Investigation of the Relative Activity of Monomer and Dimer SecYEG Translocons

Abstract

The general secretory (Sec) pathway is the principal protein transport pathway in bacteria. Two key components of this system are the protein complex SecYEG, which forms a protein‐conducting channel within the plasma membrane, and the ATPase motor protein SecA, which drives the protein translocation process. Whether the SecYEG complex functions as a monomer or requires dimerization with a neighboring SecYEG complex for activity remains unclear. To address this important question, we have made use of both photobleaching and total internal reflection fluorescence (TIRF) microscopy to assess the fluorescently labeled SecY oligomeric state, as well as an in vitro protein translocation assay to assess Sec‐dependent protein transport activity. The key component of our system is the SecYEG proteoliposome, which is a lipid vesicle reconstituted solely with SecYEG protein. In this study, we reconstituted proteoliposomes at different lipid to SecYEG protein ratios to produce distinct vesicle populations containing either mostly SecYEG monomers or containing about half SecYEG monomers and half SecYEG dimers. Next, we utilized TIRF microscopy to determine the percent of singly or doubly reconstituted proteoliposomes existing within a given population based on the ratio of single or double photobleaching events. Finally, we optimized and performed an in vitro protein transport assay to assess the overall protein translocation activity of a given population. Initial findings from theses assays suggest that the monomer SecYEG proteoliposome population is more efficient at translocating protein compared to the population with substantial dimer content. Further studies will be required to fully confirm this finding. Ultimately, we hope to elucidate any differences in the protein translocation activity between these distinct populations in order to better understand the functionality of monomer and dimer SecYEG translocons in this universally conserved system.Support or Funding InformationFunding generously provided by the NIH through the National Institute of General Medical Sciences (GM110552)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.