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Research Article

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

Jyotsna Kumar, Neila L. Kline, Daniel C. Masison
Jyotsna Kumar
aLaboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
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Neila L. Kline
aLaboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
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Daniel C. Masison
aLaboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
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DOI: 10.1128/MCB.00437-18
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  • FIG 1
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    FIG 1

    Neither Bmh1 nor Sti1 is needed for DnaJB6 to block HttQ103-GFP toxicity. Wild-type cells lacking prions (strain NK104) and expressing galactose-inducible proteins (ev, empty vector; Q, HttQ103-GFP; B6, DnaJB6) were grown overnight in SRaf medium and 5-fold serially diluted, and 5-μl drops were inoculated onto noninducing (−Gal) or inducing (+Gal) medium as indicated. Panels on the left show growth inhibition caused by HttQ103-GFP, and panels on the right show aggregation status of HttQ103-GFP in the same overnight cultures used for the toxicity assay after shifting to liquid SGal medium for 4 h. (A) Cells lacking prions; HttQ103-GFP does not form toxic aggregates. (B) As described for panel A, except cells propagate both [PSI+] and [PIN+] prions (strain NK101). HttQ103-GFP forms multiple disperse aggregates that are toxic in wild-type cells. Coexpression of DnaJB6 prevents toxicity and drives the smaller aggregates into single large foci. (C and D) As described for panel B, except cells lack Bmh1 (C) or Sti1 (D). DnaJB6 retains the ability to protect cells and assemble disperse aggregates into large foci when Bmh1 or Sti1 is absent.

  • FIG 2
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    FIG 2

    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.

  • FIG 3
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    FIG 3

    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.

  • FIG 4
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    FIG 4

    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.

  • FIG 5
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    FIG 5

    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.

  • FIG 6
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    FIG 6

    Redundant and diverse functions of DnaJB6 and DnaJB7 domains. (A) Amino acid sequence of homologous regions of DnaJB6 and DnaJB7 J-domains (differences dotted) and CTDs. Residues 245 to 309 of DnaJB7, which include a nuclear localization signal, are not shown. (B) Focus formation in cells expressing HttQ103-GFP and the indicated versions of DnaJB6 (JB6), DnaJB7 (JB7; lacks residues 227 to 234, underlined, and 245 to 309), and DnaJB6/7 hybrid proteins. JB6-7J is DnaJB6 with residues 1 to 70 replaced by those of DnaJB7; JB6-7C is DnaJB6 with residues 196 to 241 replaced by residues 192 to 244 (lacking 227 to 234) of DnaJB7. JB7-6J is JB7 with residues 1 to 70 replaced by those of DnaJB6. JB7-6C is JB7 with residues 192 to 244 replaced by residues 196 to 241 of DnaJB6. Cells were grown and images captured as described in the legend to Fig. 1.

Tables

  • Figures
  • TABLE 1

    Summary of protection from polyQ toxicity and spatial segregation of polyQ aggregates by DnaJB proteinsa

    ProteinToxicity protectionSpatial segregation
    DnaJB6++
    JB6-D33N+−
    JB6-STC only+−
    JB6-ST only−−
    JB6ΔST−−
    JB6ΔC−+
    DnaJB6-7J−+
    DnaJB6-7C+/−+
    DnaJB6-7ST−+
    DnaJB7−−
    DnaJB7-6J−−
    DnaJB7-6C−/+−
    • ↵a Figures 5 and 6 provide experimental data. +, yes; −, no; +/−, partial; −/+, noticeable but weak.

  • TABLE 2

    Yeast strains used in this studya

    StrainGenotype
    NK101MATa kar1-1 ade2-1 SUQ5 his3Δ202 leu2Δ1 trp1Δ63 ura3-52 [PSI+] [PIN+]
    NK104NK101 [psi−] [pin−]
    1611NK101 MATα sti1::HIS3
    1673NK101 hsp42::KanMX4
    1778NK101 bmh1::KanMX4
    1780NK101 btn2::TRP1
    1782NK101 cur1::KanMX4
    1784NK101 btn2::TRP1 cur1::KanMX4
    1341NK101 atg5::KanMX4
    • ↵a All strains are isogenic to NK101, a confirmed [PSI+] [PIN+] version of strain 779-6A (45).

  • TABLE 3

    Plasmids used in this study

    NameDescriptionPromoterMarkerSource or reference
    pYES2-Q103GFPQ103-GFPGAL1URA324
    pRU14DnaJB6bGAL1LEU28
    pJE201DnaJB6bGAL1TRP1This study
    pJE202DnaJB6bGAL1HIS3This study
    pMR360DnaJB6-RFPGAL1LEU28
    pNK104JB6-ST onlyGAL1LEU2This study
    pNK105JB6-STC onlyGAL1LEU2This study
    pNK106ST-RFPGAL1LEU2This study
    pNK107STC-RFPGAL1LEU2This study
    pRU29DnaJB6ΔSTGAL1LEU2This study
    pJE195DnaJB6ΔCTDGAL1LEU2This study
    pRU22DnaJB6-D33NGAL1LEU2This study
    pJE204DnaJB6D33N-RFPGAL1LEU2This study
    pDK58Btn2-RFPGAL1LEU249
    pHsp42-CherryHsp42-CherryGAL1LEU229
    pJB6-7JJB6 + JB7 J-domainGAL1LEU2This study
    pJB6-7CJB6 + JB7 CTDaGAL1LEU2This study
    pJB7DnaJB7aGAL1LEU2This study
    pJB7-6JJB7a + JB6 J-domainGAL1LEU2This study
    pJB7-6CJB7 + JB6 CTDGAL1LEU2This study
    pRS315RNQ1-GFPRnq1-GFPRNQ1LEU250
    pCUP1-DuDre-Atg8-404RFP-Atg8CUP1TRP151
    • ↵a CTD of DnaJB7 in these constructs lacks residues 227 to 234 and 245 to 309 (Fig. 6A).

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Human DnaJB6 Antiamyloid Chaperone Protects Yeast from Polyglutamine Toxicity Separately from Spatial Segregation of Aggregates
Jyotsna Kumar, Neila L. Kline, Daniel C. Masison
Molecular and Cellular Biology Nov 2018, 38 (23) e00437-18; DOI: 10.1128/MCB.00437-18

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Human DnaJB6 Antiamyloid Chaperone Protects Yeast from Polyglutamine Toxicity Separately from Spatial Segregation of Aggregates
Jyotsna Kumar, Neila L. Kline, Daniel C. Masison
Molecular and Cellular Biology Nov 2018, 38 (23) e00437-18; DOI: 10.1128/MCB.00437-18
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KEYWORDS

DnaJB6
Huntington's disease
J-protein
molecular chaperone
heat shock protein (HSP)
amyloid

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