In Escherichia coli, both secretory and inner membrane proteins initially are targeted to the core SecYEG inner membrane translocase. Previous work has also identified the peripherally associated SecA protein as well as the SecD, SecF and YajC inner membrane proteins as components of the translocase. Here, we use a cross‐linking approach to show that hydrophilic portions of a co‐translationally targeted inner membrane protein (FtsQ) are close to SecA and SecY, suggesting that insertion takes place at the SecA/Y interface. The hydrophobic FtsQ signal anchor sequence contacts both lipids and a novel 60 kDa translocase‐associated component that we identify as YidC. YidC is homologous to Saccharomyces cerevisiae Oxa1p, which has been shown to function in a novel export pathway at the mitochondrial inner membrane. We propose that YidC is involved in the insertion of hydrophobic sequences into the lipid bilayer after initial recognition by the SecAYEG translocase.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre‐proteins. SecB targets the pre‐protein to SecA that mediates pre‐protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross‐linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.

Mycobacterial pathogens use specialized type VII secretion (T7S) systems to transport crucial virulence factors across their unusual cell envelope into infected host cells. These virulence factors lack classical secretion signals and the mechanism of substrate recognition is not well understood. Here we demonstrate that the model T7S substrates PE25/PPE41, which form a heterodimer, are targeted to the T7S pathway ESX-5 by a signal located in the C terminus of PE25. Site-directed mutagenesis of residues within this C terminus resulted in the identification of a highly conserved motif, i.e., YxxxD/E, which is required for secretion. This motif was also essential for the secretion of LipY, another ESX-5 substrate. Pathogenic mycobacteria have several different T7S systems and we identified a PE protein that is secreted by the ESX-1 system, which allowed us to compare substrate recognition of these two T7S systems. Surprisingly, this ESX-1 substrate contained a C-terminal signal functionally equivalent to that of PE25. Exchange of these C-terminal secretion signals between the PE proteins restored secretion, but each PE protein remained secreted via its own ESX secretion system, indicating that an additional signal(s) provides system specificity. Remarkably, the YxxxD/E motif was also present in and required for efficient secretion of the ESX-1 substrates CFP-10 and EspB. Therefore, our data show that the YxxxD/E motif is a general secretion signal that is present in all known mycobacterial T7S substrates or substrate complexes.