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Running title: S. pombe chaperones propagate S. cerevisiae prions 1

S. pombe disaggregation machinery chaperones support S. cerevisiae growth and prion 2

propagation. 3

Michael Reidy, Ruchika Sharma and Daniel C. Masison# 4

Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and 5

Kidney Diseases, National Institutes of Health, Bethesda, MD 20892. 6

#[email protected], (301) 594-1316 7

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Hsp100 chaperones protect microorganisms and plants from environmental stress by cooperating 9

with Hsp70 and its nucleotide exchange factor (NEF) and Hsp40 co-chaperones to resolubilize 10

proteins from aggregates. The S. cerevisiae Hsp104-based disaggregation machinery also is 11

essential for replication of amyloid-based prions. E. coli ClpB can substitute for Hsp104 to 12

propagate [PSI+] prions in yeast, but only if E. coli DnaK and GrpE (Hsp70 and NEF) are co-13

expressed. Here, we tested if the reported inability of S. pombe Hsp104 (Sp-Hsp104) to support 14

[PSI+] propagation was due to similar species-specific chaperone requirements, and find that Sp-15

Hsp104 alone supported propagation of three different yeast prions. Sp-Hsp70 and Sp-Fes1p 16

(NEF) likewise functioned in place of their S. cerevisiae counterparts. Thus, chaperones of these 17

long diverged species possess conserved activities that function in processes essential for both 18

cell growth and prion propagation, suggesting S. pombe can propagate its own prions. We show 19

that curing by Hsp104 overexpression and inactivation can be distinguished, and confirm the 20

observation that, unlike Sc-Hsp104, Sp-Hsp104 cannot cure yeast of [PSI+] when it is 21

overexpressed. These results are consistent with a view that mechanisms underlying prion 22

replication and elimination are distinct. 23

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Copyright © 2013, American Society for Microbiology. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00301-12 EC Accepts, published online ahead of print on 15 March 2013

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Microorganisms and plants encode Hsp100 family protein disaggregating chaperones that 25

restore cell viability after exposure to environmental stresses by resolubilizing proteins from 26

aggregates. Protein disaggregation by Hsp100 chaperones in vivo requires cooperation with the 27

Hsp70 protein chaperone and its Hsp40 and nucleotide exchange factor (NEF) regulators (1, 2). 28

In Saccharomyces cerevisiae this machinery is also essential for replicating prions by 29

fragmenting prion polymers, which consist of amyloid, a highly organized fibrous protein 30

aggregate (3-5). The most studied yeast prions are [PSI+], [URE3] and [PIN+] (also known as 31

[RNQ+]), which are composed of the proteins Sup35p, Ure2p and Rnq1p, respectively (6-8). 32

Although yeast Hsp104 does not function in E. coli and the 45% identical E. coli homolog 33

ClpB does not function in yeast, swapping a region of these Hsp100 proteins that mediates 34

interaction with their cognate Hsp70s overcomes these species-specific restrictions (1, 2). 35

Additionally, co-expressing E. coli DnaK (Hsp70) and GrpE (NEF) with intact ClpB allows it to 36

function in place of Hsp104 to provide thermotolerance and to promote prion replication (2). 37

These findings show that amyloid remodeling activity of Hsp100 chaperone machinery is 38

conserved across life kingdoms and that specific interactions among these components of the 39

disaggregation machinery are necessary and sufficient for it to function properly in different 40

physiological contexts. 41

An earlier study reported that the 52% identical S. pombe Hsp104 (Sp-Hsp104) could function 42

in place of S. cerevisiae Hsp104 (Sc-Hsp104) to provide thermotolerance, but not propagation of 43

[PSI+] prions (9). One suggested explanation for the failure of Sp-Hsp104 to substitute for Sc-44

Hsp104 in [PSI+] propagation was that it requires interaction with cognate co-factors, which we 45

surmised would be its Hsp70/NEF system. Here, we used our system to test this hypothesis by 46

exchanging S. cerevisiae Hsp104, Hsp70 (Ssa1p, Ssa2p) and NEF (Fes1p) with their S. pombe 47


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counterparts. We find that these S. pombe disaggregation machinery components can act 48

individually in place of their S. cerevisiae counterparts to provide functions required for 49

propagation of not only [PSI+], but also [URE3] and [PIN+] prions. Our findings demonstrate 50

conservation of the functions of these components and suggest the possibility that S. pombe can 51

propagate prions. 52

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MATERIALS AND METHODS 54

Yeast strains, plasmids and media. S. cerevisiae strains 1408 ([PSI+], [PIN+]) and 1410 55

