1. Biochemistry and Chemical Biology
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In vivo experiments do not support the charge zipper model for Tat translocase assembly

  1. Felicity Alcock  Is a corresponding author
  2. Merel PM Damen
  3. Jesper Levring
  4. Ben C Berks  Is a corresponding author
  1. University of Oxford, United Kingdom
Short Report
Cite this article as: eLife 2017;6:e30127 doi: 10.7554/eLife.30127
3 figures, 1 table and 1 additional file

Figures

The charge zipper model for TatA assembly.

(A) Cartoon showing the TatA domain structure, comprising a transmembrane helix (TMH), an amphipathic helix (APH), and a densely-charged region (DCR). Below the cartoon are shown the amino acid sequences for E. coli TatA (top) and B. subtilis TatAd (bottom) starting at the beginning of the APH and with acidic (red) and basic (blue) residues indicated. The APH is assigned in each case from the corresponding NMR structures (PDB 2MN7 and 2L16) and is indicated by a gray cylinder. The E. coli TatA sequence is C-terminally truncated to residue 69. Salt bridge pairs predicted by Walther and co-workers are indicated above each sequence. For E. coli TatA the predicted salt bridge pairs tested in this study are indicated in black and the acidic DDE motif targeted in this study is underlined. For B. subtilis TatAd, the assigned intra- and inter-molecular pairs are distinguished using dotted or solid lines respectively. (B) Structure of E. coli TatA (PDB 2MN7) shown in three orientations with the charged APH side chains indicated. (C) Schematic diagram of the charge zipper model for TatA folding and assembly applied to E. coli TatA. Folding back the DCR against the APH is proposed to allow pairing of amino acids with complementary charges to form either intra-molecular or inter-molecular salt bridges. Three adjacent TatA protomers are shown with the residues of the acidic DDE motif and their potential salt bridge partners outlined in red and blue respectively. The charge zipper model does not predict which residue pairs would form inter- and intra-molecular salt bridges and one of several possible configurations is represented here. (D) The salt bridges shown in (C) are proposed to mediate self-assembly of multiple adjacent TatA molecules (1). The assembled APH/DCR units are then proposed to insert across the membrane to form the passage for substrate protein transport (2).

https://doi.org/10.7554/eLife.30127.002
Transport activity of TatA charge zipper variants.

All TatA variants were expressed from the phage lambda attachment site in the ΔtatA ΔtatE strain JARV16. Mutant strains are identified by the amino acid substitutions present in TatA where EDD is K37E/K40D/K41D, and KKK is D45K/D46K/E47K. WT refers to the ΔtatE strain J1M1, and ΔTatA refers to the parental strain JARV16. (A) Whole cell (W), periplasm (P), and spheroplast (S) fractions of cells overproducing CueO from plasmid pQE80-CueO were subject to immunoblotting with antibodies against CueO or the cytoplasmic marker protein DnaK. m is the transported form of CueO from which the signal peptide has been removed and p the precursor protein. (B) Growth of the strains when cultured in LB/glycerol/TMAO medium under anoxic conditions. Error bars represent the S.E.M of three biological replicates. (C) Serial ten-fold dilutions of log-phase cultures were spotted onto LB-agar containing the indicated amount of SDS. (D) Immunoblot of membranes isolated from the strains used in A-C, probed with TatA antibodies (upper panels) or antibodies against TatB and TatC (lower panels).

https://doi.org/10.7554/eLife.30127.003
Figure 3 with 1 supplement
TatA oligomerization behavior of charge zipper variants.

Representative fluorescence images of TatA-YFP in E. coli cells. A tatA-yfp fusion was expressed from the chromosome in three different backgrounds: ELV16 λAry which contains all other tat genes (designated AyBCE), JARV16 λAry which lacks tatE (designated AyBC), or DADE λAry which possesses no other tat genes (designated Ay). The TatA-YFP variant produced is indicated to the left of the panels, where WT is the parental protein and KKK is a D45K/D46K/E47K variant. Where indicated, CueO was overproduced from plasmid pQE80-CueO by adding IPTG to early exponential phase cultures for 30 min prior to imaging (+CueO columns). 50 μM CCCP was subsequently added, as indicated (+CCCP column), and the cells incubated for 30–45 min prior to imaging. Scale bar = 1 μm.

https://doi.org/10.7554/eLife.30127.004
Figure 3—figure supplement 1
TatA oligomerization behavior of additional charge zipper variants.

(A) Immunoblot of membranes isolated from the strains used in panel (B) and in Figure 3, probed with TatA antibodies (upper panels) or antibodies against TatB and TatC (lower panels). (B) Representative fluorescence images of strains ELV16 λAry EDD (AyEDDBCE), JARV16 λAry EDD (AyEDDBC), and MΔABC λAry EDD (AyEDDE). The scaling used to display these images differs from that employed in Figure 3 and uses 1000 a.u. as the minimum (black) and 4000 a.u. as the maximum (white).

https://doi.org/10.7554/eLife.30127.005

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional information
strain, strain background (Escherichia coli)MC4100Casadaban and Cohen, 1979
strain, strain background (E. coli)MC1061Casadaban and Cohen, 1980
strain, strain background (E. coli)J1M1Sargent et al., 1998MC4100 ΔtatE
strain, strain background (E. coli)JARV16Sargent et al., 1999MC4100 ΔtatA ΔtatE
strain, strain background (E. coli)ELV16Sargent et al., 1999MC4100 ΔtatA
strain, strain background (E. coli)DADEWexler et al., 2000MC4100 ΔtatABCD ΔtatE
strain, strain background (E. coli)MΔABCAlcock et al., 2013MC4100 ΔtatABC::apra
strain, strain background (E. coli)JARV16 λA D31KThis paperJARV16 attB::PtatAtatAD31K (kanr)
strain, strain background (E. coli)JARV16 λA K49DThis paperJARV16 attB::PtatAtatAK49D (kanr)
strain, strain background (E. coli)JARV16 λA D31K/K49DThis paperJARV16 attB::PtatAtatAD31K,K49D (kanr)
strain, strain background (E. coli)JARV16 λA K52DThis paperJARV16 attB::PtatAtatAK52D (kanr)
strain, strain background (E. coli)JARV16 λA D31K/K52DThis paperJARV16 attB::PtatAtatAD31K,K52D (kanr)
strain, strain background (E. coli)JARV16 λA EDDThis paperJARV16 attB::PtatAtatAK37E,K40D,K41D (kanr)
strain, strain background (E. coli)JARV16 λA KKKThis paperJARV16 attB::PtatAtatAD45K,D46K,E47K (kanr)
strain, strain background (E. coli)JARV16 λA EDD/KKKThis paperJARV16 attB::PtatAtatAK37E,K40D,K41D,D45K,D46K,E47K (kanr)
strain, strain background (E. coli)JARV16 λAryThis paperJARV16 attB::PtatAtatA-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)JARV16 λAry D31KThis paperJARV16 attB::PtatAtatAD31K-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)JARV16 λAry K49DThis paperJARV16 attB::PtatAtatAK49D-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)JARV16 λAry K52DThis paperJARV16 attB::PtatAtatAK52D-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)JARV16 λAry EDDThis paperJARV16 attB::PtatAtatA K37E,K40D,K41D-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)JARV16 λAry KKKThis paperJARV16 attB::PtatAtatA D45K,D46K,E47K-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)ELV16 λAry D31KThis paperELV16 attB::PtatAtatAD31K-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)ELV16 λAry K49DThis paperELV16 attB::PtatAtatAK49D-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)ELV16 λAry K52DThis paperELV16 attB::PtatAtatAK52D-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)ELV16 λAry EDDThis paperELV16 attB::PtatAtatA K37E,K40D,K41D-
EAK-eyfpA206K (kanr)
