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Figure 6.—

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Direct interactions between Hsp70 and Sup35 proteins. (A) Ssa and Ssb, but not Hsp104, interact with Sup35NM in vitro. Extracts of the E. coli strain, overproducing Sup35NM-(His)6 (“After Sup35NM-His”), and of the identical strain containing the pET-20b (+) plasmid that did not produce Sup35NM-(His)6 (“Control”), were run through a Ni2+ resin under conditions where Sup35NM-(His)6 is immobilized on this resin. Then these samples of resin were treated with extracts of the [PSI+] yeast strain OT56. Immobilized proteins were eluted from the resin, run on SDS-PAGE gel, and reacted to the appropriate antibodies. Total yeast lysates not exposed to the columns were run on the SDS-PAGE gel for comparison. The same observations were made with the extracts of strain OT55 (not shown). Similar data were obtained in at least three independent experiments for each protein. Results of one typical experiment are shown. (B) In vivo interaction of Sup35 with Ssa1 and Ssb1. Proteins were isolated from the strain GT159, transformed with the plasmid pmCUP1-SUP35-HA that expresses HA-tagged Sup35 from PCUP1 promoter. Production of Sup35-HA was induced by 100 μm CuSO4. Hsp, Sup35, and Ade2 proteins were identified by SDS-PAGE and Western blot either directly in total lysates or after immunoprecipitation with antibody against HA or Ade2 (IP). Bands reacting to Ssa and Ssb antibodies (but not to Hsp104 antibody) are visible in HA immunoprecipitates but not in Ade2 immunoprecipitates. In the cases of Ssa and Hsp104, a similar result was obtained when endogenous Sup35 was immunoprecipitated with the Sup35C antibody from the strain GT159 that did not contain PCUP1-SUP35-HA plasmid (data not shown).

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