bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.215244; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 1 AscA (YecA) is a molecular chaperone involved in Sec-dependent protein translocation 2 in Escherichia coli 3 4 Running title: AscA is a Sec chaperone 5 6 Tamar Cranford Smith1, Max Wynne1, Cailean Carter, Chen Jiang, Mohammed Jamshad, 7 Mathew T. Milner, Yousra Djouider, Emily Hutchinson, Peter A. Lund, Ian Henderson2 and 8 Damon Huber* 9 10 Institute for Microbiology and Infection; University of Birmingham; Edgbaston, 11 Birmingham, UK 12 13 1These authors contributed equally to this work 14 15 2Current address: Institute for Molecular Bioscience; University of Queensland; Brisbane, 16 Australia 17 18 *To whom correspondence should be addressed: [email protected] 19 20 Keywords: protein translocation, Sec pathway, molecular chaperone, SecB, metal binding 21 domain 22 23 ABSTRACT. 24 Proteins that are translocated across the cytoplasmic membrane by Sec 25 machinery must be in an unfolded conformation in order to pass through the protein- bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.215244; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 2 26 conducting channel during translocation. Molecular chaperones assist Sec-dependent 27 protein translocation by holding substrate proteins in an unfolded conformation in the 28 cytoplasm until they can be delivered to the membrane-embedded Sec machinery. For 29 example, in Escherichia coli, SecB binds to a subset of unfolded Sec substrates and 30 delivers them to the Sec machinery by interacting with the metal-binding domain 31 (MBD) of SecA, an ATPase required for translocation in bacteria. Here, we describe a 32 novel molecular chaperone involved Sec-dependent protein translocation, which we 33 have named AscA (for accessory Sec component). AscA contains a metal-binding 34 domain (MBD) that is nearly identical to the MBD of SecA. In vitro binding studies 35 indicated that AscA binds to SecB and ribosomes in an MBD-dependent fashion. 36 Saturated transposon mutagenesis and genetics studies suggested that AscA is involved 37 in cell-envelope biogenesis and that its function overlaps with that of SecB. In support of 38 this idea, AscA copurified with a range of proteins and prevented the aggregation of 39 citrate synthase in vitro. Our results suggest that AscA is molecular chaperone and that 40 it enhances Sec-dependent protein translocation by delivering its substrate proteins to 41 SecB. 42 43 IMPORTANCE. 44 This research describes the discovery of a novel molecular chaperone, AscA (YecA). 45 The function of AscA was previously unknown. However, it contains a small domain, 46 known as the MBD, suggesting it could interact with the bacterial Sec machinery, which 47 is responsible for transporting proteins across the cytoplasmic membrane. The work 48 described this study indicates that the MBD allows AscA to bind to both the protein 49 synthesis machinery and the Sec machinery. The previously function of the previously 50 uncharacterised N-terminal domain is that of a molecular chaperone, which binds to bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.215244; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 3 51 unfolded substrate proteins. We propose that AscA binds to protein substrates as they 52 are still be synthesised by ribosomes in order to channel them into the Sec pathway. 53 54 INTRODUCTION. 55 In Escherichia coli, most newly synthesized periplasmic and outer membrane proteins 56 are transported across the cytoplasmic membrane by the Sec machinery. During 57 translocation, protein substrates of the Sec machinery pass through an evolutionarily 58 conserved channel in the cytoplasmic membrane (composed of the integral membrane 59 proteins SecY, -E and -G) in an unfolded conformation (1, 2). In addition, translocation 60 usually requires the activity of SecA (3), an ATPase that facilitates translocation through 61 SecYEG (4). The translocation of periplasmic and outer membrane proteins typically begins 62 only after the substrate protein is fully (or nearly fully) synthesised (i.e. “posttranslationally”) 63 (5, 6). 