strain, strain background (E. coli)ELV16 λAry KKKThis paperELV16 attB::PtatAtatA D45K,D46K,E47K-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)DADE λAry D31KThis paperDADE attB::PtatAtatAD31K-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)DADE λAry K49DThis paperDADE attB::PtatAtatAK49D-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)DADE λAry K52DThis paperDADE attB::PtatAtatAK52D-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)DADE λAry KKKThis paperDADE attB::PtatAtatA D45K,D46K,E47K-EAK-eyfpA206K (kanr)
strain, strain background (E. coli)MΔABC λAry EDDThis paperMC4100 ΔtatABC::apra attB::PtatAtatA K37E,K40D,K41D-EAK-eyfpA206K (kanr)
antibodyanti-CueOThis paperRabbit poly clonal against CueO mature domain. Affinity purified (1:200)
antibodyanti-DnaKAbcamAbcam ab69617; Clone 8E2/2Mouse monoclonal (1:20000)
antibodyanti-TatAAlcock et al., 2016Rabbit polyclonal against TatA soluble domain (1:5000)
antibodyanti-TatBAlcock et al., 2016Rabbit polyclonal against TatB C-terminal peptide. Affinity purified (1:400)
antibodyanti-TatCAlcock et al., 2016Rabbit polyclonal against TatC C-terminal peptide. Affinity purified (1:1000)
recombinant DNA reagentpQE80-CueOLeake et al., 2008Synthesis of E. coli CueO with a C-terminal his6 tag
recombinant DNA reagentpKSUniAKoch et al., 2012pBluescript-based vector carrying PtatA-tatA
recombinant DNA reagentpBSTatAryAlcock et al., 2013pBluescript-based vector carrying PtatA-tatA-EAK-eyfpA206K
recombinant DNA reagentpRS552Simons et al., 1987Shuttle vector for integration of DNA at the E. coli phage lambda attachment site (attB)
recombinant DNA reagentλRS45Simons et al., 1987Phage for integration of DNA at the E. coli phage lambda attachment site (attB)
sequence-based reagentTatA D31K FSigma-Aldrich, St. Louis, MissouriOligonucleotide GGCTCCATCGGTTCCAAACTTGGTGCGTCGATC
sequence-based reagentTatA D31K RSigma-AldrichOligonucleotide GATCGACGCACCAAGTTTGGAACCGATGGAGCC
sequence-based reagentTatA K49D FSigma-AldrichOligonucleotide CAATGAGCGATGATGAACCAGATCAGGATAAAACCAGTCAGG
sequence-based reagentTatA K49D RSigma-AldrichOligonucleotide CCTGACTGGTTTTATCCTGATCTGGTTCATCATCGCTCATTG
sequence-based reagentTatA K52D FSigma-AldrichOligonucleotide GAACCAAAGCAGGATGATACCAGTCAGGATGCTG
sequence-based reagentTatA K52D RSigma-AldrichOligonucleotide CAGCATCCTGACTGGTATCATCCTGCTTTGGTTC
sequence-based reagentTatA EDD FSigma-AldrichOligonucleotide CTTGGTGCGTCGATCGAAGGCTTTGATGATGCAATGAGCGATGATG
sequence-based reagentTatA EDD RSigma-AldrichOligonucleotide CATCATCGCTCATTGCATCATCAAAGCCTTCGATCGACGCACCAAG
sequence-based reagentTatA KKK FSigma-AldrichOligonucleotide GCTTTAAAAAAGCAATGAGCAAAAAGAAACCAAAGCAGGATAAAACC
sequence-based reagentTatA KKK RSigma-AldrichOligonucleotide GGTTTTATCCTGCTTTGGTTTCTTTTTGCTCATTGCTTTTTTAAAGC
sequence-based reagentTatA zip2 FSigma-AldrichOligonucleotide CTTGGTGCGTCGATCGAAGGCTTTGATGATGCAATGAGCAAAAAG
sequence-based reagentTatA zip2 RSigma-AldrichOligonucleotide CTTTTTGCTCATTGCATCATCAAAGCCTTCGATCGACGCACCAAG

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