NR ASXZ
AU Song,Y.; Wu,Y.X.; Jung,G.; Tutar,Y.; Eisenberg,E.; Greene,L.E.; Masison,D.C.
TI Role for Hsp70 chaperone in Saccharomyces cerevisiae prion seed replication
QU Eukaryotic Cell 2005 Feb; 4(2): 289-97
PT journal article
AB 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.
MH Adenosinetriphosphatase/genetics/*metabolism; Cell Fractionation; Fluorescence Recovery After Photobleaching; Heat-Shock Proteins 70/genetics/*metabolism; Prions/genetics/*metabolism; Recombinant Fusion Proteins/genetics/metabolism; Saccharomyces cerevisiae/*physiology; Saccharomyces cerevisiae Proteins/genetics/*metabolism
AD Laboratory of Biochemistry and Genetics, National Institute of Diabetes Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
SP englisch
PO USA