64 Because proteins must be unfolded to pass through SecYEG, folding of substrate 65 proteins in the cytoplasm blocks Sec-dependent protein translocation, causing a protein to 66 become irreversibly trapped in the cytoplasm (7). Furthermore, partially folded proteins that 67 engage SecYEG can clog (or “jam”) the Sec machinery, which is toxic (8). As a result, cells 68 have evolved multiple mechanisms to prevent premature folding of substrate proteins. For 69 example, molecular chaperones can bind to unfolded Sec substrate proteins and hold them in 70 an unfolded conformation until they can be delivered to the membrane-embedded Sec 71 machinery. One such chaperone is SecB, which binds to a subset of unfolded Sec substrate 72 proteins and delivers them to SecA for translocation across the membrane (9-13). 73 Recognition of nascent substrates by SecB is dependent on SecA (14), suggesting that SecB 74 requires an intermediary to recognise its substrate proteins. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.215244; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 4 75 The interaction of SecA with SecB is mediated by a small (~20 amino acid) metal- 76 binding domain (MBD) near the extreme C-terminus of SecA (13, 15, 16). Recent work 77 indicates that the MBD also binds to ribosomes and that ribosome binding is involved in 78 coordinating binding of SecA to nascent polypeptides (17). As its name indicates, the MBD 79 binds to a transition metal (zinc and/or iron) (15, 18), and binding to the metal ion is required 80 for stable folding of the MBD (15). The amino acids responsible for metal binding are highly 81 conserved (CXCXSX6CH or CXCXSX6CC) (15, 17, 19). 82 We recently described a protein of unknown function in E. coli that contains a MBD 83 that is nearly identical to the MBD of SecA (18), YecA, which we have re-named AscA (for 84 accessory Sec component). AscA also contains a UPF0149-family domain at its N-terminus, 85 the function of which has not been described. In this work, we investigated the function of 86 AscA. The similarity of the AscA and SecA MBDs led us to investigate the interaction of 87 AscA with SecB and ribosomes and the dependence of these interactions on the MBD. 88 Genetic analysis suggested that AscA is involved in cell-envelope biogenesis and that AscA 89 could be a molecular chaperone. Further studies indicated that AscA binds to cytoplasmic Sec 90 substrate proteins and that it carries out its function in coordination with SecB in vivo. Our 91 results suggest a potential model for how AscA could facilitate Sec-dependent protein 92 translocation in E. coli. 93 94 RESULTS. 95 Binding of AscA to SecB. Many of the amino acids that mediate the interaction 96 between the SecA MBD and SecB from Haemophilus influenzae are conserved in the MBD 97 of AscA (supplemental figure S1) (18, 20). To investigate whether AscA can also bind to 98 SecB, we examined the effect of AscA on the thermophoretic mobility of SecB using 99 microscale thermophoresis. To this end, we fluorescently labelled SecB and incubated it with bioRxiv preprint doi: https://doi.org/10.1101/2020.07.21.215244; this version posted July 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 5 100 unlabelled AscA. There was a large change in the thermophoretic properties of fluorescently 101 labelled SecB at saturating concentrations of AscA (figure 1A), suggesting that SecB binds 102 to AscA. However, the presence of a truncated variant of AscA, which lacks the MBD 103 (AscAΔMBD), did not affect thermophoresis of SecB (figure 1A). Purified AscAΔMBD was 104 fully folded even in the absence of its MBD (18), indicating that the interaction between 105 SecB and AscA is dependent on the MBD. Analysis of the effect of increasing concentrations 106 of AscA on the thermophoresis of suggested an equilibrium dissociation constant (KD) of 107 approximately 150 nM (figure 1B). 108 Binding of AscA to ribosomes. We next investigated the interaction of AscA with 109 ribosomes. To this end, we incubated AscA or AscAΔMBD with purified non-translating 70S 110 ribosomes and then separated ribosome-bound AscA from unbound AscA by sedimenting 111 ribosomes through a 30% sucrose cushion by ultracentrifugation. Full-length AscA 112 cosedimented with the 70S ribosomes, indicating that it can bind to ribosomes (figure 1C). 113 Truncation of the MBD in AscAΔMBD greatly reduced its ability to cosediment with 114 ribosomes, indicating that binding is dependent on the MBD.