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.