title: Role for Hsp70 chaperone in Saccharomyces cerevisiae prion seed replication.
abstract: The Saccharomyces cerevisiae [PSI+] prion is a misfolded form of Sup35p that propagates as self-replicating cytoplasmic aggregates. Replication is believed to occur through breakage of transmissible [PSI+] prion particles, or seeds, into more numerous pieces. In [PSI+] cells, large Sup35p aggregates are formed by coalescence of smaller sodium dodecyl sulfate-insoluble polymers. It is uncertain if polymers or higher-order aggregates or both act as prion seeds. A mutant Hsp70 chaperone, Ssa1-21p, reduces the number of transmissible [PSI+] seeds per cell by 10-fold but the overall amount of aggregated Sup35p by only two- to threefold. This discrepancy could be explained if, in SSA1-21 cells, [PSI+] seeds are larger or more of the aggregated Sup35p does not function as a seed. To visualize differences in aggregate size, we constructed a Sup35-green fluorescent protein (GFP) fusion (NGMC) that has normal Sup35p function and can propagate like [PSI+]. Unlike GFP fusions lacking Sup35p's essential C-terminal domain, NGMC did not form fluorescent foci in log-phase [PSI+] cells. However, using fluorescence recovery after photobleaching and size fractionation techniques, we find evidence that NGMC is aggregated in these cells. Furthermore, the aggregates were larger in SSA1-21 cells, but the size of NGMC polymers was unchanged. Possibly, NGMC aggregates are bigger in SSA1-21 cells because they contain more polymers. Our data suggest that Ssa1-21p interferes with disruption of large Sup35p aggregates, which lack or have limited capacity to function as seed, into polymers that function more efficiently as [PSI+] seeds.
authors.affiliation: Laboratory of Biochemistry and Genetics, National Institute of Diabetes Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Youtao
authors.initials: Y
authors.lastname: Song
authors.forename: Yue-Xuan
authors.initials: YX
authors.lastname: Wu
authors.forename: Giman
authors.initials: G
authors.lastname: Jung
authors.forename: Yusuf
authors.initials: Y
authors.lastname: Tutar
authors.forename: Evan
authors.initials: E
authors.lastname: Eisenberg
authors.forename: Lois E
authors.initials: LE
authors.lastname: Greene
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Masison
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D002458:Cell Fractionation; D036681:Fluorescence Recovery After Photobleaching; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D010454:Peptide Termination Factors; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D010454:Peptide Termination Factors; D011328:Prions; D011993:Recombinant Fusion Proteins; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae