Hsp40s specify functions of Hsp104 and Hsp90 protein chaperone machines

Abstract

Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN+] prions was less sensitive than [URE3], but more sensitive than [PSI+]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.

Figure 1. Sis1/Ydj1 hybrid proteins cooperate with E. coli chaperones in distinct processes.

(A) Diagram of conserved Hsp40 domains swapped in the hybrid proteins (not to scale). Numbers indicate amino acid positions, dashed lines indicate splice joints. The regions from residues 172–352 of Sis1 and from 106–409 of Ydj1 are referred to inclusively as CTD. All alleles contain C-terminal c-myc tags (indicated as filled black circles). (B) [PSI⁺] propagation: [PSI⁺] hsp104Δ strain MR386 expressing Hsp104 on a URA3 plasmid and the E. coli chaperones ClpB, DnaK* and GrpE (BK*E) was transformed by single-copy TRP1 plasmids expressing D36N versions (*) of indicated wild type and hybrid proteins. For each hybrid, four independent transformants were grown as patches on plates containing uracil to allow loss of the plasmid encoding Hsp104. These were replica-plated onto medium containing FOA with (upper panel) or lacking (lower panel) adenine. Only [PSI⁺] cells grow on medium lacking adenine. As reported earlier , the Sis1* and Ydj1* cells show representative [PSI⁺] and [psi⁻] phenotypes, respectively, with ClpB in place of Hsp104. Control cells with Hsp104 on the TRP1 plasmid are shown below. (C) Transformants expressing Sis1* lacking its CTD (Sis1*ΔCTD) and versions of Sis1*and Y*SS lacking the dimerization domain (ΔD) were processed as in panel (B). (D) Thermotolerance: Transformants from FOA plates in upper image of panel (B) were cured of prions by growth on guanidine-containing medium and then grown in liquid medium selecting for the plasmids. Cells were diluted to similar density, heat shocked at 50°C for the times indicated at the bottom, and five microliters of five-fold serial dilutions were dropped onto YPAD plates and incubated for three days at 30°C.

Figure 2. Functions of Sis1/Ydj1 hybrids in place of Sis1.

(A) Growth and [PSI⁺] propagation: [PSI⁺] and [psi⁻] versions of strain 930 (sis1Δ) were transformed by plasmids encoding Sis/Ydj1 hybrid proteins. Transformants were grown as patches on medium containing uracil and then replica-plated onto FOA with (left panels) and without (right panels) adenine. Each patch is an individual transformant, similar results were obtained in two other transformations. (B) [URE3] propagation: [URE3] and [ure-o] versions of strain 1385 (sis1Δ) were transformed by the same plasmids and processed as in (A). Cells with prions are Ade⁺ and white while cells lacking prions require exogenous adenine and are red when grown on limiting adenine (see Materials and methods). In all panels each patch is from an independent transformant colony from one of three independent transformations. All of 3 replicated experiments gave the same results.

Figure 3. Growth complementation by Sis1/Ydj1 hybrids in place of Ydj1.

(A) Transformants of strain MR502 (ydj1Δ with YDJ1 on a URA3 plasmid) expressing the indicated hybrid proteins from the SIS1 promoter on single-copy plasmids (ev is empty vector) were taken from FOA plates and grown in liquid medium selecting for the plasmids. Cultures were normalized to the same cell density, serially diluted five-fold and dropped onto YPAD plates, which were incubated at the indicated temperatures for three days. (B) Cells expressing Sis1 or the hybrids with the Sis1 CTD that lack their dimerization domains (ΔD) were processed and grown as in (A).

Figure 4. Function of Sis1/Ydj1 hybrids in galactose induction.

Cells expressing indicated hybrid proteins (from Figure 3, panel A) were assessed for ability to induce expression of a luciferase reporter regulated by the GAL10 promoter. After adjusting cultures to the same cell density, galactose was added and the luciferase activity in aliquots of cells was then measured periodically for two hours. Cells did not grow during this time.

Figure 5. Function of Sis1, Ydj1 and Sis1-Ydj1 hybrid proteins in protein reactivation in vitro.

(A) Reactivation of heat-denatured GFP-38 by Sis1 (blue), Ydj1 (red), SSY (purple) or YYS (yellow) in combination with Hsp104 and Ssa1 was measured over time as described in Methods. Denatured GFP-38 incubated without chaperones is shown in gray. A representative plot of 3 experiments is shown. (B) Initial rates of reactivation of heat-inactivated GFP-38 by Hsp104 and Ssa1 in conjunction with Sis1, Ydj1, SSY or YYS (n = 3, ± SEM). (C) Reactivation of heat-denatured luciferase by Sis1 (blue triangles), Ydj1 (red circles), SSY (purple squares) or YYS (yellow circles) in combination with Ssa1 was measured as described in Methods. Denatured luciferase incubated without chaperones is shown in black circles. Data from three replicates are presented as the mean ± SEM.

Figure 6. Sis1 activities are important for [URE3] propagation.

(A) Diagram (not to scale) of Sis1 coding region. Numbers at top indicate amino acid positions. Domains, designated by abbreviations at top (see text) are indicated by variously shaded boxes. Mutations are indicated on the left. Deleted regions are shown as dashed lines and locations of point mutations H34Q and K199A as diamonds. Q is H34Q, A is K199A. Intact DnaJB1 (not diagrammed), is the closest human homolog of Sis1 and is indicated JB1 in the other panels. (B) Strain 1385 expressing wild type Sis1 from a URA3 plasmid was transformed by single-copy TRP1 plasmids encoding the wild type or mutant proteins (indicated on the left of each image) regulated by the SIS1 promoter. Transformants were taken from plates selecting for [URE3] and both plasmids and streaked onto plates selecting only for the plasmids. Plates were incubated for 3 days at 30°C followed by 2 days at 25°C. [URE3] cells form small white colonies, while [ure-o] cells grow into larger red colonies. (C) [URE3] cells as in panel (B) were grown as patches on plates selecting for [URE3] and both plasmids. These were replica-plated onto medium containing uracil to allow loss of the URA3 plasmid, and then onto medium containing limiting adenine and FOA (shown), which kills cells retaining the URA3 plasmid. Empty vector control is indicated as ev. (D) As in (C) except adenine was omitted from all plates to ensure growth only of cells that propagate [URE3]. (E) Cells from plate in panel (C) were streaked onto similar medium. The fainter red coloration of [ure-o] cells expressing the ‘A’ mutant is due to a slightly higher amount of adenine in the medium. (F) Cells from plate in panel (D) were streaked onto medium containing or lacking adenine, as indicated. In all panels, red colonies arose from cells that lost [URE3].

Figure 7. Curing of [URE3] by overexpression of Ydj1 and hybrid proteins.

(A) Wild type [URE3] cells (strain 1075) carrying plasmids encoding indicated hybrid proteins under control of the GAL promoter were grown in dextrose medium selecting for the plasmid, washed, and transferred to galactose medium. The proportion of [URE3] cells as a function of culture generations in galactose is shown. (B) Curing was done simultaneously using identical conditions except Sis1 and hybrid proteins lacking their dimerization domains (open symbols, as indicated) were used. For easier comparison, these data are plotted separately with the same Ydj1 data. (C) Elevating abundance of Sis1 reduces curing of [URE3] by overexpressing Ydj1, but not Hsp104. Strain 1075 was transformed simultaneously by indicated combinations of empty vectors (−) and single-copy plasmids expressing Ydj1, dominant negative Hsp104-2KT or Sis1 (+) from the GPD promoter. Images show sections of representative primary transformation plates after incubating 4 days at 30°C. Numbers below images indicate average frequency of [URE3] loss (± s.d.).

Figure 8. Some Sis1 activities are important for [PIN⁺] propagation.

[PIN⁺] [psi⁻] cells of strain 930a expressing Rnq1-GFP were transformed by plasmids encoding the mutant Sis1 proteins described in Figure 5. (A) Primary transformant colonies on medium selecting for the plasmids encoding both wild type and mutant Sis1 proteins were assessed for fluorescence. (B) Cells shown in panel (A) were transferred to plates containing uracil and then onto FOA plates to select for cells expressing the only mutant versions of Sis1. Cells from the FOA plates were then assessed for fluorescence. For each set, upper panels are fluorescent images and lower panels show the same cells in bright field. Control [pin⁻] cells carrying one copy of wild type Sis1 are shown on lower right for comparison.