| A fundamental question in biology is how proteins are translocated across membranes without interfering with the barrier function of the membrane for small ions and protons. The cytoplasmic membrane of bacteria contains a proteinaceous complex termed the ?translocase? that fulfills this function. Translocase consists of a protein conducting channel (PCC) composed of the heterotrimeric SecYEG complex and associated soluble factors that fuels the translocation reaction at the expense of nucleotide triphosphate hydrolysis. Depending on the mode of translocation (co- or post-translational), the ribosome or the motor protein SecA associates with the PCC. Translocation results from a dynamic interplay between the protein substrate, the associated soluble factor and the PCC in order to control the opening and closure of a sizeable transmembrane channel. Recent cryo-electron microscopy work ? in conjunction with previous X-ray crystallography studies ? suggests that the PCC is a dimer of front-to-front arranged SecYEG complexes. This arrangement maintains two segregated pores with different lipid accessibilities, but allows the formation of a consolidated channel necessitated. The attachment of the PCC to the ribosome occurs symmetrically via the cytosolic factor-associating domains in each heterotrimer, and suggests a mechanism for the formation and regulation of the consolidated PCC channel via nascent peptide-induced conformational changes in the ribosome. By analogy and by molecular docking of available structures of the dimeric SecA onto the PCC, we propose that SecA modulates the opening and clossure of the PCC by a similar mechanism. PCC opening/closure involves SecA conformational changes that are effected by altering the dimerization interface of SecA. The model suggests that SecA does not physically push the preprotein through but instead facilitates the directional transport of the preprotein. This project will test this novel framework for SecA-mediated protein translocation through the PCC by a combination of advanced biochemical and biophysical approaches. The study will provide novel insights in the coordinated mechanisms of channel formation and motor function of SecA. |
