JCB_201704036 3639..3654

Roughly 30% of the genome encodes membrane proteins, which are anchored to cellular membranes via at least one transmembrane domain (TMD) and play diverse physiological roles such as signaling, cell shape maintenance, and cell motility. To attain their proper structure and function, newly synthesized membrane proteins must engage dedicated protein targeting pathways by which they are delivered to the correct membrane destination in the cell (Zhang and Shan, 2014). Mislocalization of membrane proteins not only deprives cells of functional proteins, but it also disrupts cellular protein homeostasis as a result of improper exposure of the hydrophobic TMDs in the cytosol that could lead to aggregation and misfolding. This demands that the targeting processes for membrane proteins act with high efficiency to minimize the exposure of TMDs in the cytosol. The cotranslational targeting of proteins by signal recognition particle (SRP) is the most well-understood pathway for targeted delivery of integral membrane proteins. SRP recognizes hydrophobic signal sequences or TMDs near the N terminus of nascent proteins as soon as they emerge from the ribosome exit tunnel (Walter et al., 1981; Schaffitzel et al., 2006; Zhang and Shan, 2014). The TMD on the nascent protein is shielded from the cytosol by the M domain of SRP. Through the interaction between SRP and the SRP receptor (SR; termed FtsY in bacteria), the nascent protein is delivered to the SecYEG (or Sec61p) protein translocation machinery on the bacterial inner membrane (or the eukaryotic endoplasmic reticulum; Zhang et al., 2010). SRP-dependent targeting is complete before ∼130 amino acids of the nascent polypeptide C-terminal to the signal sequence or TMD is translated (Siegel and Walter, 1988; Ariosa et al., 2015), and releasing nascent proteins from the ribosome abolishes the targeting of SRP-dependent substrates (Kuruma et al., 2005). In bacteria, SRP is generally thought to mediate the targeted delivery of the majority of inner membrane proteins and several periplasmic secretory proteins that contain highly hydrophobic signal sequences (Luirink and Sinning, 2004; Schibich et al., 2016). A second major protein-targeting pathway in bacteria uses SecA, with the participation of the chaperone SecB in some cases. The SecB/A pathway targets the majority of secretory and outer membrane proteins via a posttranslational mechanism (Hartl et al., 1990). Substrates entering this pathway contain signal sequences that are less hydrophobic than those that engage SRP/SR (Neumann-Haefelin et al., 2000). These signal sequences are recognized by the preprotein cross-linking domain of SecA, which couples its ATPase cycle to the translocation of substrate proteins across SecYEG (Bauer et al., 2014). The posttranslational chaperone SecB assists in maintaining preproteins in the unfolded translocation-competent state while also delivering these proteins to SecA bound at the inner membrane (Weiss et al., 1988). The posttranslational nature of the SecB/A pathway is supported by the following observations: (A) classic SecB/A-dependent substrate proteins, such as OmpA and Protein targeting to the bacterial plasma membrane was generally thought to occur via two major pathways: cotransla‐ tional targeting by signal recognition particle (SRP) and posttranslational targeting by SecA and SecB. Recently, SecA was found to also bind ribosomes near the nascent polypeptide exit tunnel, but the function of this SecA–ribosome con‐ tact remains unclear. In this study, we show that SecA cotranslationally recognizes the nascent chain of an inner mem‐ brane protein, RodZ, with high affinity and specificity. In vitro reconstitution and in vivo targeting assays show that SecA is necessary and sufficient to direct the targeting and translocation of RodZ to the bacterial plasma membrane in an obligatorily cotranslational mechanism. Sequence elements upstream and downstream of the RodZ transmembrane domain dictate nascent polypeptide selection by SecA instead of the SRP machinery. These findings identify a new route for the targeting of inner membrane proteins in bacteria and highlight the diversity of targeting pathways that enables an organism to accommodate diverse nascent proteins. SecA mediates cotranslational targeting and translocation of an inner membrane protein