SecA interacts with ribosomes in order to facilitate posttranslational translocation in bacteria

Huber D., Rajagopalan N., Preissler S., Rocco M. A., Merz F., Kramer G., Bukau B.

Mol Cell. 2011 Feb 4;41(3):343-53.

Contributions

1. Expression and purification of SecA 2. Production of rabbit-α-SecA antibodies

3. Initial characterization of the interaction of SecA with ribosomes by in vitro rebinding experiments

4. Cloning and purification of the signal recognition particle (SRP) from E. coli 5. Discussion of the initial experiments and proofreading the manuscript

4.3.1. Objective

Although much is known about the molecular mechanism of posttranslational translocation, substrate recognition by this pathway is still a matter of debate. The current model suggests that newly synthesized secretory proteins are sequentially transferred from cytosolic chaperone SecB to SecYEG-bound motor ATPase SecA (see introduction). It was therefore proposed that SecB recognizes the substrates for posttranslational translocation (Fekkes et al, 1998; Hartl et al, 1990; Knoblauch et al, 1999). However, several observations argue against this hypothesis. Although SecB binds only a specific subset of targeted proteins in vivo (Kumamoto & Francetic, 1993), it shows very little substrate-specificity in vitro (Knoblauch et al, 1999; Randall et al, 1997). Moreover, the signal sequence is dispensable for substrate interaction with SecB (Randall et al, 1997; Randall et al, 1998). It thus remains unclear how SecB differentiates between secretory and nonsecretory polypeptides (Driessen

& Nouwen, 2008). By contrast, there is substantial evidence that SecA specifically recognizes signal sequences (Gelis et al, 2007; Kimura et al, 1991; Kourtz & Oliver, 2000;

Musial-Siwek et al, 2007; Papanikou et al, 2005). In addition, several biochemical and genetic studies indicate that substrates could be recognized already during translation. Since SecA and SecB were both shown to interact with nascent secretory polypeptides (Eisner et al, 2003; Kumamoto & Francetic, 1993; Randall et al, 1997), it is possible that SecA can contact nascent substrates before SecB. This is supported by the observation that SecA is

defects (Kumamoto & Nault, 1989). We therefore set out revisit SecA/B-dependent protein targeting and asked whether SecA is physically and functionally connected to the translation machinery.

4.3.2. Summary of the experimental data

To test whether SecA can bind directly to ribosomes, in vitro cosedimentation assays were performed. Therefore, purified SecA was incubated with ribosomes and afterwards the ribosomes were pelleted through a 30% sucrose cushion by ultracentrifugation to separate ribosome-bound from unbound SecA. By this we found that SecA cosedimented with ribosomes in an approximately 1:1 stoichiometry, indicating that there is a single SecA binding site on the ribosome. This was confirmed by fluorescence anisotropy measurements with fluorophore-labeled SecA. Moreover, ribosome binding was salt-sensitive suggesting that the interaction between SecA and ribosomes depends on electrostatic contacts. SecA normally forms dimers in solution with a KD of ∼1 µM (Akita et al, 1991; Doyle et al, 2000; Or et al, 2002; Woodbury et al, 2002). The observation that SecA binds to ribosomes as a monomer raises the possibility that the interaction involves the dimer interface. Based on the available structural data of the SecA dimer (Papanikolau et al, 2007; Vassylyev et al, 2006;

Zimmer et al, 2006) and cosedimentation experiments with truncated SecA versions, the N-terminal region of the α-helical linker domain (residues 616-668) was identified to be necessary for ribosome binding. Mutation of the lysines 625 and 633 in this region severely reduced targeting of SecA to ribosomes, indicating that these residues are critical for the interaction between SecA and the ribosome.

To identify the SecA-binding site on the ribosome, a nonspecific chemical crosslinking approach was chosen. Therefore, purified SecA was incubated with ribosomes in the presence of a crosslinking reagent. High-molecular weight crosslinking adducts, that appeared specifically in samples containing ribosomes and SecA, were affinity purified and analyzed by Western blotting against ribosomal proteins and by mass spectrometry. As a result, the ribosomal protein L23 was identified in the crosslinking adducts. These data suggested that SecA binds ribosomes close to L23, which is located on the large ribosomal subunit at the tunnel exit site. This finding was supported by a genetic screen for SecA-binding-deficient L23 mutants. Thereby, point mutations were introduced in the L23 encoding gene (rplW) and the mutants were analyzed for defects in protein translocation into the periplasm. Some of the L23 mutants that showed reduced translocation were indeed defective in SecA binding, indicating that L23 is part of the binding site of SecA on

Results and discussion

expressing ribosome-binding-deficient mutants of SecA or vice versa, SecA-binding-deficient mutants of L23. This shows that the interaction between SecA and the ribosome contributes to facilitate posttranslational translocation in vivo. Finally, the affinity of SecA was higher for ribosomes carrying nascent SecM polypeptides with an N-terminal signal sequence compared to vacant ribosomes. By contrast, a ribosome-binding-deficient SecA mutant showed reduced affinity for these ribosome-nascent chain complexes. The physical interaction between SecA and the ribosome thus increases the affinity of SecA for nascent polypeptides with translocation signals. Together the data suggest a new model for SecA/B-dependent posttranslational targeting of secretory proteins (Figure 20).

Figure 20: Model for SecA-mediated cotranslational targeting of nascent polypeptides to the posttranslational translocation pathway. Ribosome-bound SecA specifically recognizes nascent secretory preproteins (NC) via the signal sequence (SS) on their N-termini (N). Upon dissociation of SecA from the ribosome SecB is recruited via the interaction with SecA to the newly synthesized substrate to keep it in a translocation-competent state. This allows for fast and direct targeting of the substrate to the cytoplasmic membrane (CM). SecA provides the energy for posttranslational translocation through the SecYEG pore (orange) by ATP hydrolysis. Alternatively, SecB-bond substrates could be kept in an unfolded state to delay posttranslational translocation (dotted lines). Membrane-anchored signal peptidases (SPase, red) remove the SS from the secretory preprotein.

Thereby, ribosome-bound SecA scans nascent polypeptides and interacts with those

SecB SecA

ATP

SecYEG

SPase N

Periplasm

CM Cytoplasm

NC

SS

dissociate from the ribosome and allow for a fast and direct targeting to the translocon.

Alternatively, SecB could be recruited to SecA and overtake the newly synthesized substrate to keep it in a translocation-competent state and to prevent its aggregation. Cotranslational sorting of substrates to the posttranslational translocation pathway could thereby provide a mechanism to efficiently delay translocation until SecYEG pores are available (Figure 20).

Results and discussion

4.4. Components of the Ccr4-Not complex associate with polyribosomes