Non-targeted identification of prions and amyloid-forming proteins from yeast and mammalian cells

Abstract

The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.

Keywords: Aggregation; Amyloid; FUS; Mass Spectrometry (MS); Polyglutamine; Prions; Stress Granule; Yeast; rnq1; sup35.

FIGURE 1.

The workflow of the TAPI. See a detailed description under “Results” and “Experimental Procedures.”

FIGURE 2.

Purification of prion aggregates based on their SDS resistance. A, the isogenic yeast strain pair, 74D-694 [PSI+][RNQ+] and [psi-][rnq-], was lysed and treated with nucleases under identical conditions, as described under “Experimental Procedures.” After ultracentrifugation, the pellets were treated with 2% SDS at 37 °C, subjected to SDS-PAGE, and stained with Coomassie Blue. B, the same samples from A were subjected to SDS-PAGE and stained with anti-Sup35 and anti-Rnq1 antibodies. C, the isogenic yeast strain pair, differing only in its [psi] state (74D-694 [PSI+][RNQ+] and [psi-][RNQ+]), was lysed and treated as in A. Trapped proteins were eluted from the gel pieces by heating at 95 °C, concentrated, subjected to SDS-PAGE, and stained with Coomassie Blue. D, the same samples from C were subjected to SDS-PAGE and stained with anti-Sup35 and anti-Rnq1 antibodies. E, detection of Sup35 in a dilution series. 74D-694 [PSI+][RNQ+] and [psi-][rnq-] lysates were prepared and equalized by total protein concentration using a BCA assay. Four samples were prepared by diluting the prion lysate with the non-prion lysate in proportions where the final mixtures contained 100, 30, 10, and 3% of the prion lysate correspondingly. Samples were prepared by TAPI, and equal aliquots of the eluted and purified fractions (right before protease digestion) were subjected to SDS-PAGE and immunoblotting with anti-Sup35 antibodies.

FIGURE 3.

Verification of protein aggregation. A, 10 [PSI+] wild strains were analyzed by SDD-AGE (14); Sup35 was detected by immunostaining. The isogenic pair 74D-694 [PSI+][RNQ+] and [psi-][rnq-] was used as controls. HMW,- high molecular weight aggregates; LMW, low molecular weight aggregates. In the left panel, cell lysates were directly applied to SDD-AGE; in the right panel they were first pelleted (200,000 × g for 1 h), and pellets were applied to SDD-AGE (pelleting improves resolution of low molecular weight aggregates). B, the same wild strains were transformed with Sup35NM-GFP and analyzed by fluorescent microscopy. C and D, detection of polyglutamine-GFP by immunoblotting. C, yeast cells expressing HttQ103-GFP from the inducible GAL1 promoter for 5 h were lysed and subjected to TAPI. HttQ103-GFP was detected by immunoblotting with anti-GFP antibodies from crude cell lysate (lane 1) and after elution and purification before protease digestion (lane 2). No signal was detected from the uninduced control cells. D, Western analysis of the PC12 huntingtin cell model (29) with stably transfected exon 1 of the HD gene with 74 CAGs under the control of a tetracycline promoter.

FIGURE 4.

Co-localization of polyglutamine aggregates with stress granule proteins in yeast. A and B, co-expression of the GAL1-controlled HttQ103-GFP (A) or HttQ25-GFP (B) and Pub1-CherryFP in the W303. C and D, co-expression of Pbp1-GFP (C) or Pab1-GFP (D) and the GAL1-controlled HttQ103-RFP in the BY4741.

FIGURE 5.

Follow-up analysis of the identified proteins. A, immunostaining of elution fractions (before protease digestion) prepared from PC12 cells expressing either HttQ74-GFP or HttQ23-GFP. Anti-TDP-43 or anti-FUS antibodies were used. B, SDD-AGE analysis of 74D-694 [RNQ+] and [rnq-] strains that expressed the GAL1-controlled RNQ1-GFP for 5 h. As a positive control, 74D-694 [RNQ+] with overproduction of Sis1 was used. Immunostaining was performed with anti-Sis1 antibodies (Ab, left) or anti-Rnq1 antibodies (right). HMW, high molecular weight aggregates. C and D, analysis of amyloid-forming potential of the identified proteins. Eight common proteins (HA-tagged, from the yeast ORF library) were expressed in BY4741. Lysates were treated with 1% SDS and loaded on an acrylamide gel for SDS-PAGE (C) or directly on PVDF membrane (D, to estimate the expression level) followed by immunostaining with the anti-HA antibodies. In C, only a portion of the gel just below the loading wells is shown.

FIGURE 6.

Electron micrographs of the recovered SDS-resistant protein aggregates from yeast cells. A, and B, the gel-recovered material from the isogenic strain pair 74D-694 [PSI+][RNQ+] and [psi-][rnq-] acquired at 800× direct magnification on carbon/Formvar-covered copper grids; A shows a field from the strain with no prions; B shows the field from the strain harboring both [PSI+] and [RNQ+]. C and D, representative images of the proteinaceous debris from the prion-containing strain at 80,000× and 150,000× direct magnification. E, small barrel-shaped structures observed from prion-containing samples. F, example of a fiber observed after recovered material was incubated several days at room temperature. White scale bars = 10 μm; black scale bars = 100 nm. G, Western blotting and immunostaining of the gel-recovered material from A and B above with anti-Sup35 and anti-Rnq1 antibodies.