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An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

Lee HC, Portnoff AD, Rocco MA, DeLisa MP - Sci Rep (2014)

Bottom Line: As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla).The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics.Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.

ABSTRACT
The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary "hitchhiker" mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

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Tat-mediated hitchhiker export of PaoABC heterotrimer.Western blot analysis of cytoplasmic (c) and periplasmic (p) fractions prepared from E. coli MC4100 or B1LK0 cells carrying plasmid pPaoABC, which encodes the synthetic paoABC operon. Fractions were normalized to an equivalent number of cells prior to loading. Co-expression of epitope-tagged PaoA-c-Myc, PaoB-FLAG and PaoC-HA were detected using anti-c-Myc, anti-FLAG, and anti-HA antibodies, respectively. The cytoplasmic chaperone GroEL was detected using anti-GroEL antibody and served as a fractionation marker. Molecular weight (MW) markers are indicated on the left. See Supplementary Fig. S7 for uncropped versions of the images.
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f1: Tat-mediated hitchhiker export of PaoABC heterotrimer.Western blot analysis of cytoplasmic (c) and periplasmic (p) fractions prepared from E. coli MC4100 or B1LK0 cells carrying plasmid pPaoABC, which encodes the synthetic paoABC operon. Fractions were normalized to an equivalent number of cells prior to loading. Co-expression of epitope-tagged PaoA-c-Myc, PaoB-FLAG and PaoC-HA were detected using anti-c-Myc, anti-FLAG, and anti-HA antibodies, respectively. The cytoplasmic chaperone GroEL was detected using anti-GroEL antibody and served as a fractionation marker. Molecular weight (MW) markers are indicated on the left. See Supplementary Fig. S7 for uncropped versions of the images.

Mentions: E. coli PaoA carries a canonical Tat signal peptide1019 and is known to form a heterotrimeric complex with PaoB and PaoC17. Hence, we hypothesized that PaoABC might be exported out of the cytoplasm by the Tat translocase according to a hitchhiker mechanism whereby PaoB and PaoC are ferried to the periplasm by the signal peptide-bearing PaoA subunit. To test this notion, the paoABC genes were assembled into a synthetic operon under the control of a single Trc promoter in plasmid pTrc99Y using yeast-based recombineering21 (Supplementary Fig. S1). The resulting plasmid, pPaoABC, permits co-expression of the PaoABC subunits each appended with a unique C-terminal tag for specific immunodetection. Following expression of the synthetic paoABC operon in wild-type (wt) MC4100 cells, all three PaoABC subunits were detected in the periplasm, with a comparable amount of each subunit also detected in the cytoplasm (Fig. 1). In B1LK0 cells, which lack the essential TatC component of the translocase22, each PaoABC subunit accumulated only in the cytoplasm (Fig. 1), indicating that transport of PaoABC to the periplasm is dependent on the Tat pathway.


An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

Lee HC, Portnoff AD, Rocco MA, DeLisa MP - Sci Rep (2014)

Tat-mediated hitchhiker export of PaoABC heterotrimer.Western blot analysis of cytoplasmic (c) and periplasmic (p) fractions prepared from E. coli MC4100 or B1LK0 cells carrying plasmid pPaoABC, which encodes the synthetic paoABC operon. Fractions were normalized to an equivalent number of cells prior to loading. Co-expression of epitope-tagged PaoA-c-Myc, PaoB-FLAG and PaoC-HA were detected using anti-c-Myc, anti-FLAG, and anti-HA antibodies, respectively. The cytoplasmic chaperone GroEL was detected using anti-GroEL antibody and served as a fractionation marker. Molecular weight (MW) markers are indicated on the left. See Supplementary Fig. S7 for uncropped versions of the images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4273604&req=5

f1: Tat-mediated hitchhiker export of PaoABC heterotrimer.Western blot analysis of cytoplasmic (c) and periplasmic (p) fractions prepared from E. coli MC4100 or B1LK0 cells carrying plasmid pPaoABC, which encodes the synthetic paoABC operon. Fractions were normalized to an equivalent number of cells prior to loading. Co-expression of epitope-tagged PaoA-c-Myc, PaoB-FLAG and PaoC-HA were detected using anti-c-Myc, anti-FLAG, and anti-HA antibodies, respectively. The cytoplasmic chaperone GroEL was detected using anti-GroEL antibody and served as a fractionation marker. Molecular weight (MW) markers are indicated on the left. See Supplementary Fig. S7 for uncropped versions of the images.
Mentions: E. coli PaoA carries a canonical Tat signal peptide1019 and is known to form a heterotrimeric complex with PaoB and PaoC17. Hence, we hypothesized that PaoABC might be exported out of the cytoplasm by the Tat translocase according to a hitchhiker mechanism whereby PaoB and PaoC are ferried to the periplasm by the signal peptide-bearing PaoA subunit. To test this notion, the paoABC genes were assembled into a synthetic operon under the control of a single Trc promoter in plasmid pTrc99Y using yeast-based recombineering21 (Supplementary Fig. S1). The resulting plasmid, pPaoABC, permits co-expression of the PaoABC subunits each appended with a unique C-terminal tag for specific immunodetection. Following expression of the synthetic paoABC operon in wild-type (wt) MC4100 cells, all three PaoABC subunits were detected in the periplasm, with a comparable amount of each subunit also detected in the cytoplasm (Fig. 1). In B1LK0 cells, which lack the essential TatC component of the translocase22, each PaoABC subunit accumulated only in the cytoplasm (Fig. 1), indicating that transport of PaoABC to the periplasm is dependent on the Tat pathway.

Bottom Line: As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla).The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics.Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.

ABSTRACT
The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary "hitchhiker" mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

Show MeSH
Related in: MedlinePlus