The In Vivo Analysis Of Escherichia coli's SecA Membrane Topology In The Nucleotide Binding Domain I

Abstract

In Eubacteria, the ATPase SecA transports secretory proteins across the inner membrane using the membrane embedded channel, SecYEG. SecA drives the preprotein through the channel in an ATP dependant manner that promotes a series of conformational changes associated with the insertion and deinsertion of a region of SecA during the translocation cycle. To gain additional insight into the interactions between SecA and SecYEG, an in vivo sulfhydryl labeling technique was developed to probe monocysteine SecA mutants through the channel during protein translocation using N-(3-maleimidylpropionyl)biocytin (MPB). Our lab demonstrated multiple residues of SecA that are exposed to the trans side of the membrane through the SecYEG channel. These residues, which were located throughout most domains of SecA, resided on a single face within the Bacillus subtilis SecA crystal structure. This provided some insight into the regions of SecA that are in fluid contact with the channel. Interestingly, two residues located on the opposite side of the labeled face of SecA in the nucleotide-binding domain I (NBD-I) also labeled. In the present study, the NBDI domain was further investigated to determine if there were other residues in fluid contact with the SecYEG channel. Additional monocysteine SecA mutants were created in the NBDI domain and analyzed using the same methodology. After looking at the crystal structure with the newly labeled residues, the labeled face of SecA seemed less apparent. These results emphasize the dynamic nature of SecA and identified regions of SecA that may interact with the channel. The NBDI mutants will be further analyzed using an in vivo photocrosslinking technique.