SecA Cotranslationally Interacts with Nascent Substrate Proteins In Vivo

Abstract

SecA is an essential component of the Sec machinery in bacteria, which is responsible for transporting proteins across the cytoplasmic membrane. Recent work from our laboratory indicates that SecA binds to ribosomes. Here, we used two different approaches to demonstrate that SecA also interacts with nascent polypeptides in vivo and that these polypeptides are Sec substrates. First, we photo-cross-linked SecA to ribosomes in vivo and identified mRNAs that copurify with SecA. Microarray analysis of the copurifying mRNAs indicated a strong enrichment for proteins containing Sec-targeting sequences. Second, we used a 2-dimensional (2-D) gel approach to analyze radioactively labeled nascent polypeptides that copurify with SecA, including maltose binding protein, a well-characterized SecA substrate. The interaction of SecA with nascent chains was not strongly affected in cells lacking SecB or trigger factor, both of which also interact with nascent Sec substrates. Indeed, the ability of SecB to interact with nascent chains was disrupted in strains in which the interaction between SecA and the ribosome was defective. Analysis of the interaction of SecA with purified ribosomes containing arrested nascent chains in vitro indicates that SecA can begin to interact with a variety of nascent chains when they reach a length of ∼110 amino acids, which is considerably shorter than the length required for interaction with SecB. Our results suggest that SecA cotranslationally recognizes nascent Sec substrates and that this recognition could be required for the efficient delivery of these proteins to the membrane-embedded Sec machinery.IMPORTANCE SecA is an ATPase that provides the energy for the translocation of proteins across the cytoplasmic membrane by the Sec machinery in bacteria. The translocation of most of these proteins is uncoupled from protein synthesis and is frequently described as “posttranslational.” Here, we show that SecA interacts with nascent Sec substrates. This interaction is not dependent on SecB or trigger factor, which also interact with nascent Sec substrates. Moreover, the interaction of SecB with nascent polypeptides is dependent on the interaction of SecA with the ribosome, suggesting that interaction of the nascent chain with SecA precedes interaction with SecB. Our results suggest that SecA could recognize substrate proteins cotranslationally in order to efficiently target them for uncoupled protein translocation.

Protein folding and tRNA biology

“…In E. coli, SecA is an essential component of the Sec machinery, which participates in transporting proteins across the cytoplasmic membrane. SecA binds both ribosomes and nascent polypeptides that are Sec substrates (Huber et al 2016). In eukaryotes, secretory proteins posses a hydrophobic signal sequence that is recognized co-translationally by SRP (signal recognition particle), a ribonucleoprotein composed of six proteins and RNA.…”

SecA—a New Twist in the Tale

“…Schematic overview of post- and cotranslational secretion and membrane protein insertion after Huber et al (39). (A) Coupled cotranslational translocation.…”

“…And still, we have yet to solve this outstanding problem. So it is with great interest we find, in an article in the current issue of the Journal of Bacteriology (39), that the overriding view of the pathway leading to secretion and insertion may not be quite right. Therefore, it may be time to reconsider precisely how the various factors combine and operate to bring about the efficient translocation of polypeptides across and into the membrane.…”

“…Huber's new paper (39) builds on previous work on the curious interaction between SecA and the ribosome (40, 41), which does not seem to fit the classical view described above. The interaction is at the busy exit site, where it must compete for access to the nascent chain with SRP (36, 42).…”

“…For instance, SecA plays a role in SRP-dependent export of soluble protein (44), and the membrane protein RodZ is driven into the membrane by SecA (45), which smells like a cotranslational event. Huber et al demonstrate conclusively that SecA contacts a variety of nascent proteins in vivo , with a clear preference for membrane and secretory proteins (39). This sampling might help prevent aggregation in aid of efficient targeting to the Sec apparatus, but SecA must ultimately be outcompeted from membrane proteins by SRP (Fig.…”