([URE3], [PIN+]), used to monitor HSP104 complementation, were described previously (2). 56

They are isogenic to wild type strain 779-6A (MATa kar1-01 SUQ5 ade2-1 his3Δ202 leu2Δ1 57

trp1Δ63 ura3-52), which was described previously (10). Both have hsp104::KanMX and carry a 58

URA3-based plasmid encoding HSP104. Strain 1410 has ADE2 regulated by the DAL5 promoter 59

(PDAL5::ADE2) in place of ade2-1. Isogenic strains 1135 and 1161, used to monitor Hsp70 60

complementation, are the same as 1408 and 1410, respectively, but have ssa1::KanMX 61

ssa2::HIS3 ssa3::TRP1 and ssa4::ura3-f2 and carry SSA1 or SSA2 on a plasmid to maintain 62

viability (11). Isogenic strain SY346, used to monitor complementation of Fes1p, is strain 1410 63

with fes1::KanMX. Strain YKT12, which was described previously (12), is unrelated to our 64

strains and carries the [PSI+]Sc4 prion (13). It is of the same genetic background as strain 65

YJW532 used by Senechal et al. (9). Wild type strain 74-D694 (3), widely used to study [PSI+] 66

prions, is unrelated to YKT12 and our other strains. ATCC strain 38366 was the source of 67

genomic DNA used as PCR template to obtain the S. pombe chaperone genes. 68

Plasmid pMR117 (2) is a TRP1-based single-copy plasmid with the Sc-HSP104 gene and 500 69

bp of upstream and downstream flanking DNA. Plasmid pMR116 is the same, but without the 70


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HSP104 coding region. Plasmid pMR170 is pMR117 with the Sp-HSP104 ORF in place of the 71

Sc-HSP104 coding region. It was created by PCR amplifying the S. pombe HSP104 ORF using 72

primers to add SbfI and XhoI at the 5' and 3' ends, respectively. This product was then cloned 73

into pMR116 digested with PstI and XhoI. Plasmid pMR171LA2 and pMR172LA2 are the S. 74

pombe SSA1 and SSA2 ORFs, respectively, driven by the S. cerevisiae SSA2 promoter in the 75

single-copy LEU2-based plasmid pRS415 (14). They were created by first replacing the GPD 76

promoter in p415-GPD (15) with a PCR-generated fragment containing the 500 bp upstream 77

sequence of S. cerevisiae SSA2 flanked by SacI and SpeI, to give p415-PSSA2. Sp-SSA1 was 78

amplified by PCR and cloned into p415-PSSA2 via SpeI/XhoI. Sp-SSA2 was cloned similarly 79

except the restriction sites used were SpeI and PstI. Plasmid pMR173 is single-copy HIS3-based 80

pRS313 (14) with the S. pombe FES1 ORF driven by the GPD promoter. It was created by 81

replacing the GrpE ORF in pMR142H (2) with the S. pombe FES1 ORF via XbaI/XhoI. Plasmids 82

p425GalSc104 and p425GalSp104 are Sc-HSP104 and Sp-HSP104 in p425-Gal1 (16), 83

respectively. They were created by sub-cloning Sc-HSP104 and Sp-HSP104 PCR fragments cut 84

with PstI/XhoI and SbfI/XhoI, respectively, into p425-Gal1 cut with PstI and XhoI. All cloned 85

genes were verified by sequencing. 86

1/2YPD is 0.5% yeast extract, 2% peptone and 2% glucose. It contains a limiting but 87

undefined concentration of adenine. Synthetic media contain 2% glucose, 0.7% yeast nitrogen 88

base and the appropriate nutrients except those required to maintain selection of plasmids or 89

prions. Liquid media contain excess (400 mg/l) adenine. Synthetic solid media contain 2% agar. 90

When included in synthetic solid media, adenine was added at a concentration of 10 mg/l, which 91

is limiting so that ade– cells form red colonies. 92


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Monitoring prions. Our strains use the white/ade+, red/ade– phenotypes to monitor [PSI+] and 93

[URE3] prions. In strains 1408 and 1135 the presence of [PSI+] suppresses the nonsense 94

mutation in ade2-1, which allows growth in the absence of adenine and confers white/pink 95

colony color on media containing limiting adenine. [psi–] cells are adenine auxotrophs and 96

appear red on limiting adenine. [PSI+] is monitored in strain YKT12 in a similar manner except 97

the prion suppresses a nonsense mutation in ade1-14. In strain 1410 a wild type ADE2 gene is 98

regulated by the DAL5 promoter, which is repressed by Ure2p under normal growth conditions. 99

