Deletion of the Hsp70 chaperone gene SSB causes hypersensitivity to guanidine toxicity and curing of the [PSI+] prion by increasing guanidine uptake in yeast.
Yeast Ssb proteins (Ssbp) are ribosome-associated Hsp70 chaperones that function in translation. Elevated levels of Ssbp enhance the ability of over-expressed Hsp104 chaperone to eliminate the yeast [PSI+] prion, while depletion of Ssbp reduces this effect. Millimolar concentrations of guanidine in the growth medium cure yeast cells of prions by inactivating Hsp104. Guanidine is also toxic to yeast, irrespective of the status of Hsp104 and [PSI+]. Strains that lack Ssbp are hypersensitive to guanidine toxicity. Here we show that ssb- cells have normal numbers of [PSI+] "seeds", but can be cured of [PSI+] using one-sixth of the guanidine concentration required to eliminate [PSI+] from SSB cells. Correspondingly, the level of intracellular guanidine was eight-fold higher in ssb- cells than in wild-type cells, which explains all effects of Ssbp depletion on susceptibility to guanidine. The sensitivity of wild-type cells to the effects of guanidine also correlated with guanidine uptake, which was enhanced at low temperature. Guanidine sensitivity of strains mutated in any of 16 ABC membrane transporters, which are implicated in multidrug resistance, was normal. We found that an erg6 mutant that has an altered membrane lipid composition was hypersensitive to guanidine toxicity, but the lipid composition of ssb- cells was identical to that of wild-type cells. Our results suggest that Ssbp depletion does not affect prion seed regeneration, and that elevated guanidine uptake by ssb- cells may be due to increased retention rather than to an alteration in active or passive transport of the compound.
Jones
G W
GW
Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, 8 Center Drive, Rm 407, MSC 0851, MD 20892-0851, Bethesda, USA.
Song
Y
Y
Masison
D C
DC
D002455:Cell Division / Q000502:physiology; D005656:Fungal Proteins / Q000378:metabolism*; D019791:Guanidine / Q000378:metabolism / Q000633:toxicity*; D018840:HSP70 Heat-Shock Proteins / Q000172:deficiency / Q000235:genetics*; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D010454:Peptide Termination Factors; D011328:Prions; D012333:RNA, Messenger / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D010454:Peptide Termination Factors; D011328:Prions; D012333:RNA, Messenger; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase; D019791:Guanidine