FIG. 1.
The J domain of Cwc23 is dispensable. (A) Cwc23 has a conserved J domain. A sequence alignment of the predicted J domains of the indicated J proteins of S. cerevisiae is shown. The conserved histidine-proline-aspartic acid (HPD) tripeptide motif is marked (***). (B) The J domain is not critical for growth. In panels 1 and 2, equal numbers of cwc23Δ cells harboring two plasmids—URA3 CEN-Cwc23 and a vector containing no insert (−), CWC23 (WT), cwc23H50Q (H50Q), or cwc23ΔJ (ΔJ)—were spotted on medium without (−) or with (+) 5-FOA to select for cells having lost the URA3 CEN-Cwc23 plasmid, as indicated. In panels 3 to 5, 10-fold serial dilutions of equivalent numbers of cwc23Δ cells expressing either wt Cwc23 (WT), Cwc23H50Q (H50Q), or Cwc23ΔJ (ΔJ) were spotted on selective medium and incubated at the indicated temperatures for 3 days. (C) Cwc23's J domain is functional. For the left panel, 10-fold serial dilutions of an equivalent number of ydj1Δ cells expressing either wt Ydj1 from its own promoter (Ydj1), or Cwc23 (WT), or Cwc23H50Q (H50Q) from the GPD promoter or harboring only an empty vector (−) were spotted on selective medium and incubated at 30°C for 3 days. For the right panel, total protein was isolated from strains indicated in the left panel and subjected to SDS-PAGE, electroblotted, and probed with antibodies specific for Cwc23 or, as a loading control, Ssc1 (row C). (D) Cwc23 stimulates the ATPase activity of the Hsp70, Ssa1. Affinity-purified Cwc231-225-His6 (WT) or the J domain mutant (H50Q) was incubated with equimolar amounts of Ssa1-[α-32P]ATP (left panel) or Ssb1-[α-32P]ATP (right panel). Reactions were stopped after the indicated times and subjected to chromatography. ATP hydrolysis was quantified, and the data were fit to a one-phase exponential decay equation.