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Prion propagation can occur in a prokaryote and requires the ClpB chaperone.

Yuan AH, Garrity SJ, Nako E, Hochschild A - Elife (2014)

Bottom Line: Here, we demonstrate that E. coli can propagate the Sup35 prion under conditions that do not permit its de novo formation.Prion propagation in yeast requires Hsp104 (a ClpB ortholog), and prior studies have come to conflicting conclusions about ClpB's ability to participate in this process.Our demonstration of ClpB-dependent prion propagation in E. coli suggests that the cytoplasmic milieu in general and a molecular machine in particular are poised to support protein-based heredity in the bacterial domain of life.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States Whitehead Institute for Biomedical Research, Cambridge, United States.

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α-Sup35 and α-His6X antibodies are interchangeable for detecting the Sup35 NM-mCherry-His6X fusion protein.(A) Probing nitrocellulose membranes with either α-Sup35 antibody (Figure 1C) or α-His6X antibody results in the detection of similar intracellular Sup35 NM fusion protein products in the presence or absence of New1 as assessed by Western blot analysis. Full-length (FL) Sup35 NM fusion proteins are indicated by arrows. (B) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3B) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and Round 1 (R1) experimental clones as assessed by filter retention analysis. For each sample, undiluted lysate and three twofold dilutions are shown. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. (C) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3D) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and R1 control clones as assessed by filter retention analysis.DOI:http://dx.doi.org/10.7554/eLife.02949.004
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fig1s1: α-Sup35 and α-His6X antibodies are interchangeable for detecting the Sup35 NM-mCherry-His6X fusion protein.(A) Probing nitrocellulose membranes with either α-Sup35 antibody (Figure 1C) or α-His6X antibody results in the detection of similar intracellular Sup35 NM fusion protein products in the presence or absence of New1 as assessed by Western blot analysis. Full-length (FL) Sup35 NM fusion proteins are indicated by arrows. (B) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3B) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and Round 1 (R1) experimental clones as assessed by filter retention analysis. For each sample, undiluted lysate and three twofold dilutions are shown. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. (C) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3D) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and R1 control clones as assessed by filter retention analysis.DOI:http://dx.doi.org/10.7554/eLife.02949.004

Mentions: To facilitate these experiments, we fused Sup35 NM and New1 to two monomeric fluorescent proteins (mCherry bearing a C-terminal hexahistidine tag and mGFP, respectively). The two fusion proteins were produced from compatible plasmids under the control of IPTG-inducible promoters. The plasmid encoding New1-mGFP (pSC101TS-NEW1) bore a temperature-sensitive origin of replication, enabling us to cure cells of New1-encoding DNA and thereby deplete cells of the New1 fusion protein. As an initial test of our experimental system, we introduced the plasmid encoding Sup35 NM-mCherry-His6x (pBR322-SUP35 NM) together with either pSC101TS-NEW1 or an empty vector control (pSC101TS) into E. coli cells and induced the synthesis of the fusion proteins at the permissive temperature. After overnight growth, we detected SDS-stable Sup35 NM aggregates (‘Materials and methods’) only in cells producing the New1 fusion protein (Figure 1B). As New1 can independently adopt an amyloid conformation in E. coli (Garrity et al., 2010), we also detected SDS-stable New1 aggregates in cells containing both fusion proteins (Figure 1B). Western blot analysis revealed that the intracellular levels of the Sup35 NM fusion protein were comparable in the presence and absence of the New1 fusion protein (Figure 1C, Figure 1—figure supplement 1A). We also examined cells by fluorescence microscopy. In cells containing both fusion proteins, Sup35 NM formed twisted ring structures (Garrity et al., 2010) or large polar foci in 22.5% of cells (N = 258), whereas New1 formed punctate foci in 89.8% of cells (N = 258). (Figure 1D). In contrast, Sup35 NM exhibited diffuse fluorescence in 100% of cells lacking New1 (N = 532) (Figure 1D).


Prion propagation can occur in a prokaryote and requires the ClpB chaperone.

Yuan AH, Garrity SJ, Nako E, Hochschild A - Elife (2014)

α-Sup35 and α-His6X antibodies are interchangeable for detecting the Sup35 NM-mCherry-His6X fusion protein.(A) Probing nitrocellulose membranes with either α-Sup35 antibody (Figure 1C) or α-His6X antibody results in the detection of similar intracellular Sup35 NM fusion protein products in the presence or absence of New1 as assessed by Western blot analysis. Full-length (FL) Sup35 NM fusion proteins are indicated by arrows. (B) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3B) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and Round 1 (R1) experimental clones as assessed by filter retention analysis. For each sample, undiluted lysate and three twofold dilutions are shown. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. (C) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3D) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and R1 control clones as assessed by filter retention analysis.DOI:http://dx.doi.org/10.7554/eLife.02949.004
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fig1s1: α-Sup35 and α-His6X antibodies are interchangeable for detecting the Sup35 NM-mCherry-His6X fusion protein.(A) Probing nitrocellulose membranes with either α-Sup35 antibody (Figure 1C) or α-His6X antibody results in the detection of similar intracellular Sup35 NM fusion protein products in the presence or absence of New1 as assessed by Western blot analysis. Full-length (FL) Sup35 NM fusion proteins are indicated by arrows. (B) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3B) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and Round 1 (R1) experimental clones as assessed by filter retention analysis. For each sample, undiluted lysate and three twofold dilutions are shown. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. (C) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3D) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and R1 control clones as assessed by filter retention analysis.DOI:http://dx.doi.org/10.7554/eLife.02949.004
Mentions: To facilitate these experiments, we fused Sup35 NM and New1 to two monomeric fluorescent proteins (mCherry bearing a C-terminal hexahistidine tag and mGFP, respectively). The two fusion proteins were produced from compatible plasmids under the control of IPTG-inducible promoters. The plasmid encoding New1-mGFP (pSC101TS-NEW1) bore a temperature-sensitive origin of replication, enabling us to cure cells of New1-encoding DNA and thereby deplete cells of the New1 fusion protein. As an initial test of our experimental system, we introduced the plasmid encoding Sup35 NM-mCherry-His6x (pBR322-SUP35 NM) together with either pSC101TS-NEW1 or an empty vector control (pSC101TS) into E. coli cells and induced the synthesis of the fusion proteins at the permissive temperature. After overnight growth, we detected SDS-stable Sup35 NM aggregates (‘Materials and methods’) only in cells producing the New1 fusion protein (Figure 1B). As New1 can independently adopt an amyloid conformation in E. coli (Garrity et al., 2010), we also detected SDS-stable New1 aggregates in cells containing both fusion proteins (Figure 1B). Western blot analysis revealed that the intracellular levels of the Sup35 NM fusion protein were comparable in the presence and absence of the New1 fusion protein (Figure 1C, Figure 1—figure supplement 1A). We also examined cells by fluorescence microscopy. In cells containing both fusion proteins, Sup35 NM formed twisted ring structures (Garrity et al., 2010) or large polar foci in 22.5% of cells (N = 258), whereas New1 formed punctate foci in 89.8% of cells (N = 258). (Figure 1D). In contrast, Sup35 NM exhibited diffuse fluorescence in 100% of cells lacking New1 (N = 532) (Figure 1D).

Bottom Line: Here, we demonstrate that E. coli can propagate the Sup35 prion under conditions that do not permit its de novo formation.Prion propagation in yeast requires Hsp104 (a ClpB ortholog), and prior studies have come to conflicting conclusions about ClpB's ability to participate in this process.Our demonstration of ClpB-dependent prion propagation in E. coli suggests that the cytoplasmic milieu in general and a molecular machine in particular are poised to support protein-based heredity in the bacterial domain of life.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States Whitehead Institute for Biomedical Research, Cambridge, United States.

Show MeSH
Related in: MedlinePlus