The bacterial SRP receptor, SecA and the ribosome use overlapping binding sites on the SecY translocon
25.02.2010
Kuhn P, Weiche B, Sturm L, Sommer E, Drepper F, Warscheid B, Sourjik V, Koch HG.
Traffic. 2011;12(5):563-78.
Protein-protein interactions provide the driving force and specificity for a multitude of cellular processes. The identification of true interaction partners and their discrimination from co-purified contaminants is crucial to a functional understanding. We established in vivo site-directed cross-linking combined with quantitative high resolution mass spectrometry for determining protein-protein interactions in E. coli during co-translational protein targeting, an essential and universally conserved cellular process. Signal recognition particle (SRP)-dependent protein targeting ensures delivery of proteins to the bacterial cytoplasmic membrane or to the endoplasmic reticulum in eukaryotes. The transfer of the ribosome-nascent chain complex (RNC) to a vacant Sec translocon in eukaryotes is coordinated by the ?-subunit of the SRP receptor (SR). However, bacteria lack this subunit and how they coordinate RNC-transfer is still largely unknown.
For site-directed cross-linking, the UV-activatable amino acid p-benzoyl-L-phenylalanine (pBpA) was incorporated at different positions into the amino acid sequences of the binding partners FtsY, the bacterial SR? homologue, and SecY, the central component of the bacterial Sec translocon, using the amber stop codon UAG. Quantitative mass spectrometry allowed to distinguish cross-linked interaction partners from co-purified proteins as well as mapping of individual protein-protein interaction sites. We demonstrate that FtsY binds to exposed loops of SecY that serve also as binding sites for SecA and the ribosome. The binding of FtsY to SecY probably allows FtsY to sense an available translocon and to align the incoming SRP-RNC with the protein conducting channel.
