Human DnaJB6 Antiamyloid Chaperone Protects Yeast from Polyglutamine Toxicity Separately from Spatial Segregation of Aggregates

Hsp42, Btn2, and Cur1 are not needed for DnaJB6 to block HttQ103-GFP toxicity. (A to D) [PSI⁺] [PIN⁺] cells were grown and plated as described in the legend to Fig. 1. Cells lacking Hsp42, Btn2, or Cur1 (as indicated) respond to HttQ103-GFP like the wild type. DnaJB6 similarly reduces toxicity and causes assembly of the disperse aggregates into large foci. (E and F) Cells carrying plasmids encoding Hsp42-Cherry (E) or Btn2-RFP (F) were grown in SRaf, transferred to SGal, and visualized by confocal imaging after 4 h. (E) Hsp42-Cherry colocalizes with single foci of HttQ103-QGFP in the presence of DnaJB6 (88% of cells, n = 100) but only partially with smaller foci when DnaJB6 is absent (51% of cells, n = 150). (F) Btn2-RFP colocalizes with HttQ103-GFP foci in 86% of cells (n = 55) without DnaJB6 and in 78% of cells (n = 89) coexpressing DnaJB6. Qualitative confocal microscopy was used to monitor these features. CMAC (blue) stains vacuoles. Panels are sized for consistency; all scale bars are 3 μm.

DnaJB6 colocalizes with HttQ103-GFP foci adjacent to vacuole but not nucleus. (A) [PSI⁺] [PIN⁺] cells carrying plasmids encoding the indicated galactose-inducible proteins (ev, empty vector; QGFP, HttQ103-GFP; B6RFP, DnaJB6-RFP) grown in SRaf were transferred to SGal, grown for 4 h, and visualized by confocal imaging. The nucleus was visualized by DAPI and the vacuole was stained with FM464 (red) or CMAC (blue). Panels on the left show separate expression of HttQ103-GFP (upper) and DnaJB6-RFP (lower). HttQ103-GFP forms multiple foci scattered in the cytoplasm rather than adjacent to the vacuole (FM464, red). DnaJB6-RFP is diffuse in the cytoplasm. Panels on right show cells expressing both HttQ103-GFP and DnaJB6. Multiple HttQ103-GFP foci are collected into one location mostly coincident (89% cells, n = 62) with DnaJB6. (B) As described for panel A but using cells lacking prions ([psi⁻] [pin⁻]). Panels are sized for consistency; all scale bars are 3 μm.

Autophagy pathway is not involved in DnaJB6-driven HttQ103-GFP focus formation or toxicity prevention, but latrunculin A blocks focus formation. (A) Cells expressing RFP-Atg8 and empty vector (ev), HttQ103-GFP (QGFP), or nontagged DnaJB6 (B6) were visualized using confocal microscopy. RFP-Atg8 localizes at the PAS (phagophore assembly site) adjacent to the vacuole (CMAC blue) (uppermost images). In cells expressing both proteins, most foci of RFP-Atg8 (76%, n = 51) overlap HttQ103-GFP aggregates, but most HttQ103-GFP foci do not overlap RFP-Atg8. When DnaJB6 is also expressed, HttQ103-GFP forms single foci well separated from RFP-Atg8 foci in most (82%, n = 58) cells. Panels are sized for consistency; all scale bars are 3 μm. (B) An atg5 deletion strain expressing the indicated galactose-inducible proteins was grown and plated as described in the legend to Fig. 1. DnaJB6 protects cells and assembles disperse aggregates into large foci when Atg5 is absent. (C) Latrunculin A (lat A) or DMSO was added to cells expressing galactose-inducible HttQ103-GFP without (ev) or with coexpression of DnaJB6 (B6), and cells were monitored microscopically after inducing for 4 h.

STC regions are necessary and sufficient for protection from toxicity but cannot gather disperse aggregates. (A) Domain structure of DnaJB6 is diagrammed with amino acid residues delineating domains indicated above (J, J-domain; GF, glycine-phenylalanine rich; ST, serine-threonine rich; CTD, C-terminal domain). (B, left) Cells expressing galactose-inducible HttQ103-GFP together with wild-type and mutant versions of DnaJB6 as indicated (C, CTD; R, RFP). Cells carrying plasmids encoding the indicated galactose-inducible proteins (ev, empty vector; Q, HttQ103-GFP; B6, DnaJB6; D33N, point mutation in J-domain) were grown and plated as described for Fig. 1. (Right, upper) Versions of DnaJB6 without coexpressing HttQ103-GFP. (Lower) Independent replicate of the experiment shown on the left but done on a different lot of medium. (C) Microscopic observation of aggregation status of HttQ103-GFP 4 h after inducing cultures used for the toxicity assay shown in panel B. (D) Cells expressing HttQ103-GFP- and RFP-tagged versions of DnaJB6 mutants ST (top), STC (middle), and D33N (bottom) were monitored by confocal microscopy. All mutant versions of DnaJB6 are diffuse when expressed alone. All also colocalize completely with multiple HttQ103-GFP foci (91%, n = 149; 94%, n = 189; 92%, n = 153, respectively) but fail to collect them. Panels are sized for consistency; all scale bars are 3 μm.