When [URE3] is present Ure2p is depleted into prion aggregates, which activates the DAL5 100

promoter and expression of ADE2. The presence of [PIN+] prions was established by detection of 101

fluorescent foci after transforming cells with a plasmid encoding an Rnq1-GFP fusion protein 102

(17). 103

Curing of [PSI+] by overexpressing Hsp104. Curing experiments were performed as 104

described previously (18). Briefly, cells transformed by high-copy plasmids encoding various 105

alleles of galactose-inducible Hsp104 were grown for several cell divisions in galactose medium 106

and at various times were diluted and spread for 300-500 colonies on 1/2YPD plates. Entirely red 107

colonies were scored as having arisen from cells cured of prions. 108

109

RESULTS 110

S. pombe Hsp104 supports propagation of three S. cerevisiae prions. We monitored [PSI+] 111

and [URE3] prions by their effects on expression of Ade2p in strains 1408 and 1410, 112

respectively, which lack chromosomal HSP104 and express Sc-Hsp104 from a URA3-based 113

plasmid (see Methods section). In the absence of prions, the cells do not express Ade2p and 114

therefore require adenine for growth and accumulate red pigment when adenine is limiting. The 115


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presence of either prion causes enough expression of Ade2p to restore adenine protrophy and 116

white colony color. 117

To assess the ability of Sp-Hsp104 to propagate [PSI+] or [URE3] in place of Sc-Hsp104, 118

strains 1408 and 1410 were first transformed by TRP1-based plasmids encoding Sp-Hsp104, Sc-119

Hsp104 or the empty vector. The transformants were grown on medium containing uracil to 120

allow loss of the URA3 plasmid encoding Sc-Hsp104, and then replica-plated onto medium 121

containing 5-fluoro-orotic acid (FOA), which kills cells expressing Ura3p. Cells from the FOA 122

plates, which express Hsp104 only from the TRP1 plasmids, were then checked for the presence 123

of prions. 124

[PSI+] prions propagated in cells expressing either Sp-Hsp104 or Sc-Hsp104 (Fig. 1A). To 125

determine if this unexpected finding was due to our use of a different strain background, we 126

performed the same plasmid exchange using strain YKT12 (12), which has the same background 127

as the strain used in the earlier study (9). All of 20 Sp-Hsp104 transformants from three 128

independent transformations of strain YKT12 that subsequently underwent the plasmid exchange 129

propagated [PSI+]. Since [PSI+] suppresses ade1-14 in YKT12 less efficiently than ade2-1 in 130

our strains, YKT12 [PSI+] cells have a pinker color when grown under the same conditions. 131

Nevertheless, the prion phenotype of YKT12 cells expressing Sp-Hsp104 was similar to that of 132

cells expressing Sc-Hsp104. In all instances, the presence of [PSI+] in these cells was confirmed 133

using standard assays by its dominant phenotype in crosses and its elimination when cells were 134

grown in the presence of 3 mM guanidine, which inactivates Hsp104 (19, 20). 135

Using strain 1410 we found that Sp-Hsp104 also supported stable propagation of [URE3] 136

(Fig. 1A). Since [PSI+] and [PIN+] are compatible and propagate stably in cells of strain 1408 137

(2), we also examined the [PSI+] cells expressing only Sp-Hsp104 to see if [PIN+] survived the 138


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plasmid exchange. We found [PIN+] was present in all clones examined (Fig. 1B), showing that 139

Sp-Hsp104 also supported stable propagation of [PIN+] prions. Therefore, Sp-Hsp104 functioned 140

in place of Sc-Hsp104 to support propagation of three different yeast prions. 141

S. pombe Hsp70s support S. cerevisiae cell growth and propagation of [PSI+] and [URE3] 142

prions. Since our findings implied that Sc-Hsp70 cooperates with Sp-Hsp104 to fragment prion 143

polymers, we anticipated that S. pombe Hsp70s would cooperate with S. cerevisiae Hsp104 to 144

provide the Hsp70 activity required for prion propagation. To test this idea we used strains 1135 145

(for monitoring [PSI+]) and 1161 (for monitoring [URE3]), which lack all four SSA subfamily 146

genes and carry SSA1 or SSA2, respectively, on a URA3-based plasmid to support viability and 147

prions. The different Hsp70s were used because our earlier characterization of [PSI+] and 148

[URE3] prion phenotypes in cells expressing Ssa1p or Ssa2p as the only essential Ssa family 149

