Volume 41, Issue 3, Pages 343-353 (February 2011) SecA Interacts with Ribosomes in Order to Facilitate Posttranslational Translocation in Bacteria  Damon Huber, Nandhakishore Rajagopalan, Steffen Preissler, Mark A. Rocco, Frieder Merz, Günter Kramer, Bernd Bukau  Molecular Cell  Volume 41, Issue 3, Pages 343-353 (February 2011) DOI: 10.1016/j.molcel.2010.12.028 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 SecA Binds to Ribosomes in a 1:1 Stoichiometry in a Salt-Sensitive Fashion with a KD of ∼900 nM (A) SecA cosediments with ribosomes in a salt-sensitive fashion. Two micromolar purified SecA was incubated with 2 μM purified ribosomes in the presence of 50 mM or 500 mM KCl, as indicated, and the ribosome-bound SecA was separated from unbound SecA by pelleting ribosomes through a 30% sucrose cushion. The supernatant (S) and pellet (P) fractions were separated on a 10% SDS-PAGE gel and analyzed by Coomassie staining. The running positions of SecA and the ribosomal proteins are indicated. (B) Plot of the fluorescence anisotropy of 700 μM Ru(bpy)2(dcbpy)-labeled SecA versus the concentration of ribosomes and the fitted binding curve. The inset displays the Scatchard plot analysis of the anisotropy data. Both methods yielded a KD of ∼900 nM. Molecular Cell 2011 41, 343-353DOI: (10.1016/j.molcel.2010.12.028) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Ribosome Binding Is Mediated by Two Conserved Lysine Residues in the α-Helical Linker Domain (A) Ribbon representation of the E. coli SecA structure from PDB file 2VDA (Gelis et al., 2007). Nucleotide binding domain-1 (NBD1; residues 9–228 and 375–418) is colored dark blue, the polypeptide crosslinking domain (PPXD; residues 229–374) is colored cyan, NBD2 (residues 419–615) is colored green, the α-helical linker domain (residues 616–669) is colored yellow, and the C-terminal domain (CTD) is colored orange. Stick models depict residues that form intersubunit hydrophilic bonds (as calculated by PyMol v. 1.00) in structural models of the SecA dimer from PDB files 2FSF (blue), 2IPC (yellow), 2IBM (red), and both 2IPC and 2IBM (orange). (B) Two micromolar SecA418–668, which was purified as a series of C-terminally truncated protein fragments, was incubated in the presence or absence of 1 μM ribosomes. Ribosome-bound SecA418–668 was separated from unbound SecA418–668 by pelleting ribosomes through a 30% sucrose cushion. The amount of SecA418–668 in the supernatant (S) and pellet (P) fractions was analyzed by SDS-PAGE and western blotting against SecA. The masses (as determined by LC-MS) and the C-terminal five amino acids of each of the largest three protein fragments are displayed. The starred band is a ribosomal protein that cross-reacts with the anti-SecA antiserum. (C) ClustalW alignment of the α-helical linker domain (residues 622–668) from E. coli, Bacillus subtilis, and Thermus thermophilus. The proteolytic cleavage sites, which result in the two largest truncation products of SecA418–668, are noted with arrows. Conserved residues lysine-625 and lysine-633 are boxed. (D) Two micromolar Strep-SUMO-SecA616–668 was incubated in the presence or absence of 1 μM wild-type ribosomes or ribosomes containing L23(E42A). Ribosome-bound Strep-SUMO-SecA was separated from the unbound protein by pelleting ribosomes through a 30% sucrose cushion. The amount of Strep-SUMO-SecA616–668 in the supernatant (S) and pellet (P) fractions was determined by SDS-PAGE and western blotting with alkaline phosphatase-coupled Strep-Tactin, which recognizes the Strep tag. The band representing full-length Strep-SUMO-SecA616–668 is noted with an arrow. (E) One micromolar wild-type SecA, SecA(K625A), SecA(K633A), or SecA(K625A/K633A), respectively, was incubated with 1 μM ribosomes and the ribosome-bound protein was separated from the unbound protein by pelleting ribosomes through a 30% sucrose cushion. The amount of the respective SecA variant in the ribosome pellets was determined by western blotting against SecA. The percentage of the respective SecA variant that cosedimented with ribosomes compared to the amount of wild-type SecA that cosedimented with ribosome is given below. The amount of ribosomal protein L1 in the pellet fractions was determined as a loading control. Molecular Cell 2011 41, 343-353DOI: (10.1016/j.molcel.2010.12.028) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 SecA Crosslinks to Ribosomal Protein L23 (A) Binding reactions containing 1 μM SecA or 1 μM ribosomes were incubated in the absence or presence of the nonspecific crosslinker EDC (2 mM), as indicated. Samples were analyzed by SDS-PAGE and Coomassie staining. The running positions of the ribosomal proteins and full-length SecA are noted. The running position of the high-molecular weight crosslinking adduct is marked with a (∗). (B and C) SecA-biotin was crosslinked to ribosomes by using 2 mM EDC and purified by binding to streptavidin-coupled magnetic beads and washing under denaturing conditions. The purified product was resolved on a 10% SDS-PAGE gel. Gels were analyzed either by Coomassie staining (B) or by western blotting (C) with rabbit antiserum against SecA (red) and sheep antisera against the noted ribosomal proteins (green). Molecular Cell 2011 41, 343-353DOI: (10.1016/j.molcel.2010.12.028) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Mutations in the L23 Gene that Disrupt SecA Binding to the Ribosome Cause a Dominant Translocation Defect In Vivo (A) β-galactosidase activities of N48 cells (malE-lacZ) expressing the indicated L23 variants from a plasmid. Error bars represent the range of measured activities. (B) Structural model of the 50S ribosomal subunit (PDB file 2AW4; Schuwirth et al., 2005). A view is shown of the surface of L23 (light blue) distal (left) and proximal (right) to the polypeptide exit channel. The locations of conserved surface residues that affect SecA binding (yellow), TF binding (red), and the loop domain (dark blue) are indicated. Other ribosomal proteins are depicted as ribbon representations (green). The backbone trace of the ribosomal RNA is colored gray. (C) Plot of the fluorescence anisotropy of 700 μM Ru(bpy)2(dcbpy)-labeled SecA versus the concentration of the respective mutant ribosomes and the fitted binding curves. The calculated KDs for the ribosomal variants are shown (below left). (D) One micromolar SecA was incubated with 1 μM ribosomes containing the indicated mutant L23 protein. Ribosome-bound SecA was separated from the unbound SecA by pelleting ribosomes through a 30% sucrose cushion. The amount of SecA in the pellet fractions was determined by quantitative western blotting against SecA and the percentage of SecA that copelleted with the respective mutant ribosomes compared to the amount of SecA that copelleted with wild-type ribosomes is given below. As a loading control, the amount of ribosomal protein L1 in the pellet fractions was determined by using a sheep anti-L1 antibody. Molecular Cell 2011 41, 343-353DOI: (10.1016/j.molcel.2010.12.028) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Cells Expressing Ribosome-Binding-Deficient SecA or SecA-Binding-Deficient Ribosomes Are Defective for Protein Translocation (A and B) (A) Strains expressing wild-type SecA (DRH692) or SecA(K625A/K633A) (DRH693) or (B) strains expressing wild-type L23 (BB6623), L23(FEVEVE) (DRH627), L23(E42A/FEVEVE) (DRH629), or L23(FEVEVE/E89A) (DRH630) grown in M63 maltose media were pulse labeled with 35S-labeled methionine for 15 s and chased with excess unlabeled methionine. The reaction was stopped at the indicated time points by the addition of 5% trichloroacetic acid and samples were immunoprecipitated by using anti-MBP (top) or anti-β-lactamase (bottom) antisera. p, uncleaved precursor; m, mature-length protein lacking signal sequence. (C and D) (C) DRH692 and DRH693 cells or (D) BB6623 and DRH630 cells grown in M63 maltose media were separated into whole-cell (W), cytoplasm plus membranes (C), and periplasm (P) fractions with subcellular fractionation by spheroplasting. The fractions were resolved by SDS-PAGE and analyzed by western blotting against MBP or β-lactamase. (E) Processing of precursor MBP in strains expressing both wild-type SecA and wild-type L23 (DRH754; red), L23(FEVEVE/E89A) (DRH756; orange), SecA(K625A/K633A) (DRH757; green), or both SecA(K625A/K633A) and L23(FEVEVE/E89A) (DRH759; blue) was analyzed in pulse-chase experiments as described above (left). A profile of the 35S signal from each strain was generated from the lanes corresponding to the 10 min time point (right). In order to aid comparison, we normalized the 35S signal to the sum of the signals under the curves corresponding to the precursor (p) and mature (m) length peaks. Molecular Cell 2011 41, 343-353DOI: (10.1016/j.molcel.2010.12.028) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 SecA Specifically Binds to SecM-Containing RNCs with Increased Affinity (A) One micromolar SecA was incubated with 1 μM vacant ribosomes or SecM-stalled RNCs containing SecM or Strep-tagged barnase in the presence of 100 mM or 500 mM potassium acetate, as indicated. (B) One-half micromolar SecA or SecA(K625A/K633A) was incubated with 0.5 μM SecM-containing RNCs in the presence of 100 mM potassium acetate. In both cases, ribosome-bound SecA was separated from unbound SecA by pelleting ribosomes through a 30% sucrose cushion by ultracentrifugation. The pellet fractions were then analyzed by SDS-PAGE and Coomassie staining (A) or by quantitative western blotting against SecA and ribosomal protein L1 (B). In panel (B), the amount of SecA that copelleted with ribosomes was quantified by dividing the signal from SecA by the signal from L1, and the values were then normalized to the average signal for wild-type SecA. Error bars represent one standard deviation. Molecular Cell 2011 41, 343-353DOI: (10.1016/j.molcel.2010.12.028) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Model for Cotranslational Targeting of Nascent Polypeptides to the Posttranslational Translocation Pathway by SecA Schematic diagram of the proposed model for SecA-mediated cotranslational targeting to the posttranslational translocation pathway. See Discussion for detailed description. Molecular Cell 2011 41, 343-353DOI: (10.1016/j.molcel.2010.12.028) Copyright © 2011 Elsevier Inc. Terms and Conditions