Prokaryotic chaperones support yeast prions and thermotolerance and define disaggregation machinery interactions

Abstract

Saccharomyces cerevisiae Hsp104 and Escherichia coli ClpB are Hsp100 family AAA+ chaperones that provide stress tolerance by cooperating with Hsp70 and Hsp40 to solubilize aggregated protein. Hsp104 also remodels amyloid in vitro and promotes propagation of amyloid prions in yeast, but ClpB does neither, leading to a view that Hsp104 evolved these activities. Although biochemical analyses identified disaggregation machinery components required for resolubilizing proteins, interactions among these components required for in vivo functions are not clearly defined. We express prokaryotic chaperones in yeast to address these issues and find ClpB supports both prion propagation and thermotolerance in yeast if it is modified to interact with yeast Hsp70 or if E. coli Hsp70 and its cognate nucleotide exchange factor (NEF) are present. Our findings show prion propagation and thermotolerance in yeast minimally require cooperation of species-specific Hsp100, Hsp70, and NEF with yeast Hsp40. The functions of this machinery in prion propagation were directed primarily by Hsp40 Sis1p, while thermotolerance relied mainly on Hsp40 Ydj1p. Our results define cooperative interactions among these components that are specific or interchangeable across life kingdoms and imply Hsp100 family disaggregases possess intrinsic amyloid remodeling activity.

Figure 1

Prion phenotypes of parental strains and domain structure of the Hsp100 disaggregase. (A) Strains 1408 and 1410 lacking prions ([psi^–], [ure-o]) are red (lower). [PSI⁺] and [URE3] make the cells white, and [URE3] reduces growth rate. [PSI⁺] is unstable in strain 1408 due to heterogeneity of expression of the plasmid-based HSP104. Rnq1p–GFP forms foci in [PIN⁺] cells, but remains diffuse in [pin^–] cells. (B) Diagram of the domain structure of Hsp100 monomer. NTD, amino-terminal domain; NBD-1, nucleotide-binding domain 1; M, Middle region; NBD-2, nucleotide-binding domain 2. The approximate position of domain boundaries is indicated for ClpB (above) and for Hsp104 (below). The M region is imbedded within NBD-1.

Figure 2

The M region of Hsp104 allows ClpB to support propagation of yeast prions. (A) Resident wild type Hsp104-encoding plasmid in hsp104Δ strains 1408 ([PSI⁺]) and 1410 ([URE3]) was exchanged with plasmids encoding the indicated Hsp104/ClpB hybrids (KEY). Patches of cells expressing the hybrids in place of Hsp104 were replica plated onto solid media containing limiting (upper) or no (lower) adenine to assess prion phenotypes. [PSI⁺] and [URE3] cells appear white or pink on limiting adenine (upper) and can grow in the absence of adenine (lower). Punctate fluorescence of cells expressing an Rnq1–GFP fusion protein (rightmost) indicates presence of [PIN⁺]. (B) Cells from plates in A expressing the indicated Hsp104/ClpB hybrid or control protein from TRP1 plasmids were streaked onto plates containing limiting adenine and lacking tryptophan. [PSI⁺] and [URE3] cells appear white or pink, while cells lacking prions give rise to red colonies. Red sectors in white or pink colonies are progeny of cells that lost the prion during growth of the colony.

Figure 3

ClpB supports [PSI⁺] propagation if DnaK and GrpE are coexpressed. (A) The Hsp104-encoding plasmid in strain 1408 [PSI⁺] cells expressing combinations of GrpE (E) and DnaK (K) was exchanged with empty vector (ev) or plasmids encoding Hsp104 (104), ClpB, or ClpB–Y503D. Transformants were replica plated onto medium containing limiting (left) or no (right) adenine. (B) Identical samples of lysates of cells exogenously expressing combinations of chaperones (indicated above blots) were separated on four SDS–PAGE gels, blotted, and probed with antibodies to the indicated chaperones. Load is a representative portion of amido-black stained membrane of the Hsp104 blot. Asterisks indicate mutant proteins ClpB–Y503D or DnaK–R167H. (C) Cells expressing ClpB and combinations of DnaK and GrpE as indicated were grown with limiting adenine. We quantified mitotic loss of [PSI⁺] from BKE cells by adding adenine to liquid cultures grown without adenine and growing another six generations. A total of 33% (SD ± 1.7, n = 3) of them were [psi^–], while <1% of the cells expressing wild-type Hsp104 were [psi^–]. (D) The same cells were mated to [psi^–] strain 620 (horizontal streak) to test for dominant prion phenotype. Crosses grown on rich medium (top) were replica plated onto medium containing limiting adenine that selects for [PSI⁺] and [psi^–] diploids (middle) and onto similar medium lacking adenine (bottom) that allows growth of [PSI⁺] diploids only.

Figure 4

J proteins that support prion propagation. (A) As in Figure 3A, except DnaK–R167H (K*) was used in place of DnaK where indicated. (B) Replicate samples of lysates of cells lacking HSP104 (hsp104Δ), or expressing Hsp104 (HSP104) or ClpB (ClpB) from the HSP104 genomic locus were separated by SDS–PAGE, blotted, and probed using appropriate indicated antibodies; mw, molecular weight size markers. (C) As in A except DnaJ (BKEJ, [psi^–] and [PSI⁺] versions), DnaJ–D35N (J*), Sis1–D36N (S*), or Ydj1–D36N (Y*) were expressed exogenously in cells expressing ClpB from the chromosomal HSP104 locus. Wild-type (K) and R167H (K*) versions of DnaK are indicated. All panels are representative of at least three independent experiments. (D) Cells used in panel C were streaked for single colonies on medium lacking adenine and grown 3 days at 30°.

Figure 5

Thermotolerance of hsp104Δ yeast expressing combinations of E. coli proteins. (A) Cultures of the strains used in Figure 3A expressing empty vector (ev), Hsp104 (104), or the indicated combinations of ClpB (B), DnaK (K), and GrpE (E) were pretreated at 37° for 15 min and then exposed to 50° for 20 min. Fivefold serial dilutions of the cultures were transferred to rich medium and incubated 3 days. (B) Cultures of cells used in Figure 4C expressing the indicated chaperones were treated and plated similarly. Data are representative of at least three independent experiments.