Hsp70 showed that the [PSI+] phenotype is normal in cells expressing only Ssa1p, but has a 150

slightly weaker phenotype when cells express only Ssa2p (11). Conversely, [URE3] is 151

mitotically unstable in cells expressing only Ssa1p, but propagates normally in cells expressing 152

only Ssa2p. 153

After exchanging the resident URA3 plasmids encoding the Sc-Hsp70s in strains 1135 and 154

1161 with LEU2-based plasmids encoding Sp-Ssa1p or Sp-Ssa2p, we found that both of the Sp-155

Hsp70s supported robust growth of S. cerevisiae. In cells expressing the Sp-Hsp70s in place of 156

Sc-Hsp70s, [PSI+] propagated normally, but [URE3] was mitotically unstable, especially in cells 157

expressing only Sp-Ssa2p (Fig. 2A). Therefore, both Sp-Hsp70s were able to cooperate with Sc-158

Hsp104 to promote prion replication, and the latter results suggest [URE3] requires specific non-159

essential Hsp70 functions that are incompletely complemented by the Sp-Hsp70s. 160


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Sp-Ssa1p and Sp-Ssa2p are functionally distinct. In a manner similar to that seen with Sc-161

Ssa1p/Ssa2p, we observed that prion phenotypes were different in cells expressing the different 162

S. pombe Hsp70s. For example, a slight, but noticeable increase in pigmentation was seen in 163

[PSI+] cells expressing Sp-Ssa1p and Sc-Ssa2p (Fig. 2A, left center panel), which indicates Sp-164

Ssa1p behaves more like Sc-Ssa2p, and Sp-Ssa2p functions more like Sc-Ssa1p. A more obvious 165

reduction in efficiency of prion propagation was reflected by the [URE3] phenotypes. Here, 166

[URE3] was clearly less stable in cells expressing Sp-Ssa2p than in cells expressing Sp-Ssa1p 167

(Fig. 2B). In this respect, Sp-Ssa2p again functioned more like Sc-Ssa1p than Sc-Ssa2p. 168

S. pombe Fes1p supports propagation of [URE3] in place of S. cerevisiae Fes1p. Hsp70 169

NEFs are also important for propagation of yeast prions, and propagation of [URE3] depends 170

upon the NEFs Sse1p and Fes1p (2, 21-23). To assess ability of Sp-Fes1p to function in place of 171

Sc-Fes1p, we first expressed Sp-Fes1p from a single-copy (CEN) plasmid in a diploid lacking 172

one of its two chromosomal copies of FES1. We then induced the strain to undergo meiosis and 173

sporulation and analyzed the haploid progeny. CEN plasmids typically segregate to two of the 174

four spores. 175

Fig. 3 shows the four meiotic progeny of a diploid cell in which all four of the possible 176

combinations of FES1 genotypes are represented. Although our fes1Δ cells were able to maintain 177

[URE3] when grown on medium without adenine (Fig. 3A), they grew more slowly without 178

adenine and under non-selective conditions they accumulated red pigment and lost [URE3] at a 179

very high frequency (Fig. 3B). These phenotypes reflect a strong impairment of [URE3] 180

propagation in the absence of Fes1p, which is in line with earlier work showing that stable 181

propagation of [URE3] requires Fes1p (23). Cells expressing Sc-Fes1p from the chromosome 182

(FES1+) had a typical [URE3] phenotype, and when FES1+ cells expressed Sp-Fes1p from the 183


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plasmid they had a slight pink color Fig. 3A, clones c and a, respectively). When sub-cultured, 184

however, the clones expressing both Sc-Fes1p and Sp-Fes1p had a normal, stable [URE3] 185

phenotype. Although cells expressing only Sp-Fes1p propagated [URE3], there was a consistent 186

infrequent loss of [URE3] upon sub-culturing under conditions where the prion is not required 187

for growth (i.e. when adenine is present). However, [URE3] was much more stable in these cells 188

than in those lacking Fes1p (Fig. 3B). Thus, Sp-Fes1p possessed a substantial amount of the Sc-189

