Mapping the SecA-SecY Interaction Interface Using in Vivo Photocrosslinking

Abstract

In Escherichia coli (E. coli) the major secretory pathway for proteins is the “general secretion pathway” or “Sec-dependent pathway”. Its major components include the SecA ATPase, the SecB chaperone, and SecYEG: the channel-forming complex. Once bound to SecYEG, the preprotein substrate, and ATP, SecA undergoes an ATP-driven conformational cycle that drives the step-wise translocation of proteins. While cytosolic SecA specifically recognizes and binds SecYEG, the specific residues by which these two proteins interact are not known. A study from Mori and Ito (2006) analyzed SecY-SecA interaction by using an in vivo site-directed cross-linking technique developed by Schultz and co-workers. In this experimental system, an amber suppressor tRNA and a tyrosyl-tRNA synthetase from Methanococcus jannaschii are genetically engineered to allow for the charging of the tRNA with p-benzoyl-phenylalanine (pBpa), a photo-reactive phenylalanine derivative. They showed that pBpa residues introduced into the second, fourth, fifth, and sixth cytoplasmic domains of SecY could be crosslinked to SecA.We have chosen a similar approach to Mori and Ito, but we have designed sites of in vivo pBpa incorporation into SecA based on an in vivo SecA membrane topology study performed by Jilaveanu et al. (2006). The amber positions in SecA were chosen based on residues of SecA that were accessible to small molecule labeling from the exterior side of the membrane-indicating proximity to the SecYEG channel. Our results demarcate novel sites of SecA interaction with SecY. Our data provide in vivo support for the biological relevance of the recent SecA-SecYEG X-ray structure (Zimmer et al., 2008), but they also demonstrate the importance of both the SecA N-domain as well as its C-domain in stabilizing the interaction in vivo.