electrophoresis-like membrane transfer mechanism. However, it is still not known whether the observed requirement for a PMF is specific for the electrical component (externally positive electrical charge across the membrane) or the transmembrane pH component (externally acidic, resulting from the hydrogen ion concentration gradient where the concentration is higher on the outside). The aims of this proposal are to: determine whether the PMF can act directly on the membrane protein substrate to promote the spontaneous insertion of membrane proteins into liposomes; determine whether the insertion of membrane proteins requires a protein component that may mediate the PMF effects; examine the general importance of negatively charged residues in protein translocation; and determine which components of the PMF drives translocation of negatively charged residues. A combination of genetic, biochemical and biophysical methods will be used to achieve these aims. These studies are important because they will help define a basic mechanism by which hydrophilic regions of membrane proteins move across the lipid bilayer of biological membranes. This will expand our knowledge and understanding of a basic cellular function, which in turn will contribute significantly to the general foundations of biotechnology, particularly with regard to bioprocessing of genetically engineered proteins.