Fes1p functions required for propagation of [URE3]. 190

Plasmid-expressed Sp-Hsp104 does not "cure" cells of [PSI+]. Although Hsp104 is 191

necessary for prion propagation, transiently overexpressing Sc-Hsp104 "cures" cells of [PSI+] 192

prions (3). In the hsp104Δ strains where [PSI+] is supported by HSP104 on a single-copy 193

plasmid, expression of Hsp104 is modestly elevated compared with wild type cells (24), which 194

causes [PSI+] to be weaker and mitotically unstable. In contrast, overexpressing Sp-Hsp104, 195

even at higher levels, is ineffective at inhibiting [PSI+] propagation (9). The pinker color of 196

strain 1408 [PSI+] cells expressing Sc-Hsp104 compared with the whiter color of those 197

expressing Sp-Hsp104 from the plasmid (Fig. 1A, upper panel) is consistent with these 198

observations. Additionally, when we streaked these cells for isolated colonies we observed that 199

although [PSI+] was clearly weakened and mitotically unstable in the two hsp104Δ strains 200

expressing Sc-Hsp104, those expressing Sp-Hsp104 had strong and stable [PSI+] phenotypes 201

(Fig. 4). These data are consistent with the finding that overexpression of Sp-Hsp104 does not 202

inhibit [PSI+] propagation. 203

To test this conclusion more rigorously, we induced high-level expression of Hsp104 from 204

galactose-inducible promoters on a high-copy plasmid in wild type strains 779-6A and 74D-694, 205

which are from different backgrounds and have [PSI+] prions derived from different sources. Sc-206


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Hsp104 efficiently cured both strains of their [PSI+] prions, but Sp-Hsp104 did not (see Fig. 5, 207

Table 1). Taken together our data indicate that Sp-Hsp104 possessed Sc-Hsp104 activities 208

required for propagation of [PSI+], but lacked an Sc-Hsp104 function necessary for curing cells 209

of [PSI+] when overexpressed. These results are consistent with other data showing that Hsp104 210

activities important for replication and curing of [PSI+] prions are separable (17, 18, 24). 211

Overexpressing Sc/Sp-Hsp104 hybrids cures [PSI+] by impairing prion replication. Since 212

Sc-Hsp104 lacking its NTD behaves like Sp-Hsp104 in that it propagates [PSI+] but does not 213

cure [PSI+] when overexpressed (18), we considered that Sp-Hsp104 lacked an NTD activity 214

required for curing by overexpression. We therefore assessed curing ability of Sp-Hsp104 after 215

replacing its amino-terminal domain (NTD) with that of Sc-Hsp104. Overexpressing this hybrid 216

protein, named 4PPP, did not cure [PSI+] (Fig. 5, Table 1). These results suggest that a non-217

conserved Hsp104 function separate from that residing in the NTD might be necessary for the 218

curing. 219

Overexpressing Hsp104 cures cells of [PSI+] by a mechanism clearly distinct from that of 220

inactivating Hsp104. The processes differ by the kinetics of curing and by the phenotypes of 221

colonies arising from cells of cultures undergoing curing. Inhibiting Hsp104 by chemical means 222

or by expressing dominant negative Hsp104 mutants arrests replication of heritable prion 223

particles, or propagons (10, 25-28). [PSI+] cells typically have about 100-300 propagons per cell 224

(29), so after inactivating Hsp104 there is a lag of 4-6 cell divisions before [psi–] cells appear 225

while the non-replicating propagons are diluted among the dividing cells until there are too few 226

of them to be inherited by all the progeny. Beyond that point, the number of [psi–] cells roughly 227

doubles after each cell division. When assessed during the curing, many [PSI+] cells recovering 228

on plates where Hsp104 regains activity grow into colonies with several red sectors containing 229


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[psi–] progeny. In contrast, overexpressing wild type Hsp104 causes [psi–] cells to arise shortly 230

after inducing expression and at a linear rate of roughly 10% per cell division (24). [PSI+] cells 231

recovering on plates where Hsp104 abundance returns to normal give rise to colonies that are 232

entirely white or that have one or very few red sectors. 233

We tested other Hsp104 hybrids with swapped domains (P444, PPP4 and 444P) and found 234

that, unlike 4PPP, they all cured [PSI+] when they were overexpressed. However, both a lag in 235

the kinetics of curing and a highly sectored colony phenotype from cells overexpressing these 236

hybrids indicated that the curing was caused by arrested prion replication followed by dilution of 237

propagons among dividing cells (Fig. 5, Table 1). These phenotypes are characteristic of cells in 238

which Hsp104 function has been inhibited, as in cells expressing known dominant negative 239

Hsp104 mutants, such as Hsp104-KT (Fig. 5, Table 1) (3, 24, 26, 30). Thus, the hybrid proteins 240

cured [PSI+] by a dominant negative mechanism that is unlike curing by overexpression of wild 241

type Sc-Hsp104. 242

Using the plasmid shuffle described above we found that P444 and 4PPP were able to 243

propagate [PSI+] in place of Sc-Hsp104, again showing that NTD function is not critical for 244

[PSI+] propagation (Fig. 5D). The ability of hybrid P444 to support [PSI+] propagation was 245

unexpected because overexpressing it had a dominant inhibitory effect on the ability of Sc-246

Hsp104 to propagate [PSI+]. More work will be necessary to explain this unusual combination of 247

traits. Neither 444P nor PPP4 were able to propagate [PSI+]. This lack of function is consistent 248

with their curing of [PSI+] by a dominant negative effect. 249

250

DISCUSSION 251


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In contrast to earlier work (9), we find that Sp-Hsp104 supports stable propagation of [PSI+] 252

prions in S. cerevisiae. We ruled out the possibility that this finding was due to differences in 253

yeast strain background. Additionally, the variants of [PSI+] prions propagating in the 1408 and 254

YKT12 strains used here are distinct, indicating that Sp-Hsp104 can act on different 255

conformational variants of prions composed of the same Sup35p. Moreover, Sp-Hsp104 also 256

supported stable propagation of [URE3] and [PIN+] prions, which shows that it possesses general 257

Sc-Hsp104 activities required for replication of prions composed of different proteins. We 258

cannot explain the discrepancy with the earlier study, but it is possible that the earlier work was 259

done using a different S. pombe HSP104 allele. 260

We recently showed that the E. coli homolog of Hsp104 could promote propagation of yeast 261

prions only if its cognate Hsp70 and NEF are co-expressed, or if it is modified to interact with 262

yeast Hsp70. This specificity of cooperation between Hsp100 and Hsp70 indicates that the ability 263

of Sp-Hsp104 to propagate prions in place of Sc-Hsp104 must reflect its ability to cooperate 264

functionally with the S. cerevisiae Hsp70 system. Consequently, we were not surprised to find 265

that both Sp-Hsp70s, which are 94% identical to each other and 80% identical to Sc-Ssa1/2p, 266

functioned reciprocally as components of the S. cerevisiae Hsp104 disaggregation machinery to 267

promote prion replication. Additionally, our results showing that Sp-Ssa1p and Sp-Ssa2p were 268

much like Sc-Ssa1p and Sc-Ssa2p in their distinct affects on prions suggest that certain 269

differences in the functions of nearly identical Hsp70s within species are conserved across long 270

diverged species. 271

Our finding that S. pombe Hsp70s supported propagation of prions in budding yeast would not 272

have been possible if they did not also provide the essential Hsp70 functions required to support 273

viability of S. cerevisiae. The strong growth complementation by the Sp-Hsp70s reflects a high 274


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level of conservation of essential Hsp70 functions among these S. pombe and S. cerevisiae 275

chaperones. Essential cellular processes that require Hsp70, such as translation and transport of 276

proteins across membranes, depend on cooperation of Hsp70 with many regulatory co-factors. 277

For example, protein transport relies on complexes that include Hsp40s, NEFs and 278

tetratricopeptide-repeat (TPR) containing co-chaperones that recruit Hsp70 and regulate its 279

activity (31). Therefore, in addition to being able to function with the disaggregation machinery, 280

the ability of Sp-Hsp70s to support such robust growth shows that they interact functionally and 281

efficiently with these S. cerevisiae Hsp70 system components. While it can be presumed that 282

Hsp70 interactions with its obligate Hsp40 co-chaperones occur promiscuously, our finding that 283

Sp-Fes1p largely compensates for the loss of Sc-Fes1p demonstrates a specific aspect of this 284

conservation. 285

Although overexpressing Hsp104 was shown to cure cells of [PSI+] prions many years ago, 286

the molecular mechanism underlying the curing has not been resolved because many factors 287

strongly influence the curing. Some Hsp70s (Ssb) enhance curing (32) while others (Ssa) 288

antagonize it (33, 34). Moreover, Hsp40s, NEFS, Hsp90 machinery components, ubiquitin, actin 289

and factors involved in anchoring membrane proteins all influence the curing (17, 24, 35-38). 290

Here we confirm the earlier findings that Sp-Hsp104 does not cure cells of [PSI+], and that 291

swapping the C-terminal regions of Sp-Hsp104 and Sc-Hsp104 produced hybrids that cured 292

[PSI+] when overexpressed. However, we find that all our hybrid proteins that cure do so by 293

dominant negative effects. Therefore, these hybrids are not useful for identifying the regions of 294

Sc-Hsp104 that confer functions specifically required for curing of [PSI+] by Hsp104 295

overexpression. Nevertheless, the reciprocal compatibility between the interspecies Hsp100 and 296


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Hsp70 systems implies that the distinction is not likely to be related to differences in interactions 297

among these disaggregation machinery components. 298

Our CTD hybrids differed from those described earlier in that the region we swapped was 299

extended to include NBD2. Thus, it is possible that curing by the Hsp104 hybrids with the 300

smaller CTD occurs by the overexpression mechanism rather than by a dominant negative effect. 301

In line with this possibility, the inability of the 4PPP hybrid to cure [PSI+] prions suggests an Sc-302

Hsp104-specific function residing outside the NTD is important for curing. Nevertheless, further 303

work with the alleles containing swaps of the smaller CTD regions will be necessary to 304

determine the mechanism by which they cure. The ways we show to distinguish curing by 305

overexpression or dominant negative effects will be useful for such work and for other studies 306

that address how overexpressing Hsp104 cures [PSI+]. For example, recent work led to a 307

suggestion that overexpressing Hsp104 cures cells of [PSI+] by inhibiting the fragmentation of 308

prion fibers that produces propagons, or, in other words, by inhibiting prion replication (39). 309

Blocking prion replication, however, is the way that inhibiting Hsp104 causes curing, which is 310

distinct from that of overexpressing wild type Hsp104. 311

Taken together, our results show a very high degree of conservation of protein disaggregation 312

machinery functions between S. pombe and S. cerevisiae, in particular those required for 313

propagation of different prions in S. cerevisiae. These findings imply that S. pombe possesses the 314

chaperone machinery functions required to propagate its own prions. Such a capability would 315

provide another useful model system to study prion/amyloid behavior and might have important 316

implications with regard to S. pombe phenotypes. 317

318

ACKNOWLEDGEMENTS 319


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We thank Jonathan Weissman (UCSF) for strain YKT12, and our NIH colleagues for insightful 320

discussion and critical reading of the manuscript. 321

This work was supported by the Intramural Research Program of the NIH, National Institute 322

of Diabetes and Digestive and Kidney Diseases. 323

324

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Table 1. Curing of [PSI+] by Hsp104 overexpression. 432

Percent [PSI+] cells

Overexpressed

Hsp104

Strain 779-6A Strain 74D-694

3 gen 6 gen 9 gen 3 gen 6 gen 9 gen

ev 100 100 100 100 100 100

Sc-Hsp104 84 (1.2) 72 (2.6) 63 (3.1) 80 (1.0) 69 (3.4) 56 (5.5)

Sp-Hsp104 100 100 100 100 100 100

4PPP 100 100 nd 100 100 nd

P444 99 (0.4) 93 (1.1) nd 99 (.02) 98 (1.2) nd

PPP4 99 (0.2) 99 (0.2) 91 (1.7) 99 (0.3) 99 (0.4) 75 (2.9)

444P 99 (0.1) 94 (0.8) nd 99 (0.5) 99 (0.3) nd

Hsp104-KT 99 (0.3) 95 (2.0) 84 (1.3) nd nd nd

Strains carrying plasmids encoding galactose-inducible HSP104 alleles were grown to mid-log 433

phase in -Trp dextrose medium without adenine to maintain selection for prions, and then 434

transferred to -Trp galactose medium containing adenine to induce Hsp104 expression and 435

allow prion loss. When cultures reached the indicated number of generations (gen) aliquots 436

were removed and spread at a concentration of 300-500 cells per plate onto 1/2YPD. Only 437

entirely red colonies were scored as [psi–]. Values are percent [PSI+] among 1200-1500 438

colonies, with SD in parentheses; nd, not determined. 4PPP and P444 are NTD swapped 439

hybrids, PPP4 and 444P are CTD swapped hybrids (see Fig. 5A), ev is empty vector and 440

Hsp104-KT is dominant negative Hsp104 mutant. 441

442

443


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444

Fig. 1. S. pombe HSP104 supports propagation of prions in S. cerevisiae. (A) The hsp104Δ 445

strains 1408 ([PSI+]), YKT12 ([PSI+]Sc4) and 1410 ([URE3]) carry empty URA3-based and 446

TRP1-based plasmids (ev+ev), or Sc-HSP104 on a resident URA3-based plasmid plus Sc-447

HSP104 or Sp-HSP104 on TRP1-based plasmids, as indicated. Patches of cells grown on plates 448

selecting for both plasmids (not shown) were replica-plated onto -Trp medium containing uracil 449

and limiting adenine (upper panels). Cells from this plate were then replica-plated onto FOA 450

medium lacking adenine, where only cells that both express the indicated Hsp104 and carry 451

prions will grow (lower panels). Six representative transformants of each from one of three 452

independent experiments are shown. (B) Cells of strain 1408 from panel (A) carrying empty 453

vector (ev) only or expressing the indicated Hsp104 were transformed by a HIS3-based plasmid 454

encoding an Rnq1-GFP fusion protein. Cells with diffuse fluorescence are [pin–]; the presence of 455

[PIN+] prions is indicated by punctate fluorescence. 456


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457

Fig. 2. S. pombe Hsp70s complement S. cerevisiae Hsp70s for growth and prion propagation. 458

(A) Patches of cells of strains 1135 ([PSI+], left panels) and 1161 ([URE3], right panels) carrying 459

URA3-based plasmids encoding Sc-SSA1 and Sc-SSA2, respectively, and the indicated SSA1 and 460

SSA2 alleles on LEU2-based plasmids, were transferred from plates selecting for both plasmids 461

onto medium lacking leucine and containing uracil (upper panels). These were then replica-462

plated onto medium containing FOA and limiting adenine (middle panels) or FOA without 463

adenine (lower panels). Three individual transformants of each from one of three independent 464

experiments are shown. (B) [URE3] cells expressing the indicated SSA allele as the only source 465

of Ssap were taken from the FOA –Ade plate, streaked onto 1/2YPD and grown three days at 466

30º.467


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468

Fig. 3. S. pombe Fes1p possesses S. cerevisiae Fes1p function required for [URE3] propagation. 469

(A) A [URE3] fes1Δ/FES1 diploid carrying a plasmid encoding Sp-FES1 was sporulated and 470

tetrads were dissected on rich medium. Spore clones were then replica-plated onto the indicated 471

media to monitor fes1Δ (G418), [URE3] (–Ade) and Sp-FES1 (–His). A tetrad with all four 472

possible combinations of FES1 genotypes is shown. The presence of genomic Sc-FES1 and 473

plasmid-borne Sp-FES1 is indicated at the bottom. (B) Spore clones lacking FES1 (fes1Δ) or 474

expressing Sp-FES1 from the –Ade plate in panel (A) were streaked onto 1/2YPD plates and 475

grown three days at 30º to assess [URE3] phenotype. Cells expressing Sc-FES1 (lower panels) 476

show wild type [URE3] and [ure-o] phenotypes. 477

478


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479

Fig. 4. Plasmid-expressed S. cerevisiae Hsp104, but not S. pombe Hsp104, cures cells of [PSI+] 480

prions. Cells of hsp104Δ strains 1408 and YKT12 expressing the indicated Hsp104 proteins from 481

single-copy plasmids were streaked for individual colonies and grown on medium selecting for 482

the plasmids and containing limiting adenine. Cells expressing Sp-Hsp104 (right panels) have 483

normal prion phenotypes. Pinker color of [PSI+] colonies and presence of red [psi–] colonies in 484

both strains expressing Sc-Hsp104 (center panels) reflects inhibition of [PSI+] propagation by 485

Sc-Hsp104. 486

487

488

489

490

491

492

493

494

495

496

497


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498

Fig. 5 Curing of [PSI+] by overexpressing alleles of Hsp104. (A) Domain organization of S. 499

cerevisiae (Sc) and S. pombe (Sp) Hsp104s and their hybrids. Numbers indicate amino acid 500

residues that delineate domains: NTD, N-terminal domain; NBD1/M nucleotide-binding domain 501

1/M region; NBD2, nucleotide-binding domain 2; CTD, C-terminal domain. (B) Kinetics of 502

curing of [PSI+] by overexpression of Sc-Hsp104 versus its inactivation. Aliquots of cultures 503

grown in galactose medium were removed at three, six and nine generations, plated on 1/2YPD 504

and incubated at 30°C for 3 days to determine the proportion of [PSI+] cells. Dashed line is 779-505

6A expressing dominant negative Hsp104-KT. Data are from 1200-1500 colonies counted for 506

each time point in at least three independent experiments. Error bars show standard deviation. 507

(C) Representative images of colonies from experiment described in panel B. Sp-Hsp104 and 508

hybrid 4PPP did not affect [PSI+] stability. (D) Hybrids 4PPP and P444 support prion 509

propagation. Strain 1408 was transformed by single-copy TRP1 plasmids expressing indicated 510

Hsp104 proteins from the HSP104 promoter and processed as described in Fig. 1. Shown are 511

cells on –Trp plates, with and without limiting adenine, replica-plated from FOA